Datasets:

evidence_infer_treatment

like 6

TITLE: Randomized Controlled Trials of Proximal Femoral Nail Antirotation in Lateral Decubitus and Supine Position on Treatment of Intertrochanteric Fractures ABSTRACT: The objective of this study was to compare the clinical results and complications of proximal femoral nail antirotation (PFNA) on treatment of intertrochanteric fractures in 120 elderly Chinese patients using Randomized Controlled Trials (RCTs). Totaly 120 cases enrolled were randomly assigned to a lateral decubitus position group and supine position group. The hospital stay, operating time, intraoperative blood loss, length of incision, X-ray fluoroscopy time, and out-of-bed activity time in the lateral decubitus position group were significantly lower than those in the supine position group. There was not statistical significance on union time and Harris values in the two position groups. Moreover, only complications of superficial wound infection were observed in the lateral decubitus position group, but two complications of deep venous thrombosis and wound deep infection were found in the supine position group. The present findings suggested that PFNA applied in elderly patients with intertrochanteric fracture can get satisfactory effects, and the treatment of intertrochanteric fractures using lateral decubitus position showed a satisfactory clinical outcome and a lower radiological complication rate. BODY.1. INTRODUCTION: Intertrochanteric fractures are defined as "fractures involving upper end of femur through and in between both trochanters with or without extension into upper femoral shaft." An increasing incidence of intertrochanteric fractures with advancing age is well known, more in men than women, about 1.5 : 1, being outside the joint capsule fracture [1, 2]. The incidence of intertrochanteric fractures varies from country to country. An increase in the elderly population has resulted in a higher incidence of pertrochanteric fractures of the femur. The annual global number of hip fractures is expected to exceed 7 million in the next 40 to 50 years [1, 3]. These fractures will occur more often in the future. Thus, the successful treatment of these fractures is becoming increasingly important part of an orthopaedic practice. The treatment of these fractures remains a challenge to surgeons. The various treatment options for intertrochanteric fractures are operative and nonoperative. Surgical stabilization with implants is the preferred treatment method for ipsilateral intertrochanteric and femoral shaft fractures, according to most published reports. Various techniques and implants have been developed to manage these complex fractures [1]. Several clinical and biomechanical studies have analyzed the results of different implants such as the dynamic hip screw (DHS), the Gamma nail (GN), and the proximal femoral nail (PFN). Those devices have suffered cutout, implant breakage, femoral shaft fractures and subsequent loss of reduction in the clinical practice [2, 4]. Proximal femoral nail antirotation (PFNA) is a novel intramedullary device with a helical blade that is inserted by impaction, causing bone compaction around the blade; this compaction retards rotation and varus collapse [1, 5, 6]. These characteristics provide optimal anchoring and stability when the implant is inserted into osteoporotic bone. Recent studies have shown favorable clinical results for unstable intertrochanteric fractures treated with PFNA [7–10]. Based on the previous description, PFNA has shared an excellent implant for a wide variety of indications. However, adequate knowledge and experience of operative technique is imperative. Rohilla et al. reported that 41 patients of femoral shaft fractures had closed intramedullary nailing in lateral decubitus position without fracture table or image intensifier, and results suggested that this technique a safe and reliable alternative to achieve closed locked intramedullary nailing without the use of image intensifier and fracture table [11]. Kim et al. have reviewed that patients with femur fractures were treated with closed femoral intramedullary nailing in lateral decubitus position on radiolucent routine table, and results indicated that closed femoral intramedullary nailing in lateral decubitus position with the aid of intraoperative skeletal traction is safe and an effective technique with a low incidence of complications compared to the use of fracture table [12]. Based on the previous results, we hypothesized a lateral decubitus position approach as a simple and convenient technical trick for the treatment of intertrochanteric fractures in elderly patients. To our knowledge, there are few published reports of intertrochanteric fractures treated with PFNA in the lateral decubitus position. The objective of this study was to compare the clinical results and complications of lateral decubitus and supine position PFNA in the treatment of elderly intertrochanteric fracture patients using Randomized Controlled Trials (RCTs). BODY.2. MATERIALS AND METHODS.2.1. GENERAL DATA: A totally of 138 elderly patients with low-energy intertrochanteric fractures were collected and admitted into Department of Orthopaedics, Chengdu Third People's Hospital, from May 2009 to August 2010. Participants were initially screened by telephone, and full assessments were conducted only for participants who did not report any exclusion criteria during the telephone screening. All participants provided written informed consent approved by the Hospital Human Research Ethics Committee. BODY.2. MATERIALS AND METHODS.2.2. DIAGNOSIS AND INCLUSION STANDARDS: In reference to the standard for diagnosing intertrochanteric fractures, the subjects included were (1) those adult men and women of age 60 years old and/or older; (2) those who are confirmed with either by radiographs, computed tomography, or magnetic resonance imaging (MRI); (3) those whose fresh fracture occurred within 21 days; (4) those who are ambulatory prior to fracture; (5) those who signed informed Consent by themselves or their immediate family members; (6) those who have clear consciousness and no severe heart, liver kidney disorders and are able to participate in the examination and treatment. All patients in above lists were excluded. BODY.2. MATERIALS AND METHODS.2.3. EXCLUSION STANDARDS: (1) Those who have old fractures; (2) those who have pathologic fractures and/or caused by osteoporosis; (3) those who have open fractures; (4) those who have multiple injuries or multiple fractures; (5) those who were not suitable for surgery, such as severe decompensated heart, liver, or kidney failure contraindication to the surgery; (6) those who have acute or chronic infection; (7) those who are involved in chemotherapy or hormonotherapy; (8) those who have attended in other trials lately in 6 months; (9) those who are pregnant women; (10) those who are not suitable for the surgery for other reasons by investigators. BODY.2. MATERIALS AND METHODS.2.4. SURGICAL PROCEDURES: Eighteen patients were excluded from 138 elderly patients with low-energy intertrochanteric fractures. One case was excluded for old fracture, two cases were excluded for bonemetastatic tumor, four cases were excluded for multiple fractures, two cases were excluded for chemotherapy or hormonotherapy, and nine cases were not suitable for surgery. The present study is carried out by requirements of double-blind of clinical trials and to achieve separation of collectors of clinical cases, researchers, and statistics evaluators. Thus, the remaining 120 patients were randomized into a lateral decubitus position and supine position group. There was no significant difference statistically in baseline characteristics of two groups. This study was approved by Ethics Committee of Chengdu Third People's Hospital, and all study participants provided both written and verbal informed consent. This study has been registered in the Chinese Clinical Trial Register (no. ChiCTR-TRC-11001815). In the lateral decubitus position group (Figure 1), all surgical operations were carried out under general anesthesia. These patients maintained lateral decubitus position, and injured limb are upper. The disinfection soft pillow was set between the legs, and the healthy limb maintained flexion of their hip and knee during the surgical operations in order to obtain the lateral hip X-ray photos. After anesthesia, the manual traction was operated at the muscle relaxant, and then this operation was stopped. The reset degree was measured by X-ray machine, and the limb was placed in the soft pillow. The proximal trochanteric was slanted to near medial extension curved incision. The abductor muscle fiber was blunt separated, and the guide pin was pointed to the femoral canal midline and introduced in the tip of proximal trochanteric. The pin was measured in the femoral bone marrow cavity, and the hollow reamer was inserted in the proximal. After reamed, the PFNA nail was inserted by designed depth and location requirements under fluoroscopy conditions, and 130-degree aiming arm is connected to the handle. The pin was observed at anterior-posterior position and located in the lower half of the femoral neck; the pin was observed at lateral position and located at the central of femoral neck. The needle tip was located at about 5–10 mm below articular surface. Then, the screw blade was struck along the guide pin by designed location. Drill sleeve was inserted along sight distal locking hole, and the appropriate length locking screw was screwed into the demolition of sigh. Finally, the sealing cap was screwed into the end of the intramedullary nail, and the incision was flushed and closed. In the supine position group (pattern not shown), all surgical operations were also carried out under general anesthesia. These patients maintained supine position, and the torso is slightly slanted to the side slope. The feet were fixed in the boots on the extension device and maintained healthy limb abduction and limb adduction with 10–15° for obtaining lateral X-ray photos. The fractures were reset using continuous mechanical traction and fracture table and maintained this state. The following steps were similar with the lateral decubitus position group. BODY.2. MATERIALS AND METHODS.2.5. EVALUATION AFTER TREATMENTS: In the present study, all patients received prophylactic antibiotic therapy as follows. 2 grams of cefazolin were given before and after treatments. Also 0.4 mL of low-molecular-weight heparin was administered once everyday up to 14 days. After treatment, the muscles active contraction exercises were carried out by analgesia pump, such as ankle active dorsiflexion and plantar flexion and isometric contraction activity of quadriceps. After one day, joint of lower extremity passive activities was carried out by the assistance of rehabilitation therapists. After 2-3 days, the initiative straight leg and raising, and joint of lower extremity activity were carried out. After 5 days, these patients were allowed to out-of-bed activity. X-ray was measured after 2 weeks, 8 weeks, 12 weeks, 16 weeks, 6 months, 12 months, and 24 months. Patient information, mainly included the duration of hospitalisation, the intraoperative index (operative time, intraoperative blood loss, the length of incision, and the number of intraoperative X-ray), the postoperative index (out-of-bed activity time, the Harris hip score after postoperative 12 weeks, and clinic healing time of fracture), and the surgical complications. BODY.2. MATERIALS AND METHODS.2.6. STATISTICAL ANALYSIS: Data were analyzed using Pearson Chi-Square and Wilcoxon-test. For all tests, P values <0.05 were considered to be significant. Statistical analysis was performed using the SPSS statistical package, version 15.0 for Windows. BODY.3. RESULTS.3.1. BASELINE CHARACTERISTICS: The age, sex, injured side, AO type, and complication for patients are shown in Table 1. Out of the 120 elderly patients with low-energy intertrochanteric fractures, 60 patients were fixed with PFNA in the lateral decubitus position and 60 patients in the supine position. In the lateral decubitus position group, the average age was 77.3 years (range 74 to 82). The ratios of male and female patients are 43.3% (26) and 56.7% (34), respectively. The ratios of left and right are 39 : 21. Type A2 fractures were the most common in the study and were seen in 43 patients (71.7%). Type A1 fractures were found in 13 patients (21.7%) and type A3 in 4 (6.6%). The complication of hypertensive disease, diabetes mellitus, and pulmonary disease were 15, 16, and 13 patients, respectively. Similar observation in the supine position group, there was no significant difference statistically in baseline characteristics compared with the supine position group. BODY.3. RESULTS.3.2. EVALUATION AFTER TREATMENTS: As shown in Table 2, the mean time of hospital stay for patients treated with PFNA in the lateral decubitus position group was 20.2 d and was significantly lower than in the supine position group. The operative time is significantly different and is 50.6 min in Group A and 65.7 min in Group B. The intraoperative blood loss was 159.2 mL in Group A, while in Group B it was 201.5 mL. The incision length in Group A was 5.9 cm and was significantly shorter than in Group B where it was 8 cm. The number of intraoperative radiation exposure was significantly lower in Group A (8.38) than in Group B (11.5). The median out-of-bed activity time was shorter in Group A than in Group B, being 9.97 days and 12.2 days, respectively. After treatment for 12 weeks, the Harris hip score and average union time of fractures were not statistically different between the two groups. BODY.3. RESULTS.3.3. COMPLICATION AFTER TREATMENTS: As shown in the clinical results of complication, three complications were observed in the two groups. The complications were mainly as follows: deep venous thrombosis, superficial wound infection, and wound deep infection (Table 3). One patient developed a superficial wound infection in the lateral decubitus position group, which resolved with intravenous antibiotics. Two complications occurred in the supine position group. One patient developed a deep wound infection and resolved with debridement and intravenous antibiotics treatment. One patient developed deep vein thrombosis and was diagnosed by color Doppler sonography after 1 week postoperatively and treated by the vitamin K antagonist-Warfarin for 3 months. BODY.4. DISCUSSION : In the last few years, the incidence of intertrochanteric fractures has been growing as a result of longer life expectancy owing to better quality of life but also better health care. Many methods have been recommended for the treatment of intertrochanteric fractures [1, 2]. Among them, the PFNA was developed to obtain better fixation strength in the presence of osteoporotic bone, using a simpler technique in comparison with other implants. The inserted PFNA blade achieves an excellent fit through bone compaction and requires less bone removal compared to a screw. Biomechanical tests have demonstrated that the blade has a significantly higher cut-out resistance than commonly used screw systems [13–15]. In the supine group, the traction of limb was continued, and adduction and internal rotation of limb were maintained. These will help in the restoration of fractures and neck shaft angle, as well as the closer fracture chimeric and spin forward foreign greater trochanter. Thus, the trochanteric fossa is revealed, which will help in locating the guide pin. However, the limb and ipsilateral torso remained in a straight line during the actual clinical operation when the patients were supine position. Under the trunk gravitational compression, the convex body lateral soft tissue is particularly evident, especially muscular and/or obese patients. These conditions resulted in adductor and internal rotation of limb and even led anteversion change to besmaller or disappear. The supine position also resulted in the inner and deep shift of intertrochanteric fossa. These changes are often difficult to the positioning guide of main pin during the actual clinical operation. Moreover, if the tail of the guide pin is slight to the outer side, the guide pin will point at inside bone cortex, which will result in false channel formation. Thus, the false channel is needed to correct direction of the guide pin, and the operation time will extend, and intraoperative blood loss and the number of X-ray exposure also increase. Moreover, these patients were placed in the supine position on the flat radiolucent table, which can result in difficult to obtain a good lateral image of the proximal femur [16, 17]. Thus, adequate knowledge and experience of operative technique need further improvement. Nailing of the femur can be performed in both supine and lateral position. The supine position is physiologic and convenient to the anesthetist and is preferred if patients also have cervical spine injury, ipsilateral lower extremity fracture and severe pulmonary compromise. But access to greater trochanter is somewhat limited in supine position, particularly in large or obese patients in whom lateral position is preferred [16–19]. Thus, lateral positioning obviates the need for a fracture table, makes it easier to establish a starting point for an intramedullary device, and facilitates conversion to an open procedure without repositioning should this become necessary. In the present study, we designed the lateral decubitus position PFNA and investigated and compared the clinical results and complications of PFNA in the treatment of elderly intertrochanteric fracture patients. These patients were placed in the lateral decubitus position on the flat radiolucent table, and the healthy limb maintained flexion of their hip and knee during the surgical operations in order to obtain the lateral hip X-ray photos. Under anesthesia and muscle relaxation conditions, the manual traction was operated in order to restore fractures and will avoid the complications caused by the use of the fracture table, such as pudendal, sciatic and femoral nerve palsy, perineal sloughs, well leg compartment syndromes, avulsion of the inferior epigastric artery in the contralateral limb, and crush syndromes [20]. The present results suggested that only one patient developed a superficial wound infection in the lateral decubitus position group (Table 3). Compared with the supine position group, once surgical operation achieved restore fractures in the lateral decubitus position group. Most of intertrochanteric fractures are low-energy osteoporotic fractures, and primary injuries of soft tissue are lighter [1]. Thus, soft tissue hinge effect may increase antishift capacity between the fracture fragments after restore. Moreover, the lateral decubitus position will help to maintain the neck-shaft angle, restore fracture chimeric, and further improve the nail of guide needle accurately plugged in the medullary cavity. The present findings showed that the lateral decubitus position PFNA can change the overlapping shielding effect of pelvis when the guide pin was inserted, and the location of rotor nest changes shallowly compared to the supine position groups. Manual reduction is only reconsidered as simply axial traction, and it has low-energy injury. Thus, manual reduction can promote and maintain the reduction of soft tissue chain. The advantages of the lateral decubitus position group also mainly included sustained traction, mild adduction, and internal rotation bit of hips, as well as without obvious shift performance of restore fracture. After treatments for 12 weeks, the present findings showed the length of hospital stay, the intraoperative parameters (operative time, incision length, intraoperative blood loss, and the number of intra-operative radiation exposure), and out-of-bed activity time were significantly lower (P < 0.05) in patients treated with a PFNA in the lateral decubitus position group compared to those treated in the supine position group. However, there was no significant difference in the final functional outcome on treatments of intertrochanteric fractures in the elderly patients. Of course, PFNA with the lateral decubitus position also has some limitations on the treatments of intertrochanteric fractures in the elderly patients, who cannot tolerate the lateral decubitus position. Moreover, some patients have some complications, such as spine unstable fractures, severe lung damage disease, and contralateral lower extremity fractures, who are not suitable for the lateral decubitus position. BODY.5. CONCLUSION: The present results showed that PFNA with the lateral decubitus position and the supine position provided effective methods for the treatments of intertrochanteric fractures in the elderly patients. However, the PFNA with the lateral decubitus position provides a shorter operation time, less of hospital stay, blood loss, number of intraoperative X-ray, incision length and out-of-bed activity time. Therefore, PFNA fixation in the lateral decubitus position may be considered a better choice for the treatment of intertrochanteric fractures in the elderly patients. Although PFNA fixation in the lateral decubitus position was shown to be of value as the treatment of intertrochanteric fractures, further definitive research about early surgery and longer follow-up period is needed to support the use of PFNA fixation in the lateral decubitus position.

TITLE: Effects of infant massage on jaundiced neonates undergoing phototherapy ABSTRACT.BACKGROUND: Infant massage is a natural way for caregivers to improve health, sleep patterns, and reduce colic. We aimed to investigate the effects of infant massage on neonates with jaundice who are also receiving phototherapy. ABSTRACT.METHODS: Full-term neonates with jaundice, admitted for phototherapy at a regional teaching hospital, were randomly allocated to either a control group or a massage group. The medical information for each neonate, including total feeding amount, body weight, defecation frequency, and bilirubin level, was collected and compared between two groups. ABSTRACT.RESULTS: A total of 56 patients were enrolled in the study. This included 29 neonates in the control group and 27 in the experimental group. On the third day, the massage group showed significantly higher defecation frequency (p = 0.045) and significantly lower bilirubin levels (p = 0.03) compared with the control group. No significant differences related to feeding amount or body weight were observed between the two groups. ABSTRACT.CONCLUSION: Infant massage could help to reduce bilirubin levels and increase defecation frequency in neonates receiving phototherapy for jaundice. BODY.BACKGROUND: Infant massage, in which babies are massaged soon after birth, is a tradition that is common in India and many other countries. Several studies have reported that infant massage can improve weight gain, sleep patterns, growth and development, and autonomic nervous system functions, and that it can also reduce the rates of colic and infant mortality [1–6]. In addition, massage therapy can help reduce infant stress and can promote positive emotional bonding between parents and babies [7, 8]. Jaundice refers to the yellow staining of the skin and sclerae caused by an increase in serum bilirubin levels [9]. Excessive hyperbilirubinemia can lead to permanent brain damage. Jaundice affects as many as 60 % of healthy neonates and is responsible for 75 % of hospitalizations within the first week after birth [10]. Most cases of neonatal jaundice are caused by unconjugated hyperbilirubinemia, which occurs because of excessive bilirubin formation and because the neonatal liver is unable to clear bilirubin rapidly enough from the blood [10]. This type of jaundice, known as physiological jaundice, is typically harmless; although it should be monitored, it is not likely to require treatment. However, some neonates suffer from exaggerated physiological jaundice or pathological jaundice. These cases should be treated with phototherapy or they may even require exchange transfusions to reduce the risk of acute bilirubin encephalopathy or kernicterus [10, 11]. Previous research has indicated that infant massage decreases the bilirubin level of neonates suffering from hyperbilirubinemia and ameliorates neonatal jaundice [12, 13]. One study reported that full-term infants who received massage therapy had significantly lower serum total bilirubin and transcutaneous bilirubin levels than infants who did not undergo massage therapy [12]. Another study indicated that mean bilirubin levels could be significantly reduced in full-term infants by the fourth day of massage therapy, compared with infants not treated with massage [13]. Despite the fact that earlier clinical studies support the use of massage for reducing neonatal jaundice, the apparent correlation has not been extensively examined among neonates with jaundice who are receiving phototherapy. Therefore, we aimed to evaluate how infant massage affects neonates receiving phototherapy for jaundice. BODY.METHODS.STUDY PARTICIPANTS: All participants in this study were normal neonates (from birth to 5 days of age) who were receiving phototherapy for jaundice (bilirubin level greater than 15 mg/dL), according to the recommendations of The Society of Neonatology and the standard medical practice in Taiwan. Infants were recruited between August 2011 and July 2012 from a regional teaching hospital in Taichung city, located in central Taiwan. The inclusion criteria for study participation were as follows: (1) healthy full-term (gestational age, 37–41weeks) neonates, (2) birth weight of 2500–3600 g, (3) APGAR score at birth of 8–10, and (4) receiving phototherapy for hyperbilirubinemia. We excluded infants with rhesus and ABO incompatibility, subgaleal hemorrhage, congenital anomalies, infections, a glucose-6-phosphate dehydrogenase deficiency, gastrointestinal obstruction, and biliary atresia. The sample size was calculated before the study. The estimated minimum detectable difference of bilirubin in means = 2 mg/dL, expected standard deviation of residuals = 2 mg/dL, 1-β = 0.8, α = 0.05. The minimum sample size of each group was 17 subjects. We divided the participants into either a control group or a massage group by non-blinded, simple randomization. The institutional review board of our hospital approved the study, and informed consent was obtained from the parents of the neonates. BODY.METHODS.PHOTOTHERAPY: In general, healthy full-term infants were discharged at 3 days of age. If neonatal jaundice was found and phototherapy was available in the nursery, they received phototherapy there. However, if phototherapy was not available in the nursery, infants with jaundice were discharged and transferred to another hospital for further care. Phototherapy was delivered using a halogen lamp (YON DON, YD-P-222; Taiwan) at a distance of 45–60 cm from the neonate, according to the luminosity. The baby's clothing was otherwise kept to a minimum to expose as much of the neonate's skin surface to the therapy light as possible. However, the neonate's eyes and genitalia were covered to prevent damage. When the bilirubin level became less than 10 mg/dL, phototherapy was discontinued. BODY.METHODS.DATA COLLECTION: Demographic information was collected for all children, which included their age, gender, gestational age, and birth weight. We also recorded the following clinical data throughout the study period: (1) total feeding amount, (2) body weight, (3) defecation frequency, and (4) microbilirubin level. Total feeding amount, body weight, and defecation frequency were recorded from the first to the third day of hospitalization, according to the mothers' parenting logs and nursing records. Bilirubin was measured using a microassay, which is a rapid, reliable, simple, and accurate estimator of total and direct bilirubin [14]. This test required only minimal blood samples to be taken from the neonate's foot for analysis in a TBA-80FR analyzer (Toshiba, Tokyo, Japan). The primary outcome was the termination of phototherapy, and the secondary outcome was discharge after the completion of treatment. BODY.METHODS.MASSAGE PROCEDURE: All participating neonates in the intervention group received massage therapy from trained therapists. Neonates in the control group did not receive massage therapy. Each massage therapy session started on the first day of phototherapy, lasted for 15–20 min per session, and was conducted twice daily (between meals) for 3 consecutive days. Phototherapy was stopped for the 15–20 min during which neonates received massage therapy. Massage techniques were performed in accordance with International Association of Infant Massage (IAIM) guidelines. The researcher thoroughly washed his or her hands, applied massage oil (sweet almond oil, AEOMA, England), and then performed a skin test before starting the first massage therapy procedure. For the skin test, we applied sweet almond oil to the inside of baby's wrist, and after 30 min, we checked the skin for redness, a rash, or other signs of allergic reaction. None of the neonates in the massage group had an allergic reaction or experienced side effects from the sweet almond oil. After the test, we started the massage on the leg and foot (with one hand used to fix the foot), before progressing to the abdomen, hands, and finally, the back. The same researcher provided massage therapy to all neonates in the study, and the room temperature was maintained at between 26 °C and 28 °C. BODY.METHODS.STATISTICAL ANALYSES: Data were analyzed using IBM SPSS for Windows, Version 19.0 (IBM Corp., Armonk, NY, USA). Student's t-tests were used to investigate differences between the control and massage group infants with regard to feeding amount, body weight, defecation frequency, and microbilirubin levels. The Kolmogorov–Smirnov test was used to assess the normality of distribution of the investigated parameters (all parameters were shown to be normally distributed). A chi-square test was used to compare the demographic characteristics of infants in the control and massage groups. Results were considered significant at a p value of <0.05 and are presented as mean ± standard deviation. BODY.RESULTS.PARTICIPANT CHARACTERISTICS: A total of 60 neonates with jaundice were initially enrolled. However, one neonate was excluded because his bilirubin level exceeded 22 mg/dL on the second day, and he was transferred to a medical center for further care. Another three neonates were excluded because their hospital stays were less 3 days, and their discharge was against medical advice. Therefore, 56 neonates were included in the final study and randomly assigned to the control group (29 neonates; 16 males and 13 females) and the massage group (27 neonates; 11 males and 16 females). Table 1 shows the medical characteristics of participating neonates. We observed no significant differences between the two groups in terms of type of feeding, gestational age at birth, body weight at birth, body weight on the date of admission, phototherapy duration, or physical weight loss.Table 1Demographic characteristics of participating neonatesVariableItemMassage group (n = 27)Control group (n = 29) p valueSexMale11 (40.7) a 16 (55.2)0.29Female16 (59.3)13 (44.8)Kinds of feedingBreast feeding5 (18.5)5 (17.2)0.79Infant formula3 (11.1)4 (13.8)Mixed19 (70.4)20 (69.0)HematomaYes4 (14.8)2 (6.9)0.35No23 (85.2)27 (93.1)Type of deliveryVaginal delivery25 (92.6)28 (96.6)0.52Cesarean-section2 (7.4)1 (3.4)Gestational age (weeks)38.7 ± 0.7 b 39.1 ± 0.90.36Age (hours)117 ± 56109 ± 420.41Body height (cm)50.9 ± 1.750.9 ± 1.90.93Body weight at birth (gm)3069.3 ± 233.33174.5 ± 340.60.19Body weight at admission (gm)2888.9 ± 271.83008.2 ± 308.00.13Physiologic weight loss (gm)c 180.4 ± 102.3166.2 ± 114.50.63Percentage of physiologic weight loss (%)5.9 ± 3.45.1 ± 3.40.39Phototherapy duration (hours)64.8 ± 16.669.4 ± 27.30.45D1 microbilirubin level16.6 ± 1.417.1 ± 1.50.23D2 microbilirubin level14.1 ± 1.214.5 ± 1.70.38D3 microbilirubin level11.0 ± 1.111.8 ± 2.00.78Microbilirubin level at the end of phototherapy9.6 ± 1.610.0 ± 1.80.19Rebound microbilirubin level(n = 3) 10.1 ± 2.4(n = 4) 10.6 ± 1.10.55Hospital stay (hours)78.5 ± 14.087.8 ± 24.00.08 a Values are n (%) b Values are means ± standard deviation c Physiologic weight loss means the body weight loss between at birth and at admissionD1: first day of hospitalization, D2: second day of hospitalization, D3: third day of hospitalization BODY.RESULTS.TOTAL FEEDING AMOUNT: For all participating neonates, food intake increased through hospitalization. In both groups, food intake was significantly higher on the second and third days of hospitalization than on the first day (p < 0.001). However, no significant difference was observed between the groups during hospitalization (Table 2).Table 2Feeding intake among massage and control groupParameterMassage group (n = 27)Control group (n = 29)t value p valueD1 feeding amount (ml)330.7 ± 111.0 a 330.4 ± 104.40.110.99D2 feeding amount (ml)504.1 ± 79.8499.0 ± 99.00.210.83D3 feeding amount (ml)558.1 ± 74.3555.9 ± 85.80.10.92 a Values are means ± standard deviationD1: first day of hospitalization, D2: second day of hospitalization, D3: third day of hospitalization BODY.RESULTS.BODY WEIGHT: The body weights of all neonates increased through hospitalization, with the body weight being significantly higher on the third than on the first day of hospitalization (p = 0.03). However, there was no significant difference between the groups (Table 3).Table 3Body weight among massage and control groupParameterMassage group (n = 27)Control group (n = 29)t value p valueD1 body weight (gm)2,888.9 ± 271.8a 3,008.2 ± 308.0-1.530.13D2 body weight (gm)2,968.1 ± 262.73,098.3 ± 323.0-1.650.10D3 body weight (gm)3,031.9 ± 263.63,163.8 ± 342.1-1.610.11D1: first day of hospitalization, D2: second day of hospitalization, D3: third day of hospitalization a Values are means ± standard deviation BODY.RESULTS.DEFECATION FREQUENCY: Table 4 presents the defecation frequencies for the massage and control groups. The defecation frequency significantly increased for all neonates during hospitalization (p < 0.001). Although the defecation frequency was not significantly different between the control and massage groups on the first and second days of hospitalization, it was significantly higher in the massage group on the third day (p = 0.04).Table 4Defecation frequency among massage and control groupParameterMassage group (n = 27)Control group (n = 29)t value p valueD1 defecation frequency (times)3.1 ± 1.7a 3.0 ± 2.00.10.99D2 dfecation frequency (times)5.0 ± 1.54.3 ± 1.51.720.92D3 defecation frequency (times)4.6 ± 1.33.9 ± 1.32.050.04* a Values are means ± standard deviation *Values are significantly different between groups using Student's t test; P < 0.05D1: first day of hospitalization, D2: second day of hospitalization, D3: third day of hospitalization BODY.RESULTS.MICROBILIRUBIN LEVEL: The microbilirubin level was significantly decreased for all participating neonates during hospitalization (p < 0.001). Because intravenous infusions can increase the excretion of bilirubin and thereby decrease serum bilirubin levels, we excluded neonates who received intravenous infusions when comparing microbilirubin levels between the massage and control groups. We found no significant differences in the microbilirubin levels between the control and massage groups during the first and second days of hospitalization. However, on the third day of hospitalization, the microbilirubin level was significantly lower in the massage group than in the control group (p = 0.03; Table 5).Table 5Microbilirubine level of neonetes without intravenous hydration between massage and control groupParameterMassage group (n = 15)Control group (n = 11)t value p valueD1 microbilirubine level (mg/dL)15.6 ± 0.9 a 15.9 ± 1.0-1.350.19D2 microbilirubine level (mg/dL)13.9 ± 1.214.5 ± 0.8-1.360.18D3 microbilirubine level (mg/dL)10.8 ± 0.912.2 ± 1.8-2.60.03* a Values are means ± standard deviation *Values are significantly different between groups using Student's t test; P < 0.05D1: first day of hospitalization, D2: second day of hospitalization, D3: third day of hospitalization BODY.DISCUSSION.BODY WEIGHT: Our study indicated that body weight was not significantly different between the massage and control groups. This result is consistent with some reports of previous studies [15–17]. Lee also failed to identify significant differences in weight gain between infants who received massage therapy and control group infants after 4 weeks of treatment [18]. However, the recent literature has indicated that massage therapy can increase weight gain in preterm infants who receive moderate-pressure massage for 10 min three times per day over 5 days [19]. Serrano et al. also demonstrated that massaged infants weighed significantly more than control infants at 2 months of age [20], whereas Yilmaz et al. reported that both body weight and height significantly increased in massaged infants compared with control infants after 2 and 14 weeks of massage therapy [21]. Field et al. showed that preterm neonates who received massage therapy for a 5-day period had greater increases in weight gain, serum insulin levels, and insulin-like growth factor-1 (IGF-1) [16]. In this latter study, the authors speculated that weight gain following massage might have been due to increases in insulin/IGF-1 levels or vagal activity, which, in turn, could have decreased stress and gastric motility, leading to more efficient food absorption [16]. In our study, the lack of a significant increase in body weight gain after massage may have been because of the young age of our participants (average age: 4.9 ± 2.5 days in the massage group; 4.5 ± 1.7 days in the control group). In addition, it is possible that the duration of massage therapy was too short to stimulate the secretion of insulin and IGF-1. BODY.DISCUSSION.DEFECATION FREQUENCY: The defecation frequency in the massage group of this study was significantly higher than that in the control group on the third day of massage therapy, which is comparable with the results of previous studies. In the study by Seyyedrasooli et al., the defecation frequency of infants who received massage therapy was significantly higher than that in the control group by the fourth day of therapy [22]. Also, in the study by Chen et al., the mean defecation frequency of the massage group was significantly higher than that of the control group on the first 2 days of therapy [12]. However, neither of these study population included neonates receiving phototherapy for jaundice. Previous research has indicated that most neonates first pass feces within 24 h of birth, although massage therapy can stimulate the passage of meconium [12]. This may explain the significantly higher defecation frequency that we observed in the massage group by the third day of treatment. Massage therapy can increase bowel movements and the excretion of meconium [22], and an increased frequency of bowel movements might be expected to diminish the enterohepatic circulation of bilirubin in a neonate, thereby leading to increased bilirubin excretion [23]. BODY.DISCUSSION.MICROBILIRUBIN LEVEL: One previous study has suggested that early intravenous nutrition could improve neonatal jaundice by increasing urinary excretion and the metabolism of bilirubin [24]. Therefore, we excluded those neonates receiving intravenous hydration when comparing the bilirubin levels between the massage and control groups. In our study, the bilirubin level of the massage group neonates was significantly lower than that of the control group on the third day of massage therapy. This result is consistent with the study by Chen et al., who reported that in full-term neonates with jaundice, bilirubin levels were significantly decreased in the massage group compared with the control group on the fourth day of therapy [12]. In addition, Moghadam et al. indicated that the mean bilirubin level of infants with jaundice in the massage group significantly decreased on the fourth day compared with the control group [13]. In contrast to these reports, one study has indicated that transcutaneous bilirubin levels in healthy, full-term infants were not significantly different between the massage and control groups after 4 days of therapy [22]. Possible explanations for this inconsistency include differences in the massage procedure and the fact that their neonates had no hyperbilirubinemia. Massage therapy could lead to earlier reductions in bilirubin levels that might allow shorter treatments with phototherapy, resulting in earlier discharge. The most likely mechanism underlying the reduction in neonatal jaundice in the group receiving massage therapy is the stimulation of intestinal movement. This, in turn, will increase defecation frequency and allow the neonate to pass greater amounts of meconium, which contains bilirubin [12]. This is consistent with the findings of Gourley et al., who noted that stool production and serum bilirubin levels were negatively correlated in healthy term infants during the first week of life [25]. In our study, the defecation frequency was significantly higher in the massage group than in the control group on the third day of treatment. Increased defecation might therefore explain the significant reduction in bilirubin levels observed in the massage group. Moreover, massage therapy also stimulates the vagus nerve, which will increase the frequency of bowel movements and diminish the enterohepatic circulation of bilirubin, thereby increasing bilirubin excretion [22]. Furthermore, in subcutaneous tissue, physiological massage therapy can increase the flow of blood, lymph, and tissue fluids, which increases the collection and excretion of waste products such as bilirubin [12]. Nonetheless, in the current study, the number of participating neonates was small and the duration of massage therapy was short, and these limitations may have affected the reliability of statistical tests or masked other important correlations. Future research should investigate the use of massage therapy over longer periods. We also recommend that neonates receive continuous follow-up, such as home visits by a qualified healthcare professional, to ensure that massage therapy achieves the desired effects. BODY.CONCLUSION: This study shows that by the third day of intervention, the defecation frequency of neonates receiving phototherapy for jaundice was significantly higher in those also receiving massage therapy, compared with the control group not receiving massage therapy. Furthermore, microbilirubin levels were significantly lower in the massage group on the third day, but this was only assessed in those who did not receive intravenous hydration. Massage therapy is a safe and economic and no significant harmful practice that can promote bonding and interaction between the mother and infant. However, the benefits of massage therapy remain uncertain for neonatal jaundice, and although this study adds to the evidence in its favor, further research is needed to clarify the true effects of such auxiliary treatments on the outcomes of neonatal jaundice.

TITLE: TCD With Transfusions Changing to Hydroxyurea (TWiTCH): a multicentre, randomised controlled trial ABSTRACT.BACKGROUND: For children with sickle cell anaemia and elevated transcranial Doppler (TCD) flow velocities, regular blood transfusions effectively prevent primary stroke, but must be continued indefinitely. The efficacy of hydroxyurea in this setting is unknown. ABSTRACT.METHODS: TWiTCH was a multicentre Phase III randomised open label, non-inferiority trial comparing standard treatment (transfusions) to alternative treatment (hydroxyurea) in children with abnormal TCD velocities but no severe vasculopathy. Iron overload was managed with chelation (Standard Arm) and serial phlebotomy (Alternative Arm). The primary study endpoint was the 24-month TCD velocity calculated from a general linear mixed model, with non-inferiority margin = 15 cm/sec. ABSTRACT.FINDINGS: Among 121 randomised participants (61 transfusions, 60 hydroxyurea), children on transfusions maintained <30% sickle haemoglobin, while those taking hydroxyurea (mean 27 mg/kg/day) averaged 25% fetal haemoglobin. The first scheduled interim analysis demonstrated non-inferiority, and the sponsor terminated the study. Final model-based TCD velocities (mean ± standard error) on Standard versus Alternative Arm were 143 ± 1.6 and 138 ± 1.6 cm/sec, respectively, with difference (95% CI) = 4.54 (0.10, 8.98), non-inferiority p=8.82 × 10−16 and post-hoc superiority p=0.023. Among 29 new neurological events adjudicated centrally by masked reviewers, no strokes occurred but there were 3 transient ischaemic attacks per arm. Exit brain MRI/MRA revealed no new cerebral infarcts in either arm, but worse vasculopathy in one participant (Standard Arm). Iron burden decreased more in the Alternative Arm, with ferritin difference −1047 ng/mL (−1524, −570), p<0.001 and liver iron difference −4.3 mg Fe/gm dry weight (−6.1, −2.5), p=0.001. ABSTRACT.INTERPRETATION: For high-risk children with sickle cell anaemia and abnormal TCD velocities, after four years of transfusions and without severe MRA vasculopathy, hydroxyurea therapy can substitute for chronic transfusions to maintain TCD velocities and help prevent primary stroke. BODY.INTRODUCTION: Stroke is a devastating clinical event for children with sickle cell anaemia (SCA), causing severe motor and neurocognitive sequelae. Historically, stroke occurred frequently in this patient population, with an incidence of 0.61–0.76 events per 100 patient-years and cumulative childhood incidence of 7–11%. [1] After first stroke, there is a high rate of recurrence or progression even with chronic transfusion therapy; a recent publication with median transfusion duration of 5.5 years documented 17.5% of children on transfusions developed an overt stroke while an additional 27.5% had new silent cerebral infarcts. [2] Transcranial Doppler (TCD) screening of intracranial blood flow identifies children at risk for stroke; elevated time-averaged mean velocities in the distal internal carotid or middle cerebral arteries confer an increased risk, and velocities ≥200 cm/sec are associated with a 40% primary stroke rate over 3 years. [3] The multicentre STOP trial demonstrated that regular blood transfusions, with the goal of maintaining <30% sickle haemoglobin (HbS), reduced the rate of first stroke by 90% in children with SCA and abnormal TCD velocities. [4] Aiming to limit the long-term consequences of chronic transfusions, the STOP2 trial investigated whether therapy could be safely stopped in patients who normalised their TCD velocities after ≥30 months of transfusions and lacked severe vascular disease. This trial was terminated early, due to reversion to abnormal TCD velocities in 14 participants and two overt strokes in children discontinuing transfusions compared to none who continued transfusions, suggesting that indefinite therapy for primary stroke prevention was required. [5] With annual TCD screening of children with SCA now strongly recommended, [6] and evidence that transfusions successfully reduce the rate of first stroke, [7] an alternative therapy that maintains TCD velocities with fewer complications than transfusions could increase acceptance and long-term adherence, and further decrease primary stroke in this high-risk population. Hydroxyurea has emerged as an important disease-modifying treatment for SCA, with proven laboratory and clinical efficacy for children and adults. [8, 9] Through fetal haemoglobin (HbF) induction and other mechanisms of action, hydroxyurea inhibits intracellular sickle haemoglobin (HbS) polymerisation and lowers the risk of vaso-occlusive complications. [10] Hydroxyurea has the potential for lowering TCD velocities and thus reducing stroke risk, [11, 12] but definitive data are lacking regarding its efficacy in cerebrovascular disease. We hypothesized that hydroxyurea was non-inferior to transfusions for maintaining TCD velocities, after discontinuation of initial transfusion therapy to prevent primary stroke. To determine the efficacy of hydroxyurea in this setting, we conducted TCD With Transfusions Changing to Hydroxyurea (TWiTCH), a multicentre, open label, controlled Phase III randomised clinical trial. BODY.METHODS.STUDY DESIGN: TWiTCH was a non-inferiority trial (ClinicalTrials.gov NCT01425307), comparing Alternative Treatment (hydroxyurea) to Standard Treatment (transfusions) for maintaining TCD velocities as a surrogate for stroke risk, based on published data showing that elevated velocities confer an increased stroke risk. [3, 5] Patients from 26 paediatric programmes (Appendix) between 4–16 years of age with SCA and abnormal TCD velocities were eligible after ≥12 months of chronic transfusions. Documented clinical stroke, transient ischaemic attack, or severe vasculopathy were exclusions. After local IRB approval and with written informed consent, eligibility screening included original abnormal TCD verification and baseline brain MRI/MRA, TCD examination using identical non-imaging instruments (SonaraTek, Middleton, WI), liver iron concentration (LIC) by FerriScan® R2-MRI (Resonance Health, Claremont, Australia), abdominal ultrasonography and MRI, neurocognitive testing, and quality of life assessments. Participants with baseline Grade 4 or higher severe brain MRA vasculopathy, defined as moderate stenosis in >2 arterial segments or severe stenosis/occlusion in ≤2 segments, [13] or with inadequate TCD velocities from poor blood flow or bone windows, were considered screening failures and removed from the study. Children with normal or persistent conditional/abnormal screening TCD velocities on chronic transfusions remained eligible for randomisation. Participants completing screening were randomised 1:1 to study treatments. Randomisation was stratified by site and blocked, and an adaptive randomization scheme was employed to balance the covariates of baseline age and TCD velocity (See Supplement for further details). BODY.METHODS.ENDPOINTS: The primary study endpoint was the maximum TCD time-averaged mean velocity on the index side, defined as the cerebral hemisphere with the higher mean arterial velocity on baseline evaluation. TCD velocities were obtained monthly in triplicate during screening and exit, and once at 12-week intervals during the 24-month treatment period. TCD examinations were performed just before transfusions or phlebotomy, and all were read centrally by observers masked to treatment assignment and prior TCD results. Secondary endpoints included TCD velocity on the non-index side, new stroke or non-stroke neurological events, new brain MRI/MRA lesions, hepatic iron overload, sickle-related events, neuropsychological status, quality of life, growth, and treatment-related complications. BODY.METHODS.STUDY TREATMENTS: Participants randomised to Standard Treatment continued monthly transfusions to maintain ≤30% HbS, with local discretion regarding transfusion type (simple, partial exchange, or erythrocytapheresis). Deferasirox was recommended for iron overload; children on chelation maintained their current dose, while those starting chelation received deferasirox at 10–40 mg/kg/day, depending on their screening LIC value. Participants randomised to Alternative Treatment initiated hydroxyurea at 20 mg/kg/day with escalation to maximum tolerated dose (MTD), defined as moderate marrow suppression of neutrophils and reticulocytes as previously described. [14] Transfusions were slowly weaned over 4–9 months to protect against stroke during hydroxyurea dose escalation to MTD, using a standardised protocol. [15] After MTD was established and transfusions were discontinued, serial phlebotomy removed 10 mL/kg (maximum 500 mL) venous blood over 30–60 minutes at each 4-week study visit, again using a standardised protocol. [15, 16] Smaller phlebotomy volumes (5 mL/kg) were removed for haemoglobin concentrations of 8.0–8.5 gm/dL, and phlebotomy was not performed when the haemoglobin concentration was <8.0 gm/dL. The treatment period was 24 months after randomisation, with a 6-month visit after completing exit studies. BODY.METHODS.STATISTICAL ANALYSES: TWiTCH was a randomised, open label trial based on the endpoint variable of centrally determined TCD time-averaged mean arterial velocities. The primary endpoint was the 24-month TCD velocity, calculated from a general linear mixed model using all TCD velocities captured throughout the trial (Supplement), by intention-to-treat with two planned interim analyses after 33% and 67% of participants completed exit studies. The stopping guidelines for non-inferiority used the Lan-DeMets version of the O'Brien-Fleming group sequential method. [17] A one-sided non-inferiority margin of 15 cm/sec was used in the analysis, representing the biological variation of TCD examinations. [18] Sample size estimates included enrolment of 148 children, with 20% dropout during screening to yield 118 randomised participants, followed by 15% post-randomisation dropout, so that 100 participants (50 per arm) would complete the 24-month treatment period. This study design provided at least 90% power to test the non-inferiority hypothesis assuming a difference of 5 cm/sec (alternative higher than standard) and a standard deviation of 24 cm/sec. Comparisons of continuous variables were performed using t-tests, and comparisons of categorical variables were by chi-squared analyses. Analyses were "intention to treat" except for a planned on-protocol analysis of TCD velocities, which excluded participants who exited the study early. A sensitivity analysis adjusting for baseline age was also performed. All analyses were performed using STATA 14.0 (College Station, Texas, USA) or SAS 9.4 (Cary, North Carolina, USA). BODY.METHODS.STROKE ADJUDICATIONS: All new potential stroke events were evaluated with careful neurological evaluation and prompt brain MRI/MRA examinations, and then adjudicated centrally by a panel of expert reviewers. Independent and then consensus opinions were obtained from neurologists and neuroradiologists masked to study treatment. Participants with "possible" or "likely" stroke based on new neurological signs or symptoms, but without corresponding radiological changes, were scored as transient ischaemic attack. Exit brain MRI/MRA examinations allowed confirmation that no strokes had been missed by the adjudication process. BODY.METHODS.STUDY MONITORING AND SAFETY CONSIDERATIONS: An NHLBI-appointed Data and Safety Monitoring Board reviewed all enrolment, safety, toxicity, and efficacy data, including new stroke adjudications, adverse events, and interim analyses. The Principal Investigator was masked to all treatment-related results, and local investigators were masked to TCD results. Protocol-defined rescue transfusions were administered to participants in either treatment arm for perceived elevated stroke risk, such as failure to suppress %HbS on the Standard Arm and excessive toxicity or failure to achieve marrow suppression targets on the Alternative Arm. A protocol-defined alert algorithm identified participants whose TCD velocities varied substantially from baseline and might confer increased stroke risk (Supplement), and these children had additional evaluations and closer therapeutic monitoring. BODY.METHODS.ROLE OF THE FUNDING SOURCE: The study sponsor was the National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health. NHLBI did not participate in the following aspects of the research: (1) study design; (2) data collection, analysis, or interpretation; (3) writing the report; or (4) decision to submit the publication. BODY.RESULTS.SCREENING AND ENROLMENT: A total of 159 patients consented and enrolled in TWiTCH (Figure 1), including 38 ineligible for randomisation: 19 had severe vasculopathy, 10 withdrew from the study, 7 had inadequate screening TCD, 3 failed original TCD verification, and 1 had an inadequate brain MRI/MRA (2 participants failed for 2 reasons). Therefore, 121 participants were randomised, 3 more than the protocol specified, forming the Intention-To-Treat population (61 Standard Arm, 60 Alternative Arm). Most baseline demographic, clinical, and laboratory characteristics were equivalent between treatment arms, except for higher WBC, ANC, and bilirubin in the Alternative Arm. Despite the exclusion criterion of clinically overt stroke, over one-third of the participants in both treatment arms had evidence of prior silent cerebral infarctions (Table 1). BODY.RESULTS.STUDY TREATMENT: Participants assigned to Standard Treatment received primarily simple transfusions (57%), with some partial exchange transfusions (31%) or automated erythrocytapheresis (12%). The haemoglobin concentration remained steady at ~9 gm/dL (Figure 2A) with average HbS <30% throughout the study treatment period (Figure 2C). Chelation was provided to children on transfusions with hepatic iron overload, typically deferasirox starting at an average dose of 25.7 ± 6.0 mg/kg/day. On the Alternative Arm, all 60 participants started hydroxyurea at 20 mg/kg/day followed by dose escalation. MTD was achieved in 57/60 children (95%), who then discontinued transfusions per protocol; two did not reach MTD due to medication non-adherence leading to study withdrawal and one had an early adjudicated transient ischemic attack. The average time to MTD was 29 weeks (median 27, range 15–59 weeks); average hydroxyurea MTD was 26.9 ± 4.3 mg/kg/day (median 27.4, range 15.6–35.6 mg/kg/day). On hydroxyurea, the haemoglobin concentration remained stable at ~9 gm/dL, along with expected significant hematological changes in MCV, %HbF, white blood cell count, neutrophils, platelets, and reticulocytes (Figure 2). Marrow suppression targets were achieved with mean absolute neutrophil count at MTD of 3.5 ± 1.6 × 109/L (median 3.4, range 1.5–10.1 × 109/L) and final average neutrophil count of 3.6 × 109/L (Table 2). HbF responses included a mean MTD value of 27.0 ± 5.7% (median 26.2, range 15.5–38.5%) and final average HbF of 24.4%. BODY.RESULTS.PRIMARY STUDY ENDPOINT: The two treatment arms were balanced for baseline TCD velocities, but the final average velocity on the Alternative Arm was slightly lower than the average velocity on the Standard Arm (Figure 3A and 3B). After full enrolment and 37% of the participants exited, the first scheduled interim analysis revealed the stopping boundary was passed and non-inferiority was demonstrated. After 50% of participants exited, repeat analyses confirmed these findings and the study was terminated by NHLBI. Remaining participants then completed all exit studies before discontinuing protocol-directed study treatment. In total, the Standard Arm included 42 who completed study treatment, 11 truncated treatment, and 8 exited early; the Alternative Arm included 41 who completed treatment, 13 truncated treatment, and 6 exited early. The final model-based TCD velocities (mean ± standard error) on the Standard Arm versus Alternative Arm were 143 ± 1.6 and 138 ± 1.6 cm/sec, respectively, with difference (95% CI) = 4.54 (0.10, 8.98), non-inferiority p=8.82 × 10−16 and a post-hoc superiority p=0.023. The planned per-protocol analysis excluding study participants who exited the study early showed almost identical findings, with difference = 5.06 (0.56, 9.57), non-inferiority p=1.05 × 10−15 and a post-hoc superiority p=0.015. An age-adjusted analysis gave almost identical findings (not shown). Figure 3C shows baseline (enrolment) TCD velocities and final (exit) velocities for each TWiTCH study participant, illustrating that the vast majority of velocities were in the normal range at both study entry and exit. No child in either treatment arm reverted from normal to abnormal TCD velocities. Additional exit laboratory values are provided in Table 2. BODY.RESULTS.STROKE ADJUDICATIONS AND NEW NEUROLOGICAL EVENTS: Central stroke adjudication occurred for 29 possible new neurological events (12 on the Standard Arm, 17 on the Alternative Arm). No child had a positive adjudication for stroke, although 6 were labeled transient ischaemic attack (3 in each arm, see Supplement for more details). Exit brain MRI/MRA examinations revealed no new cerebral infarcts in either treatment arm, including all participants with negative stroke adjudications. Worse vasculopathy developed in one participant on the Standard Arm. BODY.RESULTS.IRON UNLOADING: Comparing baseline and exit values, average serum ferritin on the Standard Arm remained stable at 2206 and 2674 ng/mL, respectively, while average LIC rose slightly from 8.5 to 11.3 mg Fe/gm dry weight liver (p=0.052). A total of 19 adverse events were attributed to deferasirox chelation treatment, which occurred in 9 participants (15%) randomised to the Standard Arm. The most frequent events included elevated hepatic transaminases (11 in 5 children), gastrointestinal pain or other symptoms (5 in 4 children), elevated serum creatinine (1), elevated serum bilirubin (1), and rash (1). On the Alternative Arm, a mean of 7 ± 3 monthly overlap transfusions (median 6) were provided without chelation. Among 57 participants who then reached hydroxyurea MTD, 54 received phlebotomy. A total of 756 phlebotomy procedures (mean 13/child) were performed with an average total blood removal of 103 ± 54 mL/kg. Of these, 47 procedures (6.2%) did not remove the full scheduled volume, primarily due to loss of venous access (37), symptoms (7) or other reasons (3). An additional 77 phlebotomy procedures were appropriately canceled per protocol due to low haemoglobin concentration <8.0 g/dL, while another 81 were canceled for various reasons including planned anesthesia (16), provider preference (14), hydroxyurea toxicity (13), intercurrent illness (11), inadequate venous access (9), family request (5), or other (13). A total of 18 adverse events were attributed to phlebotomy procedures, which occurred in 14 participants (23%) randomised to the Alternative Arm. Comparing entry and exit values in the Alternative Arm, the average serum ferritin decreased from 3080 to 1276 ng/mL (p<0.0001), and average hepatic iron decreased from 11.3 to 9.5 mg Fe/gm dry weight liver (p=0.001). Comparing the differences between treatment arms, iron overload improved more in the Alternative Arm with ferritin difference −1047 ng/mL (−1524, −570), p<0.001 and liver iron difference −4.3 mg Fe/gm dry weight liver (−6.1, −2.5), p=0.001, Table 2. BODY.RESULTS.SAFETY MONITORING: In the Standard Arm, a total of 12 rescue transfusions were administered to 7 participants, all for >45% HbS. In the Alternative Arm, a total of 15 rescue transfusions were administered, including 9 during the initial overlap period, to 8 participants with a low hydroxyurea response. One child on the Standard Arm developed TCD velocities >240 cm/sec and was exited per the TCD Alert algorithm (Supplement). Adverse events were fairly well balanced between treatment arms but serious adverse events were more common in the Alternative Arm (Supplemental Table); there were 287 sickle-related adverse events (10 serious adverse events among 6 participants) in the Standard Arm versus 279 sickle-related adverse events (23 serious adverse events among 9 participants) in the Alternative Arm. No deaths occurred during the study treatment period. BODY.DISCUSSION: The TWiTCH trial results document the efficacy of hydroxyurea therapy for a cohort of children with SCA at high risk for primary stroke. Specifically, children on the Alternative Arm, who received an overlap period with transfusions until achieving a stable hydroxyurea MTD, successfully maintained their average TCD velocity throughout the study treatment period. The primary study endpoint (model-based TCD velocities) on the Alternative Arm was non-inferior to the Standard Arm with continued monthly transfusions, thus avoiding the frequent and unacceptable TCD velocity increases observed in STOP2 when no substitute therapy was provided. Since TCD is a direct measurement of intracerebral blood flow and an accepted surrogate for primary stroke risk in children with SCA, these findings suggest that hydroxyurea may also be effective for stroke prevention in this setting, although stroke was not the primary study endpoint of the TWiTCH trial. Importantly, no child in either treatment arm reverted from normal to abnormal TCD velocities, and no new overt, adjudicated, or silent strokes occurred on either treatment arm. Hydroxyurea has multiple therapeutic effects that should be beneficial for children with SCA and abnormal TCD velocities. The most important benefit is HbF induction, which lowers the intracellular %HbS, inhibits sickle polymer formation, and prolongs erythrocyte survival. TWiTCH participants had a robust treatment response to hydroxyurea, reaching an average 27% HbF at MTD. However, since inflammation and endothelial vasculopathy are features of cerebrovascular disease, the reduced leukocyte and reticulocyte counts from hydroxyurea should also be salutary (Figure 2). Other recognised benefits of hydroxyurea include macrocytosis with increased erythrocyte deformability, reduced adhesiveness, and lower blood viscosity with improved rheology. [10] Immediate anti-inflammatory effects of hydroxyurea on the vascular endothelium have also been reported. [19] The compelling results of the TWiTCH trial with early study termination are highly relevant to the management of this high-risk subset of children with SCA. TCD screening programmes are now well-established at most paediatric sickle cell centres and indefinite chronic transfusions are recommended by NHLBI guidelines for children with abnormal TCD velocities to prevent primary stroke. [6] However, successful comprehensive TCD screening with chronic transfusions can be difficult to implement in practice, in part due to concerns about indefinite transfusion therapy. [20–22] Children with abnormal TCD velocities now may have an alternative therapeutic option after a period of transfusions, which represents an important paradigm shift for management of stroke risk in this vulnerable patient population. The ability to screen children with SCA and offer those with abnormal TCD velocities an initial transfusion programme, followed by hydroxyurea for continued stroke prophylaxis, would likely be attractive for many families and providers. Hydroxyurea could improve implementation of primary stroke screening and treatment strategies, by reducing transfusion-associated morbidity and offering potential cost-savings. Since no new infarctions developed in either treatment arm, hydroxyurea therapy may also be relevant in the setting of silent cerebral infarctions, where transfusions have shown efficacy for preventing overt stroke and disease progression. [23] However, before considering the discontinuation of chronic transfusion therapy and starting hydroxyurea for stroke prevention, several key caveats must be noted. First, children with severe vasculopathy [13] were excluded from TWiTCH randomisation, so these children may not be suitable candidates for hydroxyurea. However, this criterion excluded only 12% of children enrolled in TWiTCH and unlike STOP2, the TWiTCH trial included children with persistently elevated TCD velocities despite chronic transfusions, which increased the number of eligible patients and ultimately should broaden applicability. [24] Second, all children received ≥12 months of transfusions (average 4 years) and then continued transfusions during an overlap period with hydroxyurea. In the setting of an abnormal TCD, transfusions should always be the initial treatment and cannot be abruptly discontinued, although the optimal duration of transfusions before considering transition to hydroxyurea has not been determined. Third, hydroxyurea was escalated to MTD to achieve target goals of myelosuppression with concomitant robust HbF responses; lower dosing regimens, infrequent monitoring, and poor adherence would reduce efficacy and potentially be associated with more sickle-related clinical events, especially during the overlap period. An additional limitation of this study is the fact that many study participants were older than the peak age of stroke incidence, although still within the age range of primary infarctive stroke. [1] Finally, the duration of hydroxyurea therapy without transfusions was relatively short; longer follow-up is clearly warranted to determine whether these findings are maintained over time. The management of transfusion-acquired iron overload is also a challenge for children on chronic transfusion therapy. Participants in the Alternative Arm, after reaching a stable hydroxyurea MTD and discontinuing transfusions, received monthly therapeutic phlebotomy to reduce their iron burden. Previously shown to be feasible and potentially beneficial in this setting, [16, 25] hydroxyurea with serial phlebotomy was superior to transfusions with chelation for managing iron overload in the TWiTCH population. With an average of 7 transfusions and 13 phlebotomy procedures, participants on hydroxyurea had significantly lower serum ferritin and LIC than those on continued transfusions with oral chelation (Table 2). Repeated phlebotomy is safe in children with SCA, and with sufficient duration can lead to full resolution of hepatic iron overload. [26, 27] Prevention of cerebrovascular disease in children with SCA is the ultimate goal, ideally through early intervention with disease-modifying therapy. Although genetic variants may influence stroke susceptibility, [28, 29] most stroke events in children with SCA remain unexplained. Data from the BABY HUG cohort suggest that early hydroxyurea therapy may prevent TCD elevation and neurodevelopmental decline. [30] New NHLBI guidelines recommend that all infants with SCA, age 9 months or above, be offered hydroxyurea treatment, regardless of disease severity. [6] If this recommendation is followed, the incidence of cerebrovascular disease will likely decline. Until then, hydroxyurea can now be considered a substitute therapy for maintaining TCD velocities and potentially preventing primary stroke in selected high-risk children with SCA after a period of transfusions. BODY.PUTTING RESEARCH INTO CONTEXT.EVIDENCE BEFORE THIS STUDY: A careful search of the literature, plus review of the recently published NHLBI evidence-based guidelines, identified several open-label trials of either blood transfusions or hydroxyurea for children with SCA and cerebrovascular disease, three randomised clinical trials of transfusions versus observation for children with SCA and cerebrovascular disease (two for abnormal TCD velocities, one for silent cerebral infarcts), and one randomised clinical trial comparing transfusions to hydroxyurea for children with SCA and previous overt stroke. To date, no study has prospectively compared blood transfusions to hydroxyurea for children with SCA and abnormal TCD velocities, although this is the largest group of children with SCA who currently receive chronic transfusions, and no alternative treatment to transfusions is currently available. BODY.PUTTING RESEARCH INTO CONTEXT.ADDED VALUE OF THIS STUDY: This multicentre randomised clinical trial provides definitive evidence that hydroxyurea treatment is non-inferior for maintaining TCD velocities, compared to continued transfusions. The results allow a major paradigm shift in the long-term management of children with SCA and established cerebrovascular disease. BODY.PUTTING RESEARCH INTO CONTEXT.IMPLICATIONS OF ALL THE AVAILABLE EVIDENCE: Children with SCA and abnormal TCD velocities are currently prescribed indefinite transfusion therapy. Now after a period of transfusions, if brain MRA does not show severe vasculopathy, a substitute regimen of hydroxyurea treatment can be considered to maintain TCD velocities and prevent primary stroke in this high-risk patient population. BODY.SUPPLEMENTARY MATERIAL: supplement

TITLE: Social Media under the Skin: Facebook Use after Acute Stress Impairs Cortisol Recovery ABSTRACT: Social media's influence on stress remains largely unknown. Conflicting research suggests that Facebook use may both enhance and undermine psychosocial constructs related to well-being. Using novel experimental methods, this study examined the impact of social media use on stress recovery. Facebook users (n = 92, 49 males, mean age 19.55 SD = 1.63) were randomly assigned to use their own Facebook profile or quietly read after experiencing an acute social stressor. All participants showed significant changes in subjective and physiological stress markers during recovery. Participants who used Facebook experienced greater sustained cortisol concentration (p < 0.05) when controlling for gender and emotional investment in the website compared to controls. Results suggest that social media use may delay or impair recovery after experiencing an acute social stressor. This novel study incorporated objective physiological markers with subjective psychosocial measures to show that Facebook use may negatively impact well-being. BODY.INTRODUCTION: To date, Facebook remains the most popular social networking site, with over 1 billion worldwide users (Facebook Company Information, 2016) and 71% of online U.S. adults naming it as their preferred platform (Duggan et al., 2015). Use of the site remains as varied as it does popular. Approximately 44% of U.S. adults now report receiving their news from Facebook (Gottfried and Shearer, 2016), while research has also found that the site serves a starkly contrasted, but still valuable, utility in coping with campus violence (Vicary and Fraley, 2010). Several studies have examined potential associations between Facebook use and outcomes related to psychosocial well-being, many of which have provided mixed and sometimes conflicting results. For example, use has been associated with increased self-esteem (Best et al., 2014), general well-being (Kim and Lee, 2011), enhanced social support (Bender et al., 2011; Liu and Yu, 2013; Troncone et al., 2015), and overall life satisfaction (Valenzuela et al., 2009; Nabi et al., 2013); just as well as with greater distress (Chen and Lee, 2013), induction of negative social comparison (Chou and Edge, 2012), and declines in subjective well-being (Kross et al., 2013; Verduyn et al., 2015). One of the first studies to examine Facebook use and health found an association between online social integration and reduced mortality rate (Hobbs et al., 2016); however, underlying mechanisms were not explored. Research has also shown that gender may play an important role in how Facebook influences well-being. Not only are females more likely to use Facebook (Anderson, 2015), they may be more susceptible to feeling threatened by specific information displayed on the site (e.g., McAndrew and Shah, 2013). The link between Facebook use and stress receives substantial public attention, and is often touted as a causal relationship (e.g., Heid, 2017). However, this relationship has not been experimentally investigated. To date, one of the few studies to objectively measure stress in relation to Facebook use found that adolescents with larger Facebook networks showed greater cortisol release compared to those who spent less time interacting with Facebook peers (Morin-Major et al., 2016). As an objective marker of the body's physiological stress response (Granger et al., 1999, 2007; Hellhammer et al., 2009; Nater and Rohleder, 2009; Birkett, 2011), cortisol can expand our understanding of the impact of Facebook use on stress. While many studies have focused on mechanisms that may buffer acute laboratory stress (e.g., Creswell et al., 2005; Arch et al., 2014; Inagaki and Eisenberger, 2016), fewer have looked at what may promote physiological recovery. Considering both the negative consequences of stress (e.g., Herbert and Cohen, 1994) and the widespread adoption of Facebook and its abundant presence in the daily lives of many, we aimed to test if social media can truly get under the skin by influencing recovery from stress. We hypothesized that should social media delay physiological and psychosocial stress recovery, participants who used Facebook immediately following an acute social stressor would show elevated and sustained salivary cortisol output, as well as subjective stress. We also explored if users highly invested in Facebook may differ in recovery, and if this effect may relate to gender. BODY.MATERIALS AND METHODS.PARTICIPANTS: Facebook users (n = 112 undergraduates) were recruited from a campus-wide participant pool system. All participants had an active Facebook account and were given course credit in exchange for participation. Self-reported medical diagnoses (e.g., anxiety, PTSD) and use of substances known to influence HPA-axis activity (e.g., steroids, hormonal contraceptives) were considered as exclusion criteria. This study was carried out in accordance with the recommendations of Expedited Review, The University of California, Merced Institutional Review Board, with written informed consent from all subjects. All subjects gave written informed consent in accordance with the declaration of Helsinki. The protocol was approved by the University of California, Merced Institutional Review Board. All data collection complied with current APA ethical standards. BODY.MATERIALS AND METHODS.EXPERIMENTAL TOOLS.STRESS INDUCTION AND PHYSIOLOGICAL MEASURES: The Trier Social Stress Test (TSST; Kirschbaum et al., 1993) has been shown to reliably induce acute stress in the majority of participants in numerous studies (Birkett, 2011). Specifically, it is known to induce a threat to social esteem and reliably induces an increase in cortisol and in negative self-related cognitions and emotions (Dickerson and Kemeny, 2004; Dickerson et al., 2004). In the current study, ~20 min after arriving in the lab, participants were instructed to spend 5 min preparing a speech that could be used in an interview for their ideal job. They then spent 5 min performing the speech in front of a disapproving committee of three presumed experts in a small laboratory room. Participants then counted backward from 1,687 by intervals of 13 for 3 min. When mistakes were made, participants were told to begin again. To further induce stress, participants were video and audio recorded during the speech and math tasks. In addition, committee members wore white lab coats and carried clipboards to enhance the illusion of being experts. The committee always consisted of mixed-gender, undergraduate members (i.e., two males and one female, or two females and one male) who were present in the laboratory room only for the duration of the stress task. BODY.MATERIALS AND METHODS.EXPERIMENTAL TOOLS.STRESS INDUCTION AND PHYSIOLOGICAL MEASURES.SALIVA SAMPLES: To control for natural cortisol fluctuations during the day (Kirschbaum and Hellhammer, 1989; Schultheiss and Stanton, 2009), all data were collected between 1:00 and 5:00 p.m. (i.e., each participant arrived for their 90-min laboratory session at either 1:00 or 3:00 p.m.). To ensure quality of saliva samples and to avoid temporary elevation of cortisol levels (Schultheiss and Stanton, 2009), participants were instructed to refrain from eating, smoking, consuming caffeine, drinking beverages other than water, brushing their teeth, or vigorously exercising in the 30 min before arriving for the study1. All samples were collected using salivette collection tubes (Sarstedt Co., Nümbretch, Germany). Participants placed a cotton roll under their tongue for 2 min of collection. To account for the natural fluctuation of cortisol in reaction to acute stress (Engert et al., 2011), saliva was collected at baseline, and at 8, 20, and 45 min post-stressor onset. Cortisol samples were immediately frozen and immunoassayed on site at a later date. All samples were placed in a –20°C freezer. Thawed samples were centrifuged and assayed in duplicate with a test volume of 25 μL. A commercially available enzyme immunoassay kit was used without modifications to the manufacturer's recommended protocol (Salimetrics, State College, PA). Sensitivity ranged from 0.007 to 3.0 μg/dL. Intra-assay and inter-assay coefficients of variation were less than 15%. BODY.MATERIALS AND METHODS.EXPERIMENTAL TOOLS.STRESS INDUCTION AND PHYSIOLOGICAL MEASURES.BLOOD PRESSURE AND HEART RATE: Blood pressure and heart rate were simultaneously measured with an Omron 10 Series digital blood pressure monitor cuff placed around the non-dominant upper arm at baseline, 8, 20, and 45 min post-stressor onset. BODY.MATERIALS AND METHODS.EXPERIMENTAL TOOLS.PSYCHOSOCIAL MEASURES.FACEBOOK USE: The Facebook Intensity Scale (FBI) measures emotional connectedness to the site and integration of site use into the lives of users (Ellison et al., 2007). The 9-item scale asks participants to rate statements such as, "Facebook has become part of my daily routine," on a five-point scale from 1 (strongly disagree) to 5 (strongly agree). The scale also measures number of Facebook friends as well as average daily time spent actively using Facebook over the past week. Intensity score is computed by averaging all items in the scale, with higher scores indicating higher intensity. Scale validity has not been established; however, the current sample showed moderate reliability (Cronbach's α = 0.77). The Facebook Activity Survey (Junco, 2012) measures frequency of specific activities within Facebook. Examples include frequency of posting status updates, sharing links, and sending private messages on a scale of 1 (never) to 5 (very frequently, 100% of the time). All participants reported their normal Facebook use habits at baseline. Experimental participants completed an adapted version of the survey regarding their specific use of the site during 30 min of recovery. In both cases, frequency of each activity was averaged across participants with higher scores indicating more frequent activity. Participants were also asked which method they most commonly used to access Facebook (i.e., mobile app, website from a computer, or both). In addition, participants in the Facebook use condition were asked how using Facebook for 30 min in one sitting compared to their normal use (i.e., they normally use it less, the same, or more), if they did anything during these 30 min that they normally would not do, and if so, what they did. All questionnaire items assessing Facebook use and stress were asked during follow-up (i.e., after participants had undergone both the acute stressor and used Facebook if they were in the experimental condition). This was done in effort not to bias participants toward the study's true purpose. Participants identified under which state they were most likely to use Facebook (lonely, bored, stressed, sad, or anxious) by rating their agreement on a 1 (strongly disagree) to 5 (strongly agree) scale for the item, "I find myself wanting to use Facebook most when feeling X" for each state. In addition, participants responded to the following statement: "Please rate how stressed using Facebook makes you feel in general," on a five-point scale ranging from 1 (not at all) to 5 (extremely). Participants also rated the following statements on five-point scales ranging from 1 (strongly disagree) to 5 (strongly agree): (1) "In general, I like to use Facebook when I am stressed," (2) "In general, using Facebook when I am stressed makes me feel less stressed," and (3) "In general, using Facebook when I am stressed makes me feel more stressed." Participants in the Facebook use condition were asked to select which statement they agreed with most after using Facebook for 30 min: (1) "Using Facebook made me feel less stressed," (2) "Using Facebook made me feel more stressed," or (3) "Using Facebook did not change my stress level." BODY.MATERIALS AND METHODS.EXPERIMENTAL TOOLS.PSYCHOSOCIAL MEASURES.MOOD: The Positive and Negative Affect Schedule (PANAS; Watson et al., 1988) measured change in mood from baseline to follow-up. The 20-item scale consists of words describing 10 negative emotions and 10 positive emotions. Participants indicated on a scale of 1 (very slightly or not at all) to 5 (extremely) how they felt in the present moment for each emotion. Higher scores for each emotion indicated higher levels of positive or negative affect respectively. The well-validated scale (Crawford and Henry, 2004) showed high internal consistency for baseline ratings of positive affect (Cronbach's α = 0.89) and negative affect (Cronbach's α = 0.79), and for 45-min post-stressor onset ratings of positive affect (Cronbach's α = 0.91) and negative affect (Cronbach's α = 0.83). In addition, mood was directly assessed after recovery for those in the Facebook use condition with the following item: "Please indicate which statement you agree with most: (1) Using Facebook increased my positive mood, (2) Using Facebook increased my negative mood, or (3) Using Facebook did not change my mood." BODY.MATERIALS AND METHODS.EXPERIMENTAL TOOLS.PSYCHOSOCIAL MEASURES.WELL-BEING: Subjective well-being was assessed at each saliva sample collection time point (see Procedure and Figure 1) with a visual analog scale anchored at "not well" and "extremely well" for the statement, "What is your overall sense of well-being right now?" Participants responded by marking along a 15 cm line. Responses were measured and rounded up to the nearest millimeter, then converted to a 15-point continuous scale with higher scores indicating greater feelings of well-being. In addition, well-being was directly assessed after recovery for those in the Facebook use condition on a scale of 1 (not at all) to 4 (a lot) with the following item: "How much did using Facebook influence your sense of well-being either positively or negatively?" Figure 1Timeline for procedural tasks and physiological sample measurements. TSST, Trier Social Stress Test; HR, heart rate; BP, blood pressure; SWB, subjective well-being; SS, subjective stress. BODY.MATERIALS AND METHODS.EXPERIMENTAL TOOLS.PSYCHOSOCIAL MEASURES.SUBJECTIVE STRESS: Subjective stress was assessed at each saliva sample collection time point with present-moment ratings of feeling tense and anxious with the items, "How tense/anxious are you feeling right now?" Each item was rated from "not at all" to "extremely" along the same visual analog scale as the well-being measure. The descriptive terms "tense" and "anxious" were used instead of "stress" for these items in effort not to bias participants to the true purpose of the study. BODY.MATERIALS AND METHODS.DESIGN AND PROCEDURE: To assess the effect of Facebook use on stress recovery, participants in the current study came into the lab believing they would be taking a survey on their Facebook use habits and providing physiological samples (i.e., saliva, blood pressure, heart rate) to assess well-being. All participants underwent an unexpected, acute social stressor before half were randomly assigned to log into their own Facebook account (experimental condition), and half were given neutral reading materials (control condition) for 30 min of recovery. Participants completed all procedures in a single, 90-min laboratory session. All procedures took place within the same laboratory room where only the individual participant and experiment leader were present (with the exception of the portion involving the TSST committee). The experiment leader (a female graduate student not involved in the TSST) explained that the study aimed to look at the influence of social media use on well-being. In the description provided by the online participant recruitment system, participants were told they would need to know their Facebook login information in order to participate in the study; however, participants randomly assigned to the Facebook use condition did not know they would be using Facebook during the study until the moment the experiment leader asked them to log into their own account (~35 min into the study). Control participants never used their login information during the study. All participants were unaware that the study involved a stress task beforehand. After the study was explained and informed consent collected, all participants completed baseline measures. Following, the experiment leader left the room, and the committee entered to conduct the TSST. The experiment leader then returned, excused the committee, and instructed participants on how to proceed. During 30 min of stress recovery, participants randomly assigned to the experimental condition (n = 42) logged into their own Facebook account on the same laboratory computer used to complete baseline and follow-up measures (a laptop stored out of sight during the TSST). They were instructed to use Facebook as they wished with the exception of disclosing any information about their current participation in the study. Participants in the control condition remained in the same room with optional reading materials (scientific journals and magazines). The experiment leader remained in the room with each participant during recovery in order to collect physiological samples and subjective stress measures; however, participants were instructed not to speak to the experiment leader during recovery. Salivary cortisol output, heart rate, blood pressure, subjective stress, and subjective well-being were assessed at baseline, and at 8, 20, and 45 min post-stressor onset (see Figure 1 for study timeline). After recovery (45 min post-stressor onset), all participants completed measures of changes in mood and reported the general influence of Facebook use on stress and well-being. In addition, participants in the experimental condition reported their Facebook activity during the recovery period and the immediate influence of Facebook use on stress, well-being, and mood. After the final saliva measure and follow-up questionnaire, all participants were debriefed about the study's true purpose of testing the effect of social media use on stress recovery. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSES: Analyses were conducted using SPSS Version 24.0 (SPSS Inc., Chicago, IL, USA). Analysis of covariance (ANCOVA) with gender and Facebook Intensity (i.e., emotional connectedness to the site) as covariates was used to test the effect of Facebook use on acute stress recovery. To check the appropriateness of assumptions for the statistical analysis, the Kolmogorov-Smirnov test, histograms, and scatter plots were used. Analyses included mean change scores from baseline to 45 min post-stressor onset for positive and negative affect, mean change scores from 8 to 20 min post-stressor onset for blood pressure and heart rate, and mean change scores from 20 to 45 min post-stressor onset for all other variables. A median split was applied to Facebook Intensity score, creating a dichotomous variable (low, high) for use as a covariate. Seven participants (3 in the Facebook condition, 2 males; 4 in the control condition, 1 male) were missing either the 20- or 45-min saliva sample and thus were not included in final analyses. To account for skewness, cortisol measures were log-transformed before analyses. Unless otherwise noted, effect sizes are presented as partial η2, which represents the proportion of explained variance between the predictors and the outcome, with values of 0.01, 0.06, and 0.14 indicating small, medium, and large effect sizes, respectively (Cohen, 1988). Significance was set at p ≤ 0.05. BODY.RESULTS.SAMPLE CHARACTERISTICS: Given our interest in stress recovery, participants who maintained stable levels or showed a decrease in cortisol output concentration in response to the stress induction (n = 10) were excluded from analyses (i.e., they did not experience an increase in physiological stress and therefore did not experience recovery). Participants who reported current use of prescription medication containing cortisol, cortisone, or hydrocortisone were excluded from analyses (n = 2). One participant was identified as an extreme outlier for cortisol (i.e., score > 4 SDs above the mean) and was excluded from analyses. The final sample of participants (n = 92; 43 females, mean age = 19.74 years, SD = 1.51, BMI = 27.31, SD = 8.42; and n = 49 males; mean age = 19.55, SD = 1.63, BMI = 21.56, SD = 7.54) identified as being Hispanic/Latino (44.6%), Asian/Pacific Islander (20.7%), biracial (14.1%), Caucasian (10.9%), or African American/Black (4.3%). The majority of the sample (68.5%) identified as first-generation college students. Average weekly alcohol consumption within normal range was permitted, however the majority of participants (73.9%) reported zero consumption. Three participants reported current use of recreational drugs while one reported current use of tobacco products. Only 15.6% of female participants (n = 7; 4 control, 3 Facebook use) reported current use of hormonal contraceptives. Three participants reported current anxiety disorder diagnosis; however, none reported current use of anti-anxiety medication. No participants reported current diagnosis of post-traumatic stress disorder nor current use of anabolic steroids. None of these participants showed extreme scores on any outcome measure, nor did their stress response patterns widely diverge from the rest of the sample. Thus, all were retained in analyses. Seven participants quit the study before or during the acute stress induction (see Design and Procedure). Independent-samples t-tests showed no significant condition differences on any measure of Facebook activity or on any baseline physiological measure (see Table 1). Compared to control participants, participants in the Facebook use condition were more likely to report that using Facebook when stressed makes them feel less stressed, t(90) = 2.06, p = 0.04, 95% CI [−0.81, −0.02], d = 0.34. Participants showed no other significant condition differences on any item regarding the general influence of Facebook use on stress and well-being. Table 1 Full sample and condition values for baseline and Facebook use measures. Full sample Control condition FB condition n 92 50 42 Females ( n ) 43 28 15 Age 19.64 (1.57) 19.88 (1.78) 19.36 (1.25) FB ACTIVITY    FB friends < 399 < 399 < 299    Years with FB account < 5 < 5 < 5    Daily use (minutes) < 44 < 44 < 44    FBI low intensity ( n ) 39 21 18    FBI high intensity ( n ) 53 29 24    Most common activities: Liking posts, following links to other websites, viewing photos Liking posts, following links to other websites, scrolling newsfeed without clicking Viewing videos, viewing photos, liking posts I FIND MYSELF WANTING TO USE FB MOST WHEN FEELING:    Lonely 45% 44% 45%    Bored 92% 94% 90%    Stressed 32% 28% 36%    Sad 18% 18% 19%    Anxious 27% 26% 29% In general, how stressed does using FB make you feel? 1.38 (0.55) 1.40 (0.57) 1.36 (0.53) In general, I like to use FB when I'm stressed 2.83 (1.03) 2.90 (0.99) 2.74 (1.08) In general, using FB when stressed makes me feel less stressed 3.32 (0.97) 3.04 (1.03) 3.45 (0.86) In general, using FB when stressed makes me feel more stressed 2.41 (0.99) 2.44 (0.97) 2.38 (1.04) PSYCHOSOCIAL STRESS    Tension 3.29 (2.78) 3.02 (2.52) 3.62 (3.07)    Anxiety 3.41 (2.86) 3.61 (2.95) 3.12 (2.76)    Well-being 10.93 (2.72) 11.29 (2.49) 10.48 (2.95)    Positive affect 29.40 (8.24) 28.22 (7.67) 30.80 (8.74)    Negative affect 15.05 (4.98) 14.76 (4.02) 15.40 (5.97) PHYSIOLOGICAL STRESS    Systolic blood pressure 112.34 (12.38) 112.18 (14.15) 112.55 (10.04)    Diastolic blood pressure 71.77 (7.87) 71.94 (8.18) 71.57 (7.58)    Heart rate 72.10 (10.86) 73.98 (11.19) 69.88 (10.14)    Cortisol 0.17 (0.11) 0.18 (0.12) 0.17 (0.10) Reported values reflect n = 92. FB, Facebook. FBI, Facebook Intensity Scale. Participants responded to number of FB Friends, Years with Facebook account, and Daily use as closed-ended questions. For these items, values represent the number, years, and time in minutes that correspond to the median responses from ordinal 1-to-5 scales. FBI low/high intensity represent number of participants in each condition after a median split was applied to the Facebook Intensity Scale. Percentages for each state (lonely, bored, etc.) represent percentage of participants who agreed or strongly agreed with each statement. All other values represent baseline condition means and standard deviations. Cortisol values represent raw salivary cortisol concentration in μg/dL. Bolded values indicate a significant difference between conditions at p ≤ 0.05 . BODY.RESULTS.EFFECT OF FACEBOOK USE ON PSYCHOSOCIAL STRESS RECOVERY: Fifty-two percent of the participants in the Facebook use condition identified the Facebook mobile app as their most common method of access, while 16.7% reported most commonly using the website on a computer, and 31% reported using both the mobile app and a computer to access Facebook equally. When asked what they did during 30 min of stress recovery, participants in the Facebook use condition identified passively scrolling newsfeed, viewing videos, and following links as the activities they spent the most time doing. The majority of participants (66.7%) indicated that they normally spend less than 30 min using Facebook in one sitting. However, the majority of participants (66.7%) also indicated that using Facebook for 30 min did not cause them to engage in any activities during one sitting in which they normally would not. Participants who reported doing something they normally would not do because of the extended use time (n = 14) almost exclusively reported that they watched videos. When asked about the effect of Facebook use on mood, 54.8% of participants reported a positive change in mood, 14.3% reported a negative change in mood, and 31.0% reported no effect on mood during recovery. In addition, 59.5% reported that using Facebook made them feel less stressed, 7.1% reported feeling more stressed, and 33.3% reported experiencing no change in stress level as a result of using Facebook during recovery. Seventy-nine percent of participants reported that using Facebook changed their sense of well-being during recovery. Participants in both conditions experienced similar changes in psychosocial stress during recovery with decreases in tension and anxiety and increases in well-being. There were no significant condition differences for tension F(1, 91) = 1.56, p = 0.21, 95% CI [–0.29, 1.25], η2 = 0.02; anxiety F(1, 88) = 0.004, p = 0.95, 95% CI [–1.01, 0.95], η2 = 0.00; or well-being F(1, 91) = 0.33, p = 0.57, 95% CI [–0.49, 0.90], η2 = 0.004. Positive and negative affect were measured at baseline and follow up. While participants showed decreases in positive affect and increases in negative affect, there were no significant condition differences for either positive affect, F(1, 91) = 2.50, p = 0.12, 95% CI [–4.78, 0.54], η2 = 0.03 or negative affect, F(1, 91) = 0.053, p = 0.82, 95% CI [–2.20, 2.77], η2 = 0.01 when controlling for Facebook Intensity and gender (see Figures 2A–D for subjective stress markers). Figure 2(A–D) Subjective stress markers and cortisol for Facebook and Control conditions. Facebook and Control conditions showed no significant differences at any time point (p < 0.05). Raw cortisol values are shown in Plot D; log-transformed scores were used for analyses. BODY.RESULTS.EFFECT OF FACEBOOK USE ON PHYSIOLOGICAL STRESS RECOVERY: Preliminary analyses confirmed that all participants experienced physiological stress in response to the TSST (see Procedure). Participants showed a significant decrease in both blood pressure and heart rate from eight to 20 min post-stressor onset, indicating that recovery of heart rate and blood pressure occurred; however, there were no significant condition effects for systolic blood pressure F(1, 91) = 0.16, p = 0.69, 95% CI [–3.60, 5.38], η2 = 0.002; diastolic blood pressure F(1, 91) = 2.11, p = 0.15, 95% CI [–0.95, 6.13], η2 = 0.023, or heart rate F(1, 91) = 1.05, p = 0.31, 95% CI [–1.65, 5.24], η2 = 0.012 when controlling for gender and Facebook Intensity score. Compared to control participants at baseline, participants in the Facebook use condition were more likely to report that using Facebook when stressed makes them feel less stressed (see Table 1). However, compared to the Facebook use condition (Mdifference = –0.35, SD = 0.37), control participants (Mdifference = –0.51, SD = 0.38) showed a significantly greater decrease in cortisol concentration from 20 to 45 min post-stressor onset when controlling for gender and Facebook Intensity score2, F(1, 84) = 5.03, p = 0.03, 95% CI [0.21, 0.33], η2 = 0.06 (see Figures 2A–D). Secondary to assessing the effect of Facebook use on stress recovery, we explored how both investment in the website and gender may influence recovery. Although sample sizes did not allow for testing interaction effects, descriptively, females in the Facebook use condition who reported high levels of Facebook Intensity showed the smallest reduction in cortisol concentration during recovery (see Figure 3). That is, based only on descriptive mean differences, they remained the most stressed compared not only to control participants with high and low Facebook Intensity, and to males who used Facebook with high and low Facebook Intensity, but also compared to females who used Facebook with low levels of Facebook Intensity. A similar pattern was reflected when females (M = 2.70, SD = 0.94) were more likely than males (M = 2.16, SD = 0.98) to report that using Facebook when stressed makes them feel more stressed t(90) = 2.65, p = 0.01, 95% CI [–0.93, –0.13], d = 0.46. This potential interaction between gender and investment in the website suggests that highly invested females may be more susceptible to social media-induced stress. Figure 3Salivary cortisol response to stress for the Facebook use condition by gender and high/low Facebook Intensity. Raw cortisol values are reported here; log-transformed values were used for analyses. BODY.DISCUSSION: The present study provides the first objective evidence of how social media may affect stress. All participants experienced significant changes in subjective and physiological stress in response to an acute laboratory stressor; however, using Facebook inhibited physiological recovery. Specifically, participants who used Facebook during recovery showed sustained cortisol levels compared to control participants, suggesting that Facebook can get under the skin. Given the mixed literature on Facebook use and well-being, several explanations for such findings exist. Social Self-preservation Theory poses that the social self-preservation system (including the HPA axis) tracks one's surroundings for threats to social status or social esteem. When a threat is present, both cortisol and negative self-related cognitions and emotions increase (Dickerson et al., 2004). Given that Facebook use has been associated with social status and social esteem (e.g., Ellison et al., 2011; Best et al., 2014), it is possible that Facebook use could be viewed as a threat to self-preservation and may induce similar physiological effects. In the present study, participants who immediately engaged with their own Facebook profile after experiencing an acute social stressor sustained significantly higher levels of objective stress compared to control participants (i.e., their recovery from stress was delayed). This extended stress response may reflect an additive effect of physiological and psychosocial arousal in response to threats to self-preservation (i.e., both the acute social stressor and Facebook were perceived as threats; therefore, participants who experienced both showed prolonged stress responses compared to those who only experienced the acute social stressor). This relationship may be particularly true for females highly invested in the site. It is also possible that immediately engaging in a stimulating activity after experiencing acute stress may have reduced the likelihood of recovering from stress. All participants were at a heightened level of arousal at the beginning of recovery. Directly beginning another task (i.e., using Facebook) may have sustained higher levels of physiological arousal. Although using Facebook sustained cortisol, it had no effect on blood pressure or heart rate. This may have been due to the natural rapid recovery rate of these more acute markers (Linden et al., 1997). However, it is also possible that Facebook differently affects sympathetic-adrenomedullary (SAM) activity (blood pressure and heart rate) and HPA activity (cortisol). Future work may benefit from including additional and more precise measurement of the SAM system (e.g., salivary alpha-amylase). Despite sustaining cortisol, Facebook did not sustain psychosocial stress. Those who used Facebook reported recovering as much as those in the control condition despite showing a sustained physiological stress response. This dissociation is consistent with findings demonstrating that the psychological experience of stress does not necessarily map on to a physiological response (e.g., Kirschbaum et al., 1995; Egloff et al., 2002; Inagaki and Eisenberger, 2016; Levi, 2016). In this specific context, this dissociation may aid in explaining mixed findings in the literature. For example, cross-sectional findings implicating associations between both Facebook use and enhanced well-being (Kim and Lee, 2011) and Facebook use and greater distress (Chen and Lee, 2013) may reflect a disconnection between what users experience and what they report. Although there were no significant effects of Facebook use on self-reported stress in this study, examining this relationship will remain important considering that the majority of participants in the Facebook use condition reported at baseline that using Facebook when stressed reduces stress. While results suggest a link between social media use and stress, the implications for overall well-being are less clear. The context of acute stress provides valuable insight into how Facebook use may influence users, especially given that a significant portion of users report not only wanting to use the site when stressed, but that using it when stressed actually reduces stress. Subjective stress reduction may in fact occur; however, our findings highlight the importance of also considering physiological stress recovery. Given the known associations between stress and negative health and well-being outcomes (Herbert and Cohen, 1994), Facebook use after experiencing acute stress may not be recommended. Despite these novel findings, limitations must be addressed. First, the majority of participants reported using the Facebook mobile app as the most common means of access. Use of the platform in such a context may have different implications for stress recovery, particularly given that mobile use implies a mobile environment (e.g., walking around in a public space). However, to best capture the effect of Facebook use on stress recovery in an experimental context, limiting platform access to a stationary laptop computer in a quiet room allowed us to rule out confounding factors. Future work assessing mobile use of the platform will require careful control of many external environmental factors. Second, in effort to provide some level of arousal for all participants, the control condition involved a stimulating, yet neutral activity. We did not assess if participants normally read magazines when feeling stressed. Third, use of a computer (vs. reading magazines) may have influenced physiological recovery. Future work may benefit from including a third condition involving complete rest. Fourth, participants may have accessed materials when using Facebook that could have differently affected arousal. As social media continues evolving, future work should consider how specific activities (e.g., posting photos, viewing videos, etc.) differently influence well-being. Similarly, the potentially variable impact of different text- and image-based platforms (e.g., Twitter, Instagram, and Snapchat) must also be considered. Finally, the broader social context of use must be acknowledged. For example, national and global-level events (e.g., the constant social media coverage of the contentious 2016 U.S. Presidential Election) may temporarily create an inherently stressful environment with otherwise undue consequences for well-being. Although much work remains to be done, the present study provides the first experimental evidence that social media may in fact get under the skin. We show that when accounting for gender and investment in the website, using Facebook after facing an acute social stressor delays physiological stress recovery in terms of cortisol. That is, using Facebook when stressed sustains physiological stress. Future work must consider with greater precision, the influence of specific Facebook activities on both psychological and physiological well-being. Particular attention should be paid to user gender and investment in the website. BODY.AUTHOR CONTRIBUTIONS: HR developed the study concept. Both authors contributed to the study design. Testing and data collection were performed by HR. Immunoassay, data analyses, and interpretation were performed by HR under the supervision of JT. HR drafted the manuscript, and JT provided critical revisions. Both authors approved the final version of the manuscript for submission. BODY.AUTHOR CONTRIBUTIONS.CONFLICT OF INTEREST STATEMENT: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

TITLE: Adequate Initial Heparin Dosage for Atrial Fibrillation Ablation in Patients Receiving Non-Vitamin K Antagonist Oral Anticoagulants ABSTRACT.BACKGROUND AND OBJECTIVE: During atrial fibrillation ablation, heparin is required and is guided by the activated clotting time (ACT). Differences in the ACT before ablation and adequate initial heparin dosing in patients receiving non-vitamin K antagonist oral anticoagulants (NOACs) were examined. ABSTRACT.METHODS: Patients who received warfarin (control, N = 90), dabigatran etexilate (N = 90), rivaroxaban (N = 90) and apixaban (N = 90) were studied. A 100 U/kg dose of heparin was administered as a reliable control dose for warfarin, and the remaining patients were randomly administered 110, 120 or 130 U/kg of heparin in each NOAC group, followed by a continuous heparin infusion. ABSTRACT.RESULTS: Periprocedural thromboembolic and major bleeding were not observed. Minor bleeding occurred rarely without significant differences among the groups examined. Baseline ACTs were longer in the warfarin (152 ± 16 s) and dabigatran (153 ± 13 s) groups than in the rivaroxaban (134 ± 13 s) and apixaban (133 ± 20 s) groups. The initial bolus heparin dosages required to produce an ACT 15 min after the initial bolus that was identical to the control (333 ± 32 s) were 120 U/kg (318 ± 29 s) and 130 U/kg (339 ± 43 s) for dabigatran, 130 U/kg (314 ± 31 s) for rivaroxaban and 130 U/kg (317 ± 39 s) for apixaban. The NOAC groups required significantly larger doses of total heparin than the warfarin group. ABSTRACT.CONCLUSION: The baseline ACTs differed among the three NOAC groups. The results of the comparison with warfarin (the control) indicated that dosages of 120 or 130 U/kg for dabigatran, and 130 U/kg for rivaroxaban and apixaban, were adequate initial heparin dosages. BODY.KEY POINTS: Adequate initial heparin dosages for atrial fibrillation ablation in patients receiving non-vitamin K antagonist oral anticoagulants (NOACs) were 10–20 % higher than those in patients receiving warfarin anticoagulation.The initial dosing of heparin needs to be adjusted in patients receiving NOACs. BODY.INTRODUCTION: Atrial fibrillation (AF) is the most common sustained arrhythmia and has a significant impact on morbidity and mortality [1]. Radiofrequency (RF) energy applied to circumferentially isolate the pulmonary veins (PVs) from the left atrium (LA), i.e. PV atrium isolation (PVAI), is the most effective treatment for AF, with a cure rate of 50–90 % [2, 3]. AF ablation is one of the most complex interventional electrophysiological procedures; thus, it is associated with several complications, most importantly thromboembolism [4]. Despite the introduction of novel ablation technologies such as open irrigation catheters, and the widespread use of systemic anticoagulation with heparin, the risk of periprocedural thromboembolism remains significant, reaching approximately 3 % in large series [5]. Over the past 5 years, non-vitamin K antagonist oral anticoagulants (NOACs), also known as novel oral anticoagulants, such as dabigatran etexilate, rivaroxaban and apixaban, have been approved for long-term oral anticoagulation, and their safety, efficacy and quality of anticoagulation in patients with non-valvular AF have been demonstrated [6]. NOACs offer several advantages, including short half-lives, ease of administration, fewer interactions and no need for laboratory monitoring. Although continuation or short-term interruption of NOACs is a safe strategy for most invasive procedures, patients with cardiovascular risk undergoing major procedures may benefit from heparin bridging [7]. We hypothesized that the baseline activated clotting time (ACT), i.e. just before transseptal puncture, and the initial bolus and total heparin required during an ablation procedure, based on the ACT, may differ among NOACs and warfarin anticoagulation therapies. The Heart Rhythm Society's scientific statement recommends 100 U/kg of standard heparin administered as an initial bolus before transseptal puncture in patients who have been administered warfarin anticoagulation therapy, and a target ACT of 300–350 s [4]. Another worldwide survey has reported that many studies have maintained an ACT of at least 230–350 s [5]. No study regarding changing the initial heparin dosing has been reported, as the relationship between the initial heparin dosage and the ACT for determining an adequate initial heparin dosage has not been evaluated. On the basis of the aforementioned scientific statement, a 100 U/kg initial bolus of heparin under uninterrupted warfarin anticoagulation therapy was set as a reliable standard control dose. In the present study, we evaluated the relationship among three different initial heparin bolus doses of anticoagulation therapies and the ACT 15 min after the initial bolus of heparin was administered, and we compared the results with those in patients receiving warfarin (the control) to determine the adequate dosage of initial heparin for each NOAC. BODY.METHODS.STUDY SUBJECTS: The present study was conducted at a single-centre, Okayama Heart Clinic (Okayama, Japan), and it included patients who underwent their first AF ablation between September 2013 and December 2014. Patients with previous AF ablation and decreased renal function (creatinine clearance rate <30 mL/min) were excluded. We analysed 360 patients (age 64 ± 10 years; 274 men and 86 women; 224 with paroxysmal AF and 136 with non-paroxysmal AF). Patients were classified into a control (warfarin) group and three NOAC groups: dabigatran, rivaroxaban and apixaban. Each group included 90 consecutive patients. On the basis of the Heart Rhythm Society's scientific statement described above, 100 U/kg of an initial bolus heparin dose in patients receiving warfarin anticoagulation therapy was used in the control group [4]. The primary and second endpoints were set as the ACT measured 15 min after the initial bolus heparin administration (15-min ACT) and thromboembolic complications. A preliminary test to determine the range of the heparin dosage in the NOAC groups indicated that 100 U/kg did not provide >50 % of patients with 15-min ACTs >300 s. Therefore, for each NOAC group, the dosage of the initial heparin bolus administration was randomly assigned, i.e. 110, 120 and 130 U/kg, as stated below. The anticoagulant regimens in the three groups are shown in Fig. 1. The selection and dosages of NOAC were left to the physician's discretion, considering the patient's characteristics (including renal function) and the drug manufacturer's directions [8]. Dabigatran and apixaban were administered twice a day in the morning and evening. Rivaroxaban was administered once a day in the morning.Fig. 1Schematic presentation of the anticoagulation medication regimens administered in the three non-vitamin K antagonist oral anticoagulant (NOAC) and control (warfarin) groups. AF atrial fibrillation, PT-INR prothrombin time–international normalized ratio After the completion of AF ablation, heparin was discontinued. Protamine was administered intravenously (20 or 30 mg, 2800–3600 anti-heparin IU) just after removal of all sheaths to reverse heparin, depending on whether the ACT was 300–350 s or >350 s, respectively). When bleeding at the puncture site did not stop after the initial administration of protamine sulfate, additional doses of it (10–20 mg, 1400–2800 anti-heparin IU) were administered, depending on the bleeding status. Haemostasis at the catheter insertion site was confirmed 3 h after completion of the AF ablation, which was the removal of all devices and sheaths from the vessels, then a single dose of each NOAC was administered. Data obtained from 120 days of anticoagulation therapy (from 30 days before to 90 days after) were analysed. The examination procedure complied with the rules of the Declaration of Helsinki [9], and the study was approved by the Institutional Ethics Committee for Human Research of Okayama Heart Clinic. Written informed consent was obtained from all patients. BODY.METHODS.CATHETER ABLATION FOR ATRIAL FIBRILLATION: Details of the present AF ablation procedure have been published elsewhere [10]. In brief, for electrical mapping and ablation, five venous accesses were obtained as follows. Two standard electrophysiology catheters were positioned: a 4-French (F) catheter (Japan Lifeline Co., Ltd, Tokyo, Japan) at the His bundle region via a femoral vein and a 6-F catheter in the coronary sinus via the right intrajugular vein. Three catheters were positioned in the LA by use of the Brockenbrough technique, which requires two decapolar ring catheters (Japan Lifeline Co., Ltd) and an open irrigated ablation catheter (CoolFlexTM, St Jude Medical, Inc.; CoolPath DuoTM, St Jude Medical, Inc.; or Safire BLUTM, St Jude Medical, Inc.). PVAI was performed in all patients by use of an open irrigated ablation catheter inserted via the transseptal sheath with an electroanatomic integration mapping system (Ensite-NavX System, St Jude Medical, Inc.). The temperature of the oesophagus was continuously monitored by a catheter with a temperature sensor (SensiThermTM, St Jude Medical, Inc.) during the ablation. The endpoint of PVAI was defined as [1] the elimination of PV potentials recorded by the two ring catheters within the ipsilateral PVs and the lack of LA capture during intra-PV, isthmus and PV atrium pacing at least 30 min after isolation; and [2] no recurrence of PV spikes within all of the PVs after intravenous administration of 20–40 mg of adenosine triphosphate during sinus rhythm or coronary sinus pacing. Acute success of the ablation was defined as satisfaction of the endpoint criterion noted above. In patients with paroxysmal AF, only PVAI was performed. In patients with persistent and long-standing persistent AF, additional ablation was performed in combination with PVAI. After PVAI, an LA roof line was created first, and ablation of complex fractionated atrial electrograms in the right atrium, LA and coronary sinus, and linear ablation, were also performed at the operator's discretion. Further ablation of the superior vena cava and cavotricuspid isthmus was also performed. If the AF did not terminate, direct current cardioversion was performed to achieve normal sinus rhythm. BODY.METHODS.HEPARIN ADMINISTRATION: In each group, when AF ablation was performed in the afternoon, heparin (5000 U) was administered subcutaneously in the morning on the day of the AF ablation procedure. Before transseptal catheterization, a bolus of heparin (100 U/kg) was administered in the warfarin group as the control. For each NOAC, a bolus of intravenous heparin, in which the dosage was randomly assigned, i.e. 110, 120 and 130 U/kg, was administered. After bolus heparin administration, a continuous heparinized saline infusion was administered via a peripheral vein to maintain the ACT within 300–350 s to avoid thrombus formation. The ACT was measured in 30-min intervals. BODY.METHODS.POSTABLATION CARE AND FOLLOW-UP: After the procedure, anticoagulation therapy was continued for at least 3 months after AF ablation in all groups (Fig. 1). All patients were followed monthly at our centre for at least 90 days after AF ablation. BODY.METHODS.COMPLICATIONS: Cerebrovascular accidents and transient ischaemic attacks were considered thromboembolic complications after intracranial haemorrhage was ruled out. Pulmonary embolism and deep venous embolism were also defined as thromboembolic complications. Cardiac tamponade, pericardial effusion and bleeding were considered bleeding complications. Cardiac tamponade was defined by characteristic clinical features with a considerable pericardial effusion that required drainage. Pericardial effusion was defined as an effusion determined in the pericardial space by routine follow-up echocardiography without any haemodynamic disturbance. Major bleeding was defined as bleeding requiring blood transfusion, haematomas requiring surgical intervention and cardiac tamponade requiring drainage. Late cardiac tamponades were those occurring >48 h after the procedure. Minor bleeding complications included small haematomas and pericardial effusions not requiring drainage (non-tamponade). The primary safety outcome was a composite of bleeding and thromboembolic complications. Miscellaneous non-anticoagulation-related events were also recorded. BODY.METHODS.STATISTICAL ANALYSIS: We used SPSS version 17 for statistical analysis. Data were expressed as mean ± standard deviation. For comparison of two groups, a student's t test and Chi squared test were used for continuous and categorical variables, respectively. For multiple comparisons of continuous variables, including ACT levels among the warfarin control and three NOAC groups, we used one-way analysis of variance (ANOVA) or a Kruskal–Wallis test, and Scheffe's F test or a Mann–Whitney U test with Bonferroni correction as post hoc tests to compare two groups in multiple groups, when appropriate. Chi squared tests with m × n contingency tables and two-tailed tests for categorical variables were used to evaluate the differences among the three NOAC groups. Differences at P < 0.05 were considered significant. BODY.RESULTS.PATIENT CHARACTERISTICS: Patient characteristics in the control and three NOAC groups are summarized in Table 1. No significant differences were found in the patients' background characteristics, such as age, sex and associated disorders, among the control and three NOAC groups. Similarly, echocardiographic parameters did not differ among the control and three NOAC groups. Furthermore, no significant differences were seen in the AF conditions among the four groups.Table 1Patient characteristics in the control (warfarin) and three non-vitamin K antagonist oral anticoagulant groupsCharacteristicWarfarinDabigatranRivaroxabanApixaban P value N = 90 N = 90 N = 90 N = 90Age (years)66 ± 963 ± 962 ± 1065 ± 100.17Sex, female, N (%)24 (27 %)24 (27 %)15 (17 %)23 (26 %)0.32Type of AF Paroxysmal, N (%)52 (58 %)54 (60 %)56 (62 %)62 (69 %)0.36 Persistent, N (%)27 (30 %)22 (24 %)28 (31 %)21 (23 %) Long-standing persistent, N (%)11 (12 %)14 (16 %)6 (7 %)7 (8 %)Duration of AF (years)4 ± 36 ± 84 ± 34 ± 30.12Echocardiography parameters LVEF (%)65 ± 765 ± 966 ± 765 ± 80.41 LA diameter (mm)41 ± 541 ± 741 ± 542 ± 50.15CHADS2 score0.6 ± 0.70.5 ± 0.70.6 ± 0.70.5 ± 0.70.69 045 (50 %)52 (58 %)51 (57 %)55 (61 %)0.69 135 (39 %)28 (31 %)27 (30 %)23 (26 %) ≥210 (11 %)10 (11 %)12 (13 %)12 (13 %)CHA2DS2-VASc score1.4 ± 1.21.4 ± 1.31.5 ± 1.21.6 ± 1.00.30Cr (mg/dL)0.88 ± 0.420.81 ± 0.190.85 ± 0.200.87 ± 0.210.18CCr (mL/min)90 ± 3188 ± 2190 ± 3380 ± 230.12PT-INR2.3 ± 0.3Dabigatran (mg)267 ± 41Rivaroxaban (mg)14.2 ± 1.8Apixaban (mg)9.1 ± 2.0Values are presented as mean ± standard deviation AF atrial fibrillation, CCr creatinine clearance, Cr creatinine, LA left atrium, LVEF left ventricular ejection fraction, PT-INR prothrombin time–international normalized ratio No significant differences were found in the clinical characteristics, echocardiographic parameters and AF status obtained among patients who were administered 110, 120 and 130 U/kg of an initial bolus of heparin in each NOAC group (Table 2).Table 2Comparison of characteristics among patients receiving three different initial bolus heparin dosages for each non-vitamin K antagonist oral anticoagulantDabigatranRivaroxabanApixabanBolus heparin dosage:110 U/kg120 U/kg130 U/kg P value110 U/kg120 U/kg130 U/kg P value110 U/kg120 U/kg130 U/kg P valueNumber of patients303030303030303030Age (years)63 ± 1064 ± 863 ± 100.9861 ± 1262 ± 964 ± 100.3465 ± 1163 ± 1066 ± 110.77Sex, female, N (%)9 (30 %)7 (23 %)8 (27 %)0.857 (23 %)5 (17 %)3 (10 %)0.576 (20 %)6 (20 %)12 (40 %)0.13Type of AF Paroxysmal, N (%)18 (60 %)17 (57 %)19 (63 %)0.9818 (60 %)19 (63 %)19 (63 %)0.9421 (70 %)22 (73 %)19 (63 %)0.41 Persistent, N (%)8 (27 %)7 (23 %)7 (23 %)9 (30 %)10 (33 %)9 (30 %)6 (20 %)5 (17 %)10 (33 %) Long-standing persistent, N (%)4 (13 %)6 (20 %)4 (14 %)3 (10 %)1 (4 %)2 (7 %)3 (10 %)3 (10 %)1 (4 %)Duration of AF (years)8 ± 125 ± 44 ± 40.364 ± 23 ± 24 ± 40.975 ± 44 ± 33 ± 20.42Echocardiography parameters LVEF (%)66 ± 665 ± 1165 ± 100.9965 ± 765 ± 666 ± 90.8065 ± 967 ± 764 ± 70.13 LA diameter (mm)41 ± 641 ± 741 ± 70.9841 ± 541 ± 540 ± 60.9942 ± 542 ± 441 ± 50.64CHADS2 score0.4 ± 0.60.5 ± 0.60.7 ± 0.80.540.5 ± 0.70.7 ± 0.80.4 ± 0.60.360.6 ± 0.80.4 ± 0.70.6 ± 0.70.71 019 (63 %)17 (57 %)16 (53 %)0.2917 (57 %)15 (50 %)19 (63 %)0.8918 (60 %)20 (67 %)17 (57 %)0.82 19 (30 %)11 (36 %)8 (27 %)10 (33 %)8 (27 %)9 (30 %)7 (23 %)7 (23 %)9 (30 %) ≥22 (7 %)2 (7 %)6 (20 %)3 (10 %)7 (23 %)2 (7 %)5 (17 %)3 (10 %)4 (13 %)CHA2DS2-VASc score1.3 ± 1.31.4 ± 1.31.6 ± 1.50.771.2 ± 1.21.5 ± 1.31.2 ± 1.10.641.6 ± 0.91.5 ± 0.91.7 ± 1.10.75Cr (mg/dL)0.83 ± 0.230.84 ± 0.190.76 ± 0.150.230.85 ± 0.230.83 ± 0.210.88 ± 0.150.320.89 ± 0.240.89 ± 0.180.82 ± 0.220.18CCr (mL/min)87 ± 2687 ± 1688 ± 200.6697 ± 4294 ± 3379 ± 170.1479 ± 2782 ± 2279 ± 200.81Values are presented as mean ± standard deviation AF atrial fibrillation, CCr creatinine clearance, Cr creatinine, LA left atrium, LVEF left ventricular ejection fraction BODY.RESULTS.PROCEDURAL PARAMETERS AND ABLATION SUCCESS: No significant differences were seen in the procedural parameters, including the procedure time and RF energy supply time, between the control and dabigatran, rivaroxaban and apixaban groups. All patients in the control and three NOAC groups reached the endpoint of PVAI, and the initial success rate did not differ among the four groups (Table 3). When comparisons were made among patients who received an initial bolus of 110, 120 and 130 U/kg, no significant differences in the procedural parameters and ablation success were found among the three NOAC groups (Table 4).Table 3Comparison of procedural parameters among the control (warfarin), dabigatran, rivaroxaban and apixaban groupsProcedural variablesWarfarinDabigatranRivaroxabanApixaban P value N = 90 N = 90 N = 90 N = 90Presenting rhythm Sinus rhythm59 % (53/90)57 % (51/90)58 % (52/90)62 % (56/90)0.88 AF/AFL41 % (37/90)43 % (39/90)42 % (38/90)38 % (34/90)Acute success98 % (88/90)98 % (88/90)98 % (88/90)99 % (89/90)0.99Procedure time (min)110 ± 24106 ± 24105 ± 18105 ± 200.41Fluoroscopy time (min)33 ± 933 ± 1033 ± 633 ± 80.21RF time (min)35 ± 833 ± 833 ± 633 ± 80.20Intraprocedural cardioversion32 % (29/90)29 % (26/90)32 % (29/90)31 % (28/90)0.96PVAI success100 % (90/90)100 % (90/90)100 % (90/90)100 % (90/90)1Values are presented as mean ± standard deviation AF atrial fibrillation, AFL atrial flutter, PVAI pulmonary vein atrium isolation, RF radiofrequencyTable 4Procedural parameters among patients receiving three different initial bolus heparin dosages for each non-vitamin K antagonist oral anticoagulantDabigatran P valueRivaroxaban P valueApixaban P valueHeparin dosage:110 U/kg120 U/kg130 U/kg110 U/kg120 U/kg130 U/kg110 U/kg120 U/kg130 U/kgNumber of patients303030303030303030Procedural variables Presenting rhythm Sinus rhythm17 (57 %)16 (53 %)18 (60 %)0.8717 (57 %)17 (57 %)18 (60 %)0.9919 (63 %)20 (67 %)17 (57 %)0.72 AF/AFL13 (43 %)14 (47 %)12 (40 %)13 (43 %)13 (43 %)12 (40 %)11 (37 %)10 (33 %)13 (43 %) Acute success97 % (29/30)97 % (29/30)100 % (30/30)0.9497 % (29/30)100 % (30/30)97 % (29/630)0.94100 % (30/30)97 % (29/30)100 % (30/30)0.88 Procedure time (min)102 ± 22112 ± 26105 ± 220.47107 ± 13103 ± 22105 ± 180.83103 ± 19105 ± 19107 ± 210.59 Fluoroscopy time (min)30 ± 736 ± 1333 ± 100.2333 ± 733 ± 533 ± 50.9132 ± 732 ± 634 ± 110.76 RF time (min)33 ± 933 ± 933 ± 70.8832 ± 534 ± 533 ± 60.3033 ± 432 ± 534 ± 60.12 Intraprocedural cardioversion9 (30 %)10 (33 %)7 (23 %)0.6910 (33 %)11 (37 %)8 (27 %)0.709 (30 %)8 (27 %)11 (37 %)0.70 PVAI success100 % (30/30)100 % (30/30)100 % (30/30)1100 % (30/30)100 % (30/30)100 % (30/30)1100 % (30/30)100 % (30/30)100 % (30/30)1Values are presented as mean ± standard deviation AF atrial fibrillation, AFL atrial flutter, PVAI pulmonary vein atrium isolation, RF radiofrequency BODY.RESULTS.ACTIVATED CLOTTING TIME 15 MIN AFTER THE INITIAL BOLUS HEPARIN ADMINISTRATION: No significant differences in the ACT before the AF ablation procedure were found between patients who underwent AF ablation in the morning and afternoon (Table 5). Rather, the ACT was identical between the morning and afternoon sessions. Further, none of the parameters evaluated was significantly different between the morning and afternoon sessions (data not shown). Thus, the present study performed the analysis using combined data from the morning and afternoon sessions.Table 5Comparison of baseline activated clotting times (ACTs) between patients who underwent atrial fibrillation (AF) ablation in the morning or afternoon N AF ablation time P valueMorningAfternoon N ACT (s) N ACT (s)All360148141 ± 18212145 ± 180.06Warfarin9040152 ± 1650151 ± 180.74Dabigatran9022153 ± 1368157 ± 160.28Rivaroxaban9040134 ± 1350135 ± 110.58Apixaban 9046133 ± 2044132 ± 140.79Values are presented as mean ± standard deviation The baseline ACTs in the control and dabigatran groups (151 ± 17 and 156 ± 15 s, respectively) were significantly longer than those in the rivaroxaban (135 ± 12 s) and apixaban (132 ± 17 s) groups (Fig. 2). Figure 3 shows the ACTs 15 min after the initial bolus heparin administration (defined as the 15-min ACT). Generally, the ACT was longer as the dosage of heparin increased in each NOAC group. In the dabigatran group, the 15-min ACTs for the initial heparin doses of 120 U/kg (318 ± 29 s) and 130 U/kg (339 ± 43 s) were significantly longer than those for the initial heparin dose of 110 U/kg (295 ± 21 s). In the rivaroxaban group, the 15-min ACT for the initial heparin dose of 130 U/kg (314 ± 31 s) was longer than that for the initial heparin doses of 120 U/kg (295 ± 21 s) and 110 U/kg (295 ± 32 s). In the apixaban group, the 15-min ACTs for the initial heparin dose of 120 U/kg (313 ± 39 s) and 130 U/kg (317 ± 39 s) were longer than that for the initial heparin dose of 110 U/kg (298 ± 36 s).Fig. 2Baseline activated clotting times (ACTs), i.e. just before transseptal perforation, among the control (warfarin) and three non-vitamin K antagonist oral anticoagulant groups. The P values indicate significant differences, and the vertical bars indicate one standard deviationFig. 3Activated clotting time (ACT) 15 min after the initial bolus heparin administration (15-min ACT) responses to 100 U/kg heparin in the warfarin control group and to three different initial heparin dosages for each non-vitamin K antagonist oral anticoagulant (NOAC). The P values indicate significant differences between two groups, the percentages inside the bars indicate the incidence rates of patients with 15-min ACTs >300 s and the vertical bars indicate one standard deviation The initial bolus heparin dosages that produced a 15-min ACT identical to that of the control group (333 ± 32 s) were 120 U/kg (318 ± 29 s) and 130 U/kg (339 ± 43 s) for dabigatran, 130 U/kg (314 ± 31 s) for rivaroxaban and 130 U/kg (317 ± 39 s) for apixaban (Fig. 3). These initial heparin dosages of NOACs showed that >70% of patients had a 15-min ACT > 300 s (Fig. 3; percentages are shown in the columns). These percentages were not significantly different from that of 100 U/kg initial heparin dosage in the warfarin control group. Although actual values of 15-min ACT was statistically not different between the initial heparin dose of 120 and 130 U/kg in the apixaban group, 120 U/kg showed 53% of patients with 15-min ACT > 300 s, whereas 130 U/kg, > 70% of patients. In the warfarin control group, the initial 100 U/kg heparin administration achieved 15-min ACTs >300 s in 83 % of patients. Regarding the total heparin required, no significant differences were observed among the three different initial heparin dosages for each NOAC. When the control and three NOAC groups were compared, the total heparin required was first 128 ± 22 U/kg in the warfarin (control) group, second 153 ± 29 and 156 ± 26 U/kg in the dabigatran and rivaroxaban groups, respectively, and third 168 ± 34 U/kg in the apixaban group, in statistically significant increasing order (Fig. 4).Fig. 4Total heparin required during the atrial fibrillation ablation procedure among the control (warfarin) and three non-vitamin K antagonist oral anticoagulants groups. The vertical bars indicate one standard deviation No significant differences in protamine usage for haemostasis were found among the control and three NOAC groups (control, 13 ± 20; dabigatran, 26 ± 23; rivaroxaban, 25 ± 25; apixaban, 28 ± 28 mg). BODY.RESULTS.COMPLICATIONS: From 30 days before ablation, no patients in the dabigatran, rivaroxaban and apixaban groups had any thromboembolic or bleeding complications. During the procedural and periprocedural periods, no major bleeding complications were observed among the four groups (Table 6). The incidence of minor bleeding complications was low, and no significant differences were found among the four groups. No significant differences in minor bleeding were found among patients who were administered 110, 120 and 130 U/kg of heparin for each NOAC.Table 6Comparison of complications and safety outcomesWarfarinDabigatranRivaroxabanApixaban P valueInitial dosage of heparin (U/kg):100Total110120130Total110120130Total110120130Number of patients90903030309030303090303030Complications Thromboembolic complications Stroke/TIA00000000000001 DVT00000000000001 Pulmonary embolism00000000000001 Bleeding complications Major bleeding complications (N) Periprocedural cardiac tamponade00000000000001 Late cardiac tamponade00000000000001 Retroperitoneal bleeding00000000000001 Decreased haemoglobin level >4 g/dL00000000000001 Blood transfusion required00000000000001 Minor bleeding complications (N) Pericardial effusion1 (1 %)3 (3 %)0212 (2 %)1102 (2 %)1010.92 Groin hematoma1 (1 %)2 (2 %)0112 (2 %)1101 (1 %)0100.99 Haematuria2 (2 %)2 (2 %)1103 (3 %)1112 (2 %)1100.99 Other Prolonged hospitalization00000000000001Safety outcome (composite of bleeding and thromboembolic complications)4 (4 %)7 (8 %)1427 (8 %)3315 (6 %)2210.92 DVT deep venous thrombosis, TIA transient ischaemic attack After discharge, late thromboembolic and bleeding complications were not observed in any of the patients during a follow-up period of at least 90 days after AF ablation. Late pericardial effusion was not observed in any patients. BODY.RESULTS.SAFETY OUTCOMES: Overall, the safety outcomes did not differ among the four groups (Table 6). Similarly, no significant differences in the safety outcomes were found among patients who were administered 110, 120 and 130 U/kg of heparin for each NOAC. BODY.DISCUSSION: The present study's results showed that in patients with NOAC therapy who underwent AF ablation, differences in the baseline ACT were observed among NOACs, indicating that the adequate initial heparin dosage was different for each NOAC. The results of the comparison between the 15-min ACTs seen with the NOACs and that seen with the control (warfarin) indicated that initial heparin dosages of 120 or 130 U/kg for dabigatran and 130 U/kg for rivaroxaban and apixaban were adequate. For patients who underwent AF ablation in the afternoon, administration of 5000 U of heparin subcutaneously in the morning was appropriate, because the time course of the ACTs was not identical between the morning and afternoon sessions. Our methods for catheter ablation for AF were essentially the same as those recently described in previous studies with improved methods [11, 12]. The fluoroscopic and procedure times in AF ablation were comparable or superior to those in recent reports [13, 14]. Major and minor bleeding complication rates in the three different NOAC groups and in the morning and afternoon ablation subgroups were similar or slightly lower than those in other reported studies [15, 16]. However, the reasons for these lower complication rates were unclear. Recent developments in the ablation systems and equipment may have partially accounted for the shorter procedure times, resulting in slightly fewer bleeding complications. Furthermore, the initial AF ablation success rates were identical to those in recent studies [12, 14]. The present procedural parameters and clinical outcomes indicate that these methods for AF ablation were satisfactory. Furthermore, the clinical and echocardiographic parameters and AF conditions did not differ among the three groups. These considerations validate the comparisons made among the groups. For patients who underwent AF ablation in the afternoon, 5000 U of heparin was administered subcutaneously in the morning. The efficacy of administering 5000 U of heparin subcutaneously to prevent thromboembolism for up to 12 h has been reported [17, 18]. The present study found no differences; rather, the ACTs before ablation between morning and afternoon ablation in each NOAC group were similar. No differences in thromboembolic and bleeding complications were found between patients who underwent AF ablation in the morning and afternoon (data not shown). Therefore, the subcutaneous administration of 5000 U of heparin in the morning for AF ablation performed in the afternoon was considered adequate. For warfarin anticoagulation therapy, many studies have used 100 U/kg of heparin for the initial heparin bolus administration, and the efficacy and safety of this dosage have been well demonstrated [4, 19, 20]. Thus, we compared the ACT seen with each NOAC therapy and that seen with warfarin to determine the adequate initial dose of heparin for each NOAC. To the best of our knowledge, no reports have examined the effects of different dosages of the initial heparin administration on the ACT in comparison with warfarin. The present results regarding adequate initial heparin dosing thus cannot be compared with those of previous reports. Several studies have recommended an ACT during the AF ablation procedure. Although an ACT >300 s throughout the ablation procedure has been recommended [21, 22], no report has evaluated the optimal 15-min ACT. One study reported that 80 % of 777 centres worldwide used ACT-guided administration of heparin with a range between 250 and 300 s [5]. The present study used 100 U/kg of heparin in the warfarin (control) group, which resulted in a 15-min ACT >250 s; this indicated that administering 100 U/kg of heparin in a warfarin group as a reliable control was valid. The present results indicated that a higher dosage of the initial bolus heparin dose was required for NOACs in comparison with warfarin (the control) [23]. The reasons for this difference are obscure. However, a few studies have examined the heparin requirements and ACTs seen with NOACs and warfarin [24–26], and they reported that NOACs require a higher dose of heparin and more time to reach the target ACT than uninterrupted warfarin. The reported studies used variable dosages for the initial heparin administration, and they did not directly compare the results. These studies also did not recommend a fixed dosage for the initial heparin administration. In our study, all NOACs required higher doses of initial heparin, and a lower percentage of patients receiving NOACs reached a 15-min ACT of >300 s than those treated with uninterrupted warfarin; these findings are consistent with the results of the other recent studies. The safety and efficacy of NOACs as anticoagulants have been demonstrated by large randomized studies [19, 20, 23, 27]. Our results for the pre-ablation period are consistent with those studies' findings. During the pre-ablation period, no patients who received any of the three different NOACs exhibited any thromboembolic complications or bleeding episodes. As the pre-ablation period was not long and the number of patients was relatively small, these results cannot be considered conclusive. Thromboembolic complications were also not observed in any patients who received 100, 120 and 130 U/kg of an initial bolus of heparin for each NOAC. The accumulation of experience and improved methods, including ablation devices, has reduced the incidence of thromboembolic complications [28]. The AF ablations were performed at a single, high-volume centre (performing 600 cases/year) by well-experienced operators (performing at least 250 cases/year) and this would, at least partly, account for no major complications being observed in the present study. The small number of patients in each group in addition to this would explain why the differences in thromboembolic complication in relation to the initial heparin dosage were masked. Nevertheless, the significance of controlling the ACT during AF ablation procedures has been well demonstrated [4, 21, 22]. Similarly, the overall bleeding complications were fewer or comparable to those reported in other studies [16, 29, 30]. Again, no significant differences in these complications were observed among patients who received 110, 120 and 130 U/kg of an initial bolus of heparin for each NOAC. The results indicated that the dosage of the initial heparin administration was safe at least up to 130 U/kg for each NOAC. The baseline ACT and 15-min ACT in the dabigatran group were longer than those in the rivaroxaban or apixaban groups, and the increase corresponded to each dosage. The reasons for the differences were obscure. Dabigatran is a direct thrombin inhibitor, and both rivaroxaban and apixaban are factor Xa inhibitors. Variable sensitivity of the ACT in response to dabigatran has been reported. In contrast, dose-dependent increases in the ACT in response to rivaroxaban and apixaban have been reported. The action site of the drug and the sensitivity of the ACT to the drug may have accounted for the differences. The present study had some limitations. First, our study included a relatively small number of patients. Second, it was a single institutional experience with a relatively homogeneous patient group. Although the clinical backgrounds did not differ among the groups, and careful statistical analysis suggested that an increase in the number of patients was not likely to produce different results, multicentre studies with more patients are required to confirm the present study's results. BODY.CONCLUSION: The present study showed that the baseline ACTs differed in patients receiving the three NOACs. The results of the comparison with warfarin (the control) indicated that initial bolus heparin dosages of 120 or 130 U/kg for dabigatran, and 130 U/kg for rivaroxaban and apixaban, were thought to be adequate.

TITLE: Endometrial safety of ospemifene: results of the phase 2/3 clinical development program ABSTRACT.ABSTRACT.OBJECTIVE: This study aims to assess the endometrial safety of ospemifene based on phase 2/3 clinical trials of postmenopausal women with up to 52 weeks of exposure to ospemifene 60 mg/day versus placebo. ABSTRACT.ABSTRACT.METHODS: Endometrial safety was evaluated in a development program of six randomized, double-blind, placebo-controlled, parallel-group studies of postmenopausal women aged between 40 and 80 years who had vulvar and vaginal atrophy. Participants were randomized 1:1 to ospemifene 60 mg/day or placebo in one 6-week trial and three 12-week trials; one of the 12-week trials had a 40-week extension study. In a separate 52-week trial, women were randomized 6:1 to ospemifene 60 mg/day or placebo. Endometrial safety was assessed by endometrial histology (biopsy), transvaginal ultrasound, and gynecologic examination. ABSTRACT.ABSTRACT.RESULTS: In these trials, 1,242 women who received ospemifene 60 mg/day and 924 women who received placebo were evaluable for safety. Endometrial hyperplasia occurred in less than 1% of women treated with ospemifene; no endometrial cancer was reported. The mean (SD) increase in endometrial thickness among women treated with ospemifene was 0.51 (1.54) mm at 12 weeks, 0.56 (1.61) mm at 6 months, and 0.81 (1.54) mm at 12 months. Women who received placebo had a mean (SD) increase of 0.07 (1.23) mm at 12 months. ABSTRACT.ABSTRACT.CONCLUSIONS: These clinical trial data indicate that up to 52 weeks of treatment with oral ospemifene 60 mg/day was safe for the endometrium. There was no increase in the incidence of endometrial cancer or hyperplasia among postmenopausal women treated with ospemifene compared with placebo. BODY: Ospemifene, an estrogen receptor agonist/antagonist with tissue-selective effects that is sometimes referred to as a selective estrogen receptor modulator (SERM), was recently approved for the treatment of moderate to severe dyspareunia, a symptom of vulvar and vaginal atrophy (VVA) due to menopause.1 Although SERMs lack the steroidal structure of estrogens, certain SERMs have been reported to elicit tissue-specific responses, such as positive effects on the vaginal epithelium, prevention of breast cancer, and treatment and prevention of osteoporosis with an acceptable benefit/risk profile.2 However, SERMs have been reported to have a spectrum of effects on the endometrium. Tamoxifen, a first-generation SERM, increases the risk of endometrial cancer; this finding is not associated with raloxifene, a second-generation SERM.3,4 Clinical development of other SERMs, such as levormeloxifene and idoxifene, was discontinued because of unacceptable safety profiles, where adverse effects on gynecologic tissues and elsewhere outweighed the benefits. Thus, gynecologic evaluation of a SERM is an essential component for establishing its overall benefit/risk profile in postmenopausal women.5 Ospemifene has been shown in preclinical and clinical studies to exert positive effects on the vaginal epithelium and minimal effects on the endometrium.6-8 The only other approved prescription medications for the treatment of VVA include systemic estrogens, estrogen plus progestogen, and local vaginal estrogens.1 The endometrial effects of unopposed systemic estrogens have long been known.9 The Postmenopausal Estrogen/Progestin Interventions trial found that, after 12 months, 25 of 119 women (21.0%) receiving conjugated equine estrogens (CEE) alone had endometrial hyperplasia, 12 women (10.1%) had complex hyperplasia, and 3 women (2.5%) had hyperplasia with atypia. Three women treated with CEE/medroxyprogesterone acetate (MPA) were reported to have simple hyperplasia (n = 2) or complex hyperplasia (n = 1) at 12 months; there were no reports of hyperplasia with atypia.10 Progestin has been added to oral estrogens to protect against endometrial proliferation. Endometrial outcomes in the Women's Health, Osteoporosis, Progestin, Estrogen trial were reported for women receiving CEE alone (0.3-0.625mg/d) or a CEE/MPA combination (0.3/1.5 to 0.625/2.5 mg/d). Thirty-two of 2,153 women (1.5%) predominantly in the CEE-alone 0.45- and 0.625-mg treatment groups, developed endometrial hyperplasia by the 12-month evaluation. The incidence of hyperplasia was low (≤0.4%) in all CEE/MPA groups. One case of endometrial hyperplasia was identified in each of the CEE 0.3 mg/MPA 1.5 mg and CEE 0.45 mg/MPA 1.5 mg groups. Groups treated with CEE/MPA had a significantly lower (P ≤ 0.05) incidence of endometrial hyperplasia than the groups treated with corresponding doses of CEE alone, with the exception of the lowest dose (CEE 0.3 mg/MPA 1.5 mg and CEE 0.3 mg), where there was one case of hyperplasia in each group.11 Vaginally administered estrogens have been shown to be effective and well-tolerated for the treatment of VVA. Symptom relief is achieved with low doses of estrogen; however, systemic effects have been reported.12 In a recent study of 10-μg estradiol vaginal tablets,13 one case of endometrial hyperplasia (without atypia) and one case of endometrioid carcinoma were reported among women who were treated for up to 52 weeks, resulting in an incidence rate of 0.52% among 386 women who were reported to have undergone an endometrial biopsy. The mean endometrial thickness was not reported to have increased with treatment in this study. The endometrial safety of conjugated estrogens vaginal cream 0.3 mg applied once daily or twice weekly was evaluated in 155 participants with endometrial biopsies and revealed no endometrial hyperplasia or carcinoma. Transvaginal ultrasound (TVUS) results obtained at week 52 or at early termination showed endometrial thickness of at least 5 mm in approximately 10% of participants.14 The Food and Drug Administration (FDA) draft guidance for vasomotor symptoms and VVA clinical trials recommends evaluating the incidence rate of endometrial hyperplasia at 12 months: "We recommend that the results from the clinical trial demonstrate a hyperplasia rate that is ≤1% with an upper bound of the one-sided 95 percent confidence interval for that rate that does not exceed 4 percent. The frequency of atypical hyperplasia and cancer are important additional factors to be considered in determining approvability of the drug product." The FDA guidance also advises that "the incidence of hyperplastic polyps and associated atypia would be considered in the safety review."15 This report will present and discuss the gynecologic effects of a recently approved nonestrogen oral product, ospemifene 60 mg/day. BODY.METHODS: The phase 2/3 randomized, double-blind, placebo-controlled, parallel-group studies (one 6-wk study, two 12-wk studies, one 12-wk study with a 40-wk extension study, and one 52-wk safety study) compared ospemifene 60 mg/day and placebo in the treatment of postmenopausal women.16-20 Participants were aged 40 to 80 years. Baseline criteria for VVA included 5% or less superficial cells on vaginal smear (maturation index), vaginal pH higher than 5.0, and at least one moderate or severe symptom of VVA. One 12-week study (N = 79), the 40-week long-term extension study (N = 118), and the 52-week long-term safety study (N = 426) required participants to have an intact uterus. The 40-week extension study required women to remain on the same therapy they were randomized to receive during the 12-week study. Only women who completed the 12-week study could qualify for the 40-week extension study. The three 12-week studies and the 6-week study randomized participants 1:1 to receive ospemifene or matching placebo, whereas in the 52-week long-term safety study, women were randomized 6:1 to receive ospemifene or matching placebo. Ospemifene (or matching placebo) was taken orally each morning with food. Women were excluded if they had abnormal endometrial histology other than atrophy based on baseline biopsy, uterine bleeding of unknown origin, clinically significant abnormal gynecologic findings, endometrial thickness of 4 mm or more on centrally read TVUS, pathologic findings on endometrial biopsy or Papanicolaou test, or clinically significant findings on physical examination. Participants were not permitted to take other hormonal products, including progestins, during the course of the investigation. Endometrial thickness was measured on TVUS at baseline (screening/visit 1); on weeks 12, 26, and 52; or at the end of therapy. For consistency of data, TVUS images and videos were read at a central laboratory. Endometrial biopsy data were only reported from studies of 12 weeks' duration or longer. Endometrial biopsies in the 52-week trial were obtained at baseline and on week 52, and those in the 12-week studies were obtained at baseline and on week 12. Endometrial evaluation of biopsy-obtained tissues was performed on weeks 12 and 26 if TVUS assessment demonstrated an endometrial thickness of 4 mm or more. Endometrial biopsy samples were collected using a suction curette and analyzed by two independent pathologists in a central laboratory. Pathologists were blinded to study treatment and to each other's readings of the histology slides. If there was disagreement over the endometrial histology, a third pathologist evaluated the samples. The final diagnosis was determined by concurrence between two of the three independent pathologists; if there was no agreement among the three pathologists, the most severe histopathologic diagnosis was reported. Histology was summarized as number (percentage) in the following categories: no tissue, tissue insufficient for diagnosis, atrophic, inactive, proliferative (weakly proliferative, actively proliferative, disordered proliferative), secretory pattern (cyclic type, progestational type including stromal decidualization), menstrual type, simple hyperplasia without atypia, simple hyperplasia with atypia, complex hyperplasia without atypia, complex hyperplasia with atypia, and carcinoma. Blaustein's criteria were used to classify endometrial hyperplasia.21 Endometrial histopathologic characterization of endometrial polyps was performed after identification with TVUS based on regulatory guidance. Investigative sites were asked to locally confirm the polyps identified on TVUS because views of the suspected polyps identified on TVUS that were sent for central reading were limited compared with real-time dynamic views available locally. If the local site confirmed the central reader's finding of a polyp, the participant was discontinued from the study, and hysteroscopy was performed to obtain tissue for histologic diagnosis. The tissue—when available and believed to represent a uterine polyp—was then sent for external expert review. All studies were carried out in accordance with the Declaration of Helsinki (2000) and current Good Clinical Practice outlined in the International Conference on Harmonisation for Good Clinical Practice (E6) and in compliance with local regulatory requirements. Before study initiation, all participants provided written informed consent forms using forms approved by the independent ethics committee. The protocols, amendments, and informed consent forms were reviewed and approved by the independent ethics committee before study initiation. BODY.METHODS.STATISTICAL METHODS: We reported safety data on the intent-to-treat population, which included all participants who had taken at least one dose of the study drug. Treatment-emergent adverse events (TEAEs) were tabulated by system organ class and preferred term according to the Medical Dictionary for Regulatory Activities (MedDRA, version 10), causality, and severity of event (mild, moderate, or severe). Baseline and time point assessments for clinical chemistry and laboratory safety variables were summarized by descriptive statistics. All statistical tests were two-sided tests; P < 0.05 was considered statistically significant. One-sided 95% CI (upper limit) was calculated to assess serious endometrial outcomes (endometrial hyperplasia, cancer, or both). McNemar's test for correlated proportions was used to examine changes in endometrial thickness at two different time points. Changes in endometrial thickness were converted from continuous data into a binary categorical variable using the following definitions: "increase" was defined as a change of more than 1 mm, whereas "unchanged" or "decrease" was defined as a change of less than 1 mm. Differences in categorical variables between ospemifene and placebo were tested using the Fisher exact test. Incidence rates and 95% CIs were based on the Poisson distribution, with the comparison of incidence rates based on the maximal likelihood ratio test. Hazard ratios were also computed. For certain parameters, data from both the 12-week study and its 40-week extension study were used. For example, for women in both the 12-week study and its corresponding 40-week study, duration of therapy was the combined sum of therapy (range, 12-52 wk) and completion of therapy was determined by the 40-week extension, as all women in the 40-week extension were required to complete the 12-week study. All analyses were performed using SAS version 9.2 (SAS Institute, Cary, NC). BODY.METHODS.VAGINAL BLEEDING: Participants with any of the following terms were included in the statistical analysis for vaginal bleeding: coital bleeding, postmenopausal hemorrhage, genital hemorrhage, vaginal hemorrhage, uterine hemorrhage, metrorrhagia, dysfunctional uterine bleeding, bleeding anovulatory, menorrhagia, and polymenorrhagia. Cases having a higher-level group term of "menstrual cycle and uterine bleeding disorders" were also included. For all adverse events (AEs), if a woman had more than one TEAE that coded to the same preferred term, the woman was counted only once for that preferred term. Preferred terms are sorted in descending frequency in the ospemifene group. Post–endometrial biopsy bleeding/spotting cases (with the preferred term "postprocedural hemorrhage") were not included. BODY.RESULTS: A total of 2,166 women were randomized to ospemifene 60 mg/day or placebo in the phase 2/3 double-blind, placebo-controlled, clinical trials: 1,242 women were randomized to the ospemifene 60 mg/day treatment group, and 924 women were randomized to the placebo group. Of the 1,394 randomized women with an intact uterus, 851 received ospemifene and 543 received placebo. One 6-week study, two 12-week studies, one 12-week study with a 40-week extension study, and one 52-week study with a specific focus on endometrial safety were included in this analysis (Fig.). All participants were required to remain on the randomized treatment throughout each of the studies, including the 40-week extension study; no participants were rerandomized. FIG.Disposition diagram of phase 2/3 double-blind, placebo-controlled studies included in this analysis: all participants. The participant's demographics—age, race, and body mass index (BMI)—at baseline were comparable among women with or without an intact uterus in the ospemifene and placebo treatment groups (Table 1). The mean age at entry for ospemifene versus placebo was 59.4 versus 58.9 years; 93.3% versus 90.6% of participants were white; and the mean (SD) BMI was 25.7 (4.0) versus 26.0 (4.2) kg/m2 among the women enrolled (Table 1). In three of the studies, participants were required to have an intact uterus; in one of these studies, there were approximately six times more actively treated participants than placebo participants because of the prespecified randomized allocation ratio, with a mean (SD) treatment duration of 199 (144) days for ospemifene 60 mg/day versus 124 (110) days for placebo (Table 2). Thus, in the overall analysis population, more women treated with ospemifene had an intact uterus (851 of 1,242 [68.5%]) than women treated with placebo (543 of 924 [58.8%]). There were baseline differences in weight and BMI in participants with an intact uterus versus participants without an intact uterus, regardless of treatment group randomization (two-way analysis of variance, P < 0.0001; Table 1). TABLE 1 Demographic characteristics in phase 2/3 double-blind, placebo-controlled studies: total population and participants with an intact uterus TABLE 2 Disposition in phase 2/3 double-blind, placebo-controlled studies: participants with an intact uterus The percentage of participants who were considered study completers was similar for the ospemifene 60 mg/day (1,061 of 1,242 [85.4%]) and placebo (802 of 924 [86.8%]) groups. The most common reason for discontinuation in the ospemifene group was AE; 95 (7.6%) of 1,242 participants in the ospemifene 60 mg/day group discontinued because of AEs. In the ospemifene 60 mg/day group, the AEs that most frequently led to study discontinuation were hot flushes (13 of 1,242 [1.0%]), muscle spasms (7 of 1,242 [0.6%]), headache (6 of 1,242 [0.5%]), and vaginal discharge (6 of 1,242 [0.5%]). In the placebo group, hot flushes (3 of 924 [0.3%]) and diarrhea (3 of 924 [0.3%]) were the most common AEs leading to discontinuation. The most common reason for discontinuation in the placebo group was "other" (60 of 924 [6.5%]). "Other" included reasons such as withdrawal of consent, lack of efficacy, and noncompliance. In the ospemifene group, 718 of 851 participants (84.4%) with an intact uterus were study completers; in the placebo group, 466 of 543 participants (85.8%) with an intact uterus were study completers (Table 2). BODY.RESULTS.VAGINAL BLEEDING: Vaginal bleeding or spotting was reported in 10 of 851 women (1.2%) with an intact uterus in the ospemifene 60 mg/day group (incidence rate, 2.17 per 100 patient-years) and in 5 of 543 women (0.9%) in the placebo group (incidence rate, 2.72 per 100 patient-years; P = 0.7; Table 3). One of 391 women who had had a hysterectomy in the ospemifene 60 mg/day group experienced a TEAE related to vaginal spotting; this event occurred on day 56 of treatment and was not associated with vaginal lesions on visual inspection on day 79. None of the vaginal bleeding or spotting TEAEs led to discontinuation. TABLE 3 Incidence rates of vaginal bleeding for ospemifene 60 mg/day versus placebo BODY.RESULTS.ENDOMETRIAL THICKNESS: The mean (SD) increase in endometrial thickness based on TVUS was 0.51 (1.5) versus 0.06 (1.2) mm at 12 weeks, 0.56 (1.6) versus 0.05 (1.3) mm at 6 months, and 0.81 (1.5) versus 0.07 (1.2) mm at 12 months for participants in the ospemifene and placebo groups, respectively. The differences between participants receiving ospemifene and participants receiving placebo were statistically significant (P ≤ 0.001, Welch's test) at all three evaluations. We assessed for a possible association between endometrial thickness and vaginal bleeding, which demonstrated no statistically significant correlation (ospemifene 60 mg/day: Spearman ρ = 0.0625, P = 0.2527; placebo: Spearman ρ = 0.3152, P = 0.0847; Table 4). One woman with vaginal bleeding and endometrial thickness greater than 10 mm had a diagnosis of endometrial polyp with simple hyperplasia without atypia. TABLE 4 Endometrial thickness (by TVUS) at the final visit and vaginal bleeding among participants with an intact uterus An endometrial thickening of 5 mm or more during a participant's last visit demonstrated no statistically significant increase in incidence rate (hazard ratio, 1.58; P = 0.2; Table 5). The mean (95% CI) incidence rate per 100 patient-years for an endometrial thickening of 5 mm or more, based on the total duration of randomized therapy, was 11.3 (8.4-14.8) for ospemifene 60/day mg versus 7.1 (3.7-12.5) for placebo. TABLE 5 Endometrial thickness in participants with an intact uterus The consistency of small changes in endometrial thickness (≤1 mm) was evaluated. Changes in each participant's endometrial thickness from baseline to week 12 and from week 12 to month 12 were examined among participants receiving ospemifene and placebo to determine whether there was any significant difference in the two correlated proportions. Endometrial thickness was evaluated with TVUS in two studies in which women had endometrial thickness evaluation at baseline, at week 12, and at month 12. Using McNemar's test for correlated proportions, we compared endometrial ultrasounds with increases or decreases at week 12 with endometrial ultrasounds with increases/decreases at month 12. Table 5 summarizes the findings for participants in the ospemifene and placebo groups. In both the ospemifene and placebo groups, there was no statistically significant difference in the proportion of participants whose endometrial thickness increased between the two periods (from baseline to week 12 and from week 12 to month 12); there was also no significant difference between the proportion of participants whose endometrial thickness changed direction (increased in the first period, then decreased in the second period) and the proportion of participants whose endometrial thickness changed in the reverse direction (first decreased, then increased). There was no statistically significant difference in the correlated proportions for the ospemifene group (94 of 176 [53.4%] vs 82 of 176 [46.6%]) or for the placebo group (19 of 32 [59.4%] vs 13 of 32 [40.6%]). Thus, the chance of having a small increase or a decrease in endometrial thickness on week 12 or week 52 was randomly distributed. BODY.RESULTS.UTERINE POLYPS: Among women with an intact uterus, 5 of 851 participants (0.6%) in the ospemifene 60 mg/day group and 1 of 543 participants (0.2%) in the placebo group experienced a uterine polyp–related TEAE. Post hoc assessment revealed that the histologic diagnosis of polyp in the absence of a visible polyp on TVUS could be associated with the shape of the suction curette, which created the impression that the biopsy sample was a polyp even in the absence of pathology. Among the six women with a report of polyp, one woman (in the ospemifene 60 mg/d group) experienced vaginal bleeding and had an endometrial thickness greater than 10 mm on TVUS. BODY.RESULTS.ENDOMETRIAL HISTOLOGY: There were no cases of endometrial cancer observed with exposure of up to 52 weeks in the ospemifene clinical trials. One woman had an endometrial biopsy consistent with simple hyperplasia without atypia and a polyp that resolved (Table 6). This woman presented with vaginal bleeding, which led to the diagnostic biopsy 3 months after therapy with ospemifene. This one case of simple hyperplasia (of 342 biopsies) at 12 months in a participant taking ospemifene 60 mg/day met the FDA criterion of 1% or less incidence. TABLE 6 Summary of endometrial biopsy findings: ospemifene 60 mg/day versus placebo At baseline, most endometrial samples had a histologic interpretation of no tissue, tissue insufficient for diagnosis, or atrophic or inactive endometrium (ospemifene, 98.1%; placebo, 95.9%). In the ospemifene 60 mg/day treatment group, 1.2% of women had weakly proliferative endometrial histology and 0.3% of women had actively proliferative endometrial histology. In the placebo group, 3.2% of endometrial tissue was reported as weakly proliferative, 0.2% of endometrial tissue was reported to be of proliferative pattern (disordered type), and 0.2% of endometrial tissue was reported to be of secretory pattern (progestational type; Table 6). Of the nine ospemifene participants with reports of weakly proliferative endometrium at baseline, two women had weakly proliferative endometrium present at 12 weeks, three participants did not have a follow-up biopsy, and the other four participants had follow-up findings of an atrophic or inactive endometrium. None of these participants had vaginal bleeding or spotting. Endometrial biopsies were performed on follow-up in women who participated in the clinical trials for 12 months and received ospemifene (n = 342) or placebo (n = 83). The histologic pattern was similar to that at baseline, with 96.5% and 100.0% of participants receiving ospemifene and placebo, respectively, having reports of tissue insufficient for diagnosis or atrophic or inactive endometrium. Of these participants receiving ospemifene, 2.0% had weakly proliferative endometrial histology, 0.3% had actively proliferative endometrial histology, and 0.3% had proliferative pattern (disorder type) tissue; 0.9% had other (various types of polyps). About 3.5% of ospemifene participants had histologic findings other than inactive, atrophic, or insufficient tissue at 12 months, similar to the baseline endometrial biopsy results of placebo participants (4.1%). There were no endometrial carcinomas, complex hyperplasias, or simple hyperplasias with atypia in either the ospemifene group or the placebo group with up to 1 year of study completion. BODY.RESULTS.PELVIC ORGAN PROLAPSE: There were rare cases of pelvic organ prolapse in both the ospemifene and placebo groups. Two women had bladder prolapse (one ospemifene participant and one placebo participant), and one ospemifene participant had a report of cystocele. Each of the two ospemifene participants had delivered two infants vaginally; the placebo participant had given birth vaginally once. Both women who received ospemifene were aged 63 years; the woman who received placebo was aged 53 years. BODY.DISCUSSION: Ospemifene is a tissue-selective estrogen receptor agonist/antagonist that exerts a beneficial effect on vaginal epithelial tissue. Ospemifene also displayed an acceptable endometrial safety profile in studies of VVA treatment in postmenopausal women evaluated up to 52 weeks. The phase 2/3 studies of ospemifene were powered to provide substantial safety data, allowing a prospectively defined assessment of potential treatment effects on endometrial hyperplasia. In these studies, only one woman who received ospemifene treatment was diagnosed with endometrial hyperplasia (simple hyperplasia without atypia). She also experienced vaginal bleeding and was found at study exit to have a thickened endometrium, which on follow-up biopsy 3 months later was confirmed to be endometrial simple hyperplasia. Based on histologic findings, a similar incidence of proliferative endometrial changes was present at baseline and 12 months in the ospemifene and placebo groups. Although the safety studies of ospemifene were up to 12 months in duration, many women may receive considerably longer treatment in practice. Further studies are needed to evaluate the longer-term risks of endometrial hyperplasia and malignancy in such women. FDA guidance for VVA trials recommends that clinical trials demonstrate an endometrial hyperplasia rate of 1% or less, with an upper bound less than 4% of the one-sided 95% CI for that rate. With a single case of simple hyperplasia without atypia (0.3%) reported at 12 months, our results are well within the FDA criterion for endometrial safety. The prevalence of endometrial polyps is dependent on age, menopause status, and hormone therapy use.22 A similar proportion of women with suspected endometrial polyps, based on histology, was found in the ospemifene and placebo groups in the phase 2/3 studies. All polyps were found in the single 52-week study (ospemifene, 5 of 364 [1.4%]; placebo, 1 of 62 [1.6%]). Thus, no increase in the incidence of endometrial polyps was observed in the 1-year clinical trial. Slight mean increases in endometrial thickness, as assessed on TVUS, were reported in both treatment groups. However, the mean increase was less than 1 mm, with large SDs warranting caution in drawing conclusions. The imprecision of endometrial ultrasounds in assessing very small changes seems consistent with the findings that a woman could have a report of an increase in endometrial thickness at 12 weeks with a subsequent decrease reported at study completion without a change in her therapy. Although, numerically, more participants treated with ospemifene reported endometrial thickness measurements of 5 mm or more, women treated with ospemifene had longer treatment durations than women receiving placebo. There were no statistically significant differences in endometrial thickness between groups (P = 0.2) when the duration of study participation was considered. The effects on the endometrium were consistent across the phase 2/3 studies. The observed small increases in endometrial thickness after ospemifene treatment were without concomitant cellular proliferation. Such changes in endometrial thickness have also been reported with raloxifene and tamoxifen.23 SERMs may cause glandular cystic atrophy, which can appear to cause endometrial thickening on ultrasound but without evidence of cellular proliferation.23,24 Data from the use of Premarin vaginal cream found an increase in endometrial thickness of 5 mm or more in approximately 10% of women,14 and the incidence of similar increases in endometrial thickness with ospemifene treatment was well within this finding. In all studies, a statistically significant effect on physiologic vaginal parameters (increased proportion of superficial cells, decreased proportion of parabasal cells, reduced vaginal pH, and improved visual evaluation) was found with ospemifene 60 mg/day compared with placebo.16,17,20 These positive findings are sustained through 52 weeks of treatment. Subjective improvements in VVA symptoms were reported, with consistent improvement in moderate to severe dyspareunia. SERMs such as tamoxifen and raloxifene have not demonstrated similar favorable estrogen agonist effects on vaginal tissue. A limitation of the current analysis is the combination of disparate studies. Although all studies were undertaken in a generally similar fashion, they differed in some aspects of design, such as requirement for women to have an intact uterus at enrollment, randomization ratio, and study duration. It can be difficult to compare outcomes between short-term and long-term studies; however, we addressed this limitation by using patient-year analyses. BODY.CONCLUSIONS: Although ospemifene exerts beneficial effects on the vagina, endometrial safety is maintained, suggesting that the effects of ospemifene on estrogen receptors vary among the different components of the genital tract. This is in contrast to oral estrogen alone, which has a full agonist effect on both vaginal and endometrial tissues.10 No exogenous progestin use was permitted during the ospemifene clinical trials; thus, the favorable endometrial profile of ospemifene is further distinguished from that of oral steroidal estrogens. The endometrial data in these ospemifene studies, including histology and ultrasound results, seem to be approximately consistent with data for the SERM raloxifene.25 In conclusion, ospemifene exerts positive effects on vaginal tissue in clinical trials,16,17,19,20 yet displays an acceptable endometrial safety profile with up to 1 year of daily oral treatment.

TITLE: A randomized controlled trial of multi-session online interpretation bias modification training: Short- and long-term effects on anxiety and depression in unselected adolescents ABSTRACT.INTRODUCTION: Negatively biased interpretations play an important role in anxiety and depression, which are highly prevalent in adolescence, and changing such biases might thus reduce or prevent emotional disorders. We investigated the short- and long-term effects of an online interpretation bias modification training in unselected adolescents to explore its potential in preventing anxiety and depression. ABSTRACT.METHODS: Participants (N = 173) were randomly allocated to eight online sessions of interpretation or placebo training. Interpretation bias was assessed pre- and post-training. Primary outcomes of anxiety and depression, and secondary measures of emotional resilience were assessed pre- and post-training and at three, six, and twelve months follow-up. ABSTRACT.RESULTS: Compared to placebo, interpretation training marginally increased positive interpretations. Irrespective of training condition, symptoms of anxiety and depression showed a decline post-training and at follow-up, and indices of resilience showed an increase. Change in interpretation bias, baseline interpretation bias, stressful life events, or number of training sessions completed did not moderate the effects on anxiety or depression. ABSTRACT.CONCLUSIONS: Results suggest that interpretation training as implemented in this study has no added value in reducing symptoms or enhancing resilience in unselected adolescents. BODY.INTRODUCTION: Cognitive models of anxiety and depression assume that biases in information processing play an important role in the aetiology of these disorders (e.g. [1], [2]). Recently developed cognitive training paradigms directly target such cognitive vulnerabilities and could be employed online as a possible low barrier early intervention or prevention program [3]. Adolescents seem a particularly relevant target group for this type of preventive interventions for two reasons: first, this age-group is the most vulnerable for the development of anxiety and depression [4], and second, it is also in a period of heightened brain plasticity [5]. When a positive information processing style could be acquired at this age, this might protect against the development or worsening of emotional problems. The aim of the current study was to investigate the short- and long-term effects of a specific type of cognitive training: Cognitive Bias Modification for Interpretations (CBM-I), on the following outcomes: interpretation bias, symptoms of anxiety and depression (primary outcomes), and secondary measures of emotional resilience. There is ample evidence indicating that individuals with anxiety disorders or depression are characterized by a tendency to interpret ambiguous information in a negative way (for a review, see [2]). It has been shown that experimentally increased negative interpretation bias also strengthens people's emotional responding to experimental stressors [6], supporting the relevance of such negative interpretation bias as a causal agent in the development of emotional disorders. These findings fuelled research into potential therapeutic applications of this CBM-I paradigm. In the most-often used CBM-I training paradigm, participants read ambiguous scenarios, which are consistently disambiguated in a positive way by completing a word fragment. Recent meta-analyses of CBM-I studies in adults [7], [8] showed consistent positive effects on interpretation bias, while findings on mood, stress-reactivity, and anxiety or depressive symptoms were more mixed. Mood effects seemed to be larger when CBM-I was used with imagery instructions and with more training sessions [8]. In the context of depression, positive effects were observed with a scenario-based paradigm with more emphasis on imagery (e.g. [9], [10], [11]). A first meta-analysis on CBM-I in youth [12] revealed a comparable pattern: a significant effect on interpretation bias (with moderate effect size), but no significant overall effects on anxiety and depression. Effects were found to be larger in unselected youth and when training was performed at school. Note that in this meta-analysis, both CBM-I and CBM for attention studies were included. In CBM for attention procedures, participants are trained to focus their attention on positive or neutral information instead of negative or threatening information, in order to reduce a negative attentional bias. Focusing on CBM-I only, a recent re-analysis of six youth studies [13] reported significant mood effects when comparing positive and negative training. Given the large variability in study design (number of sessions, sample, type of training, and assessment tasks) and the small number of included studies and participants, the results from both meta-analyses are difficult to interpret. Important steps forward are performing larger studies with more power to detect effects and explore for whom training works best. Earlier research suggested that training might be especially effective in those adolescents with a more negative interpretation bias [14], [15], but some studies employing multiple sessions of CBM-I training in healthy adolescents also showed changes in stress responses [16], [17]. The current study focused on unselected adolescents, varying from no symptoms to a clinical level of anxiety or depressive symptoms. This provides the possibility to examine which adolescents profit most, and to test the effects on symptomatology as well as on resilience (e.g. stress-reactivity, self-esteem), thus exploring the potential of CBM-I as a universal or targeted prevention program. Based on meta-analytic findings, training was optimized by the use of multiple sessions and more emphasis on imagery. CBM-I training has a clear hypothesized mechanism of change; potential emotional effects should be mediated by change in interpretations. Only a few studies have directly tested the hypothesized mediational path and found that change in interpretations indeed (partly) mediated change in depressive symptoms [11], trait anxiety [18], and social anxiety [19]. Note that it is also quite likely that for the changed interpretation bias to affect emotional functioning, time is needed to apply the new processing style in daily life. That is, to interpret (stressful) life experiences in a more positive way and to change behaviour correspondingly [20]. Therefore, to assess processes of change and to fully appreciate the potential of CBM-I, multiple assessments over a longer period are crucial. Until now, most research has focused on short-term effects (pre-post-design), with only a handful of studies including follow-up assessments after several weeks or months. In adults, marginally significant effects on social anxiety were observed four weeks after CBM-I training [21] and significant effects at seven weeks follow-up [22]. However, Salemink, Kindt, Rienties, and van den Hout [23] found no effects at three months follow-up, but also no short-term effects were observed in that study. The only RCT that investigated CBM-I as an early preventive intervention in adolescents focussed on youngsters with heightened levels of social and/or test anxiety and used a 10-week internet-based multi method approach including both CBM-I and an attentional bias training. Although the multi method CBM intervention showed a positive effect on interpretation bias that was still evident at two-year follow up, this study failed to find convincing evidence for the efficacy of the combined training to reduce symptoms of social and test anxiety [24][25]. In the current study, adolescents were randomized over one of two training groups: a CBM-I training or a placebo-control training, consisting of eight online sessions, completed over four weeks. Interpretation bias was assessed during training, and pre- and post-training (recognition task). Emotional measures were administered both pre- and post-training, and at three, six, and 12 months follow-up. Our first, and primary hypothesis was that CBM-I would reduce symptoms of anxiety and depression compared to a placebo training, both at the short- and long-term. Second, compared to placebo, we expected a stronger reduction in negative interpretation bias in the CBM-I group. Our third hypothesis was that symptom change would be larger for participants who showed a larger change in interpretation bias. Fourthly, we examined other factors that moderated training effectiveness. More specifically, we tested whether stronger training effects would be observed in adolescents with a more negative baseline interpretation bias, or in adolescents who experienced a relatively large amount of real life stress. Also, we investigated whether training effects would be stronger when completing a larger number of training sessions (cf. [26]). Finally, to further explore the preventive potential of CBM-I in increasing emotional resilience, we assessed immediate effects on stress-reactivity, as well as short and long-term effects on secondary emotional measures of self-esteem, perseverative negative thinking, test anxiety, and social-emotional, and behavioral problems. BODY.METHODS.DESIGN AND ETHICS: The current study was approved by the ethics committee of the psychology department of the University of Amsterdam and carried out in accordance with the provisions of the Declaration of Helsinki. It was part of a larger study, which was registered in the Dutch trial register with number NTR3950 (http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=3950), and also included two types of attentional bias training, an emotional working memory training and three corresponding placebo groups. The focus of the current manuscript is on the CBM-I and CBM-I placebo training and results of the other paradigms are reported in separate papers [27], [28]. Manipulations and measures not used in this specific study are described in S1 Appendix. The trial was registered after the start of participant recruitment but before the end of data collection, as trial registration of experimental preventive trials was not yet standard policy at our department. Randomization was performed for the larger study, stratified by school, gender and age group (under/above 15 years), and followed a 4:4:4:4:1:1:1:1 ratio, with four experimental and four placebo conditions respectively. Fewer participants were randomized to the placebo conditions (a total of 20%), to increase the appeal of the project to schools, and because we originally planned to combine them into one control condition (resulting in five conditions). The computerized randomization procedure was written by a programmer not involved in the study, and both participants and test assistants were blind to allocation. An a-priori power analysis was performed for the larger study in G*Power 3.1 [29]. The required sample size to detect a within-between interaction in a repeated measures ANOVA was computed with the following parameters: a small effect size of f = .10 (based on [30], [31], [32]), a power of 90%, an alpha of .05, five groups, five measurements, a correlation of 0.5 between measurements, and a nonsphericity correction of 0.375. The power analysis revealed that 470 participants were needed in order to detect a Condition (five groups: CBM-I, combined control condition, and the three other experimental conditions, see above) x Time interaction effect in predicting anxiety or depression scores (our primary outcome measures). Anticipating drop-out, we aimed for 600 participants. Since training compliance was relatively low in the first ten schools (five out of eight sessions completed on average), four more schools were invited to increase the expected number of completers (resulting in a total of 14 schools). Recruitment stopped after including participants from those four schools. Note that the a-priori power analysis was performed for five groups, as we initially planned to analyze all placebo groups together as one combined control group and compare it with all experimental groups. As we decided to focus in this paper on interpretive bias only and thus perform analyses for the CBM-I and CBM-I Placebo group specifically, with stringent corrections for multiple comparisons, this has reduced our power. A sensitivity analysis with 2 groups, a sample size of 78 (based on our smallest group), 5 measurements, a correlation of 0.5, a nonsphericity correction of 0.25 (unstructured covariances), an alpha of .0045 (after Bonferroni Holm correction), and a power of .90, revealed that we were able to detect a Condition x Time interaction with a medium effect size of f = .30. BODY.METHODS.PARTICIPANTS: In total, 2312 adolescents from 14 regular high schools in the Netherlands were invited for the study between January and September 2013. The last follow-up assessments were completed in November 2014. Inclusion criteria were: Scholars in the 1st to 6th grade (aged 11–19) of a regular high school (all levels except special education), and parental consent. A total of 733 participants and their parents provided written informed consent (see Fig 1 for flow diagram of the larger study) and were randomized. Four participants dropped out and were excluded since they requested removal of their data and 48 participants were excluded because they missed the first assessment, resulting in a total of 681 participants for the larger study. For the CBM-I and CBM-I Placebo (from now on referred to as 'Placebo') group, 173 participants (134 and 39 respectively) remained for intention-to-treat analyses (60.7% female, mean age 14.35, SD = 1.11). Background variables of these groups can be found in Table 1. 10.1371/journal.pone.0194274.g001Fig 1Flow chart of the larger study. 10.1371/journal.pone.0194274.t001 Table 1 Demographic characteristics per training condition. CBM-I (n = 134) Placebo (n = 39) Age, mean ( SD ) 14.31 (1.10) 14.49 (1.16) Female, n (%) 82 (61.19) 23 (58.97) School level, n (%)     - Lower 32 (23.88) 11 (28.21)     - Middle 28 (20.90) 6 (15.38)     - Higher 74 (55.22) 22 (56.41) Sessions, mean ( SD ) 5.54 (2.33) 5.38 (2.35) High life events group, n (%) 40 (29.90) 16 (43.20) BODY.METHODS.INTERPRETATION TRAINING (CBM-I): The CBM-I paradigm from Mathews and Mackintosh [6] was used to manipulate interpretation bias. Participants read 3-line ambiguous scenarios, with a missing word in the last sentence, presented as a word-fragment. Participants had to indicate the first missing letter with the corresponding key, after pressing the spacebar as quickly as they recognized the word. Completing the word-fragment disambiguated the scenario in a positive way in experimental trials. The correct word was displayed after a correct response and the interpretation was reinforced by a "yes" or "no" comprehension question about the scenario, followed by feedback. An example scenario might be: "You are playing a solo as part of a concert. As you are playing you know you are making some mistakes. At the end you think back to the bits that you played well and feel pl—sed (pleased)". "Do you feel happy when you think about the bits you played well? (Yes)". Each training session consisted of three blocks of 14 trials, with 10 training scenarios and two positive and two negative probe scenarios (disambiguated in a positive or negative way respectively). The order of scenarios was randomized beforehand, with the same order applied to all participants. Probe scenarios were used to assess interpretation bias during training and also obscured the goal of training [6]. A relative reduction in reaction times to positive probes compared to negative probes would indicate a decrease in negative interpretation bias. Participants were asked to imagine the scenarios as happening to themselves and as vividly as possible, as the use of mental imagery has been found to increase training effects [25]. After each 4th trial, participants rated to what extent they were able to imagine the outcome of the scenario on a 4-point scale. In the Placebo condition, the 10 training scenarios were in the same context and started with the same sentence, but ended in a neutral way. Comprehension questions focused on factual information. The same probe scenarios as in the experimental condition were used. In total, 576 unique ambiguous scenarios (with 288 positive, 240 neutral and 48 negative resolutions) were created, based on previous studies in the context of anxiety and depression [9], [15], [23], [33], [34]. Scenarios previously used with adults were adapted for adolescents and English scenarios were translated and adapted to Dutch culture where appropriate. For scenarios including gender-specific words (e.g., your boyfriend/girlfriend) male and female versions were created. New scenarios were also developed for situations specific to panic, generalized anxiety, and depressed mood, as most original scenarios focused on social situations. A progress bar indicated how many trials were left in each block. Between blocks, short breaks were provided with feedback, consisting of the number of points earned based on performance (one point for each correct answer, to word fragments and comprehension questions). At the end of each session, points of this and previous session(s) were presented in a graph. We expected this feedback to improve motivation and engagement (cf. [35]). BODY.METHODS.PRIMARY OUTCOME MEASURES: Anxiety symptoms were assessed with the Screen for Child Anxiety Related Emotional Disorders (SCARED, [36]), a 41-item (rated 0–2) self-report questionnaire assessing social phobia, separation anxiety, generalized anxiety, panic/somatic symptoms and school phobia. Depressive symptoms were assessed with the Children's Depression Inventory (CDI, [37]), a 27-item self-report questionnaire with items consisting of three statements indicating varying levels of depressive symptomatology (0–2). BODY.METHODS.SECONDARY COGNITIVE OUTCOME MEASURES: Interpretation bias was assessed with the Recognition Task (REC-T, [6]), where participants read ambiguous scenarios, completed word-fragments and answered comprehension questions as in the CBM-I training. However, here the scenario remained ambiguous also after completing the word fragments. After presentation of eight scenarios, titles of these scenarios were presented again in random order, once with a negative interpretation and once with a positive interpretation (randomized). Participants rated the extent to which the interpretation corresponded to the scenarios on a 4-point scale. An interpretation bias index was computed by subtracting ratings for positive interpretations from ratings for negative interpretations; a higher score thus indicated a negative interpretation bias. Two stimuli sets were created to use pre- and post-training, and they were counterbalanced across participants. The REC-T has been used repeatedly to assess effects of CBM-I in adolescents (e.g., [14], [15], [16], [17], [24]), and has been shown to differentiate between high and low neuroticism in adults, while scores are not affected by mood state [38]. BODY.METHODS.SECONDARY EMOTIONAL OUTCOME MEASURES: Self-esteem was assessed with the Rosenberg Self-Esteem Scale (RSES, [39]), a 10-item (rated 1–4) self-report questionnaire. Test anxiety was assessed with a Dutch self-report questionnaire, the "performance motivation test for children" (Prestatie Motivatie Test voor Kinderen, PMT-K, [40]). Only the 14-item (rated 0–1) subscale assessing negative test anxiety was used. The Perseverative Thinking Questionnaire (PTQ, [41]) was used to assess worry and rumination. The PTQ is a 15-item (rated 1–5) self-report questionnaire assessing key features of repetitive negative thinking (repetitive, intrusive and difficult to disengage from) and the unproductiveness of and mental capacity captured by this thinking. The Strengths and Difficulties Questionnaire (SDQ, [42]) is a 25-item (rated 0–2) self- and parent-report questionnaire assessing emotional problems, conduct problems, hyperactivity-inattention and peer problems as well as pro-social behaviour. The total difficulties score, computed based on all problem subscales, was used in this study. Stress reactivity was assessed by using Cyberball [43], [44] as a social stressor. In this task, participants are led to believe that they play an online ball-tossing game, which is programmed such that after two own tosses, the participant is excluded from the game. To assess changes in mood in response to the stress-task, participants had to indicate how anxious, nervous, sad, happy, confident, and enthusiastic they felt on a scale from 0–100 (not at all–very much) before and after the task. Ratings were combined into a positive and negative mood scale respectively. Internal consistency for all emotional outcome measures was adequate to excellent in the larger study sample (SCARED α = .92, CDI α = .86, RSES α = .86, PTQ α = .95, PMT-K α = .81, SDQ α = .71, SDQ-parent α = .71, positive mood α = .72, negative mood α = .65). BODY.METHODS.STRESSFUL LIFE EVENTS: Stressful life events were assessed with the Dutch "TRAILS events scale" ('TRAILS Gebeurtenissen vragenlijst', [45]), a self-report questionnaire assessing the occurrence and impact of 25 stressful events (e.g., parental divorce, severe illness/death of a family member, victimization). Participants had to indicate whether the event occurred either during the past three months, during the last two years or never/longer ago and how stressful (rated 0–3) the experienced event was. A stressful life events index was calculated by adding the impact scores for all life events that had been experienced in the previous period. Next, based on [45] we dichotomized this index into "high stress" (scores > 6) and "low or average stress" for each time point. Finally, since we were interested in the long-term interaction between stress and training, groups were created separating those who were in the "high stress" group at least at one time point and those who never were. BODY.METHODS.PROCEDURE: Adolescents of invited classes received oral instructions about the content and aim of the study, explained as "investigating a training to make adolescents more resilient to stress and negative emotions, by learning to worry less and have a more positive view on your environment". Both adolescents and parents received information letters and had to provide written informed consent. The first assessment (T1), the first training and the post-training assessment (T2) were completed under supervision and took place during regular school hours in a computer classroom. Assessments started with the REC-T and some other computer tasks, followed by the online questionnaires, and participants were automatically directed to the next task. Apart from interpretation bias, also attentional bias and working memory were assessed, and questionnaires on attentional control, alcohol-related problems and high sensitive personality were administered, but not used for the current study. See S1 Appendix for a description of these materials. Both assessments took about 80 minutes. One to seven days after T1, the first training session was performed at school. For the remaining training sessions, participants received a reminder by e-mail and text message twice a week. Each session took approximately 15 minutes and had to be completed within two days. Reminders were sent after missing two sessions, offering technical assistance where needed. T2 was almost identical to T1, except for the inclusion of the Cyberball stress-task, and took place 1–7 days after the last training sessions. At the end of this session, participants were fully debriefed on Cyberball and compensated by vouchers and participation in a lottery, based on the number of sessions completed (<six sessions: one lottery ticket, six or seven sessions: five euros and two lottery tickets, eight sessions: 10 euros & three lottery tickets). Three (FU1), six (FU2) and 12 (FU3) months after T2, participants received a text message and an e-mail with a link to complete the follow-up assessments, consisting of the same questionnaires as T1. Reminders were sent after one week, and test-assistants made phone calls to non-responders after two weeks. BODY.METHODS.DATA ANALYSES: To examine whether the CBM-I and Placebo group differed on demographic characteristics or baseline scores on outcome measures, chi-square tests and independent t-tests were performed. To assess potential treatment effects, mixed regression analysis was performed. This method takes into account repeated assessments and uses all available data without discarding participants with missing data at specific time points. For all outcome measures, a mixed model with Participant as the grouping variable and Time as a repeated measure variable was tested. With regard to the covariance between time points, we verified (based on AIC and BIC criteria) whether these were structured according to compound symmetry, or first order autoregressive, or whether these were unstructured. The latter was the case for all analyses. School could have been added as another grouping variable, but was not included, as preliminary analyses indicated that this did not improve model fit, and that school explained less than 0.6% of the variance in our primary outcome measures. To test our first and second hypothesis, for both anxiety and depressive symptoms, and interpretation bias (REC-T and RTs to probe scenarios) respectively, a model including the factors Time and Condition (CBM-I or Placebo), and their interaction, was created. The factor Time had two levels for short-term outcome measures (T1 and T2), five levels for long-term measures (T1, T2, FU1, FU2, and FU3) and eight levels for probe RTs (one for each training session). The best model was selected in a backward elimination procedure, in which parameters were excluded from the model based on AIC and BIC criteria and significance level of the parameters. To test our third hypothesis, i.e. that symptom change would be larger for participants who showed a larger change in interpretation bias, a model was created including the factors Time and Condition, the covariate bias change and all possible interaction terms. Note that while conceptually we hypothesized change in interpretation bias to be a mediator of emotional effects, in mixed regression this was implemented as a moderating factor, as this analysis method is more suitable here, and mediation also implies that emotional effects will be observed specifically in those participants who display a change in bias. To test our fourth hypothesis, we assessed the moderating role of baseline interpretation bias, stressful life events, or number of completed training sessions with separate models using the same approach. The effects of interest in these analyses were the three-way interactions between Time, Condition, and Moderator. To explore training effects on stress reactivity, and the other secondary outcome measures (RSES, PTQ, PMT-K, SDQ, SDQ-P, mood scales), the same procedure as for the primary outcome measures was used, starting with a model including Time, Condition and their interaction. To control for Type I errors related to the number of outcome measures, Bonferroni-Holm correction was applied for the full set of 11 outcome measures. Effects with p <05 that did not survive this correction were defined as marginal. BODY.RESULTS.PRELIMINARY ANALYSES: The CBM-I and Placebo group did not differ on demographic characteristics nor outcome measures at baseline, all p's > .320. On average, participants completed 5.51 sessions (SD = 2.32), and 41 adolescents (23.7%) completed all eight training sessions. Girls completed significantly more training sessions than boys, t (131.71) = -3.34, p = .001. Missing data ranged between 7.5% at T2 (17.9% for parent-report), and 54.3% at FU3 (41.6% for parent-report). The number of completed assessments was not related to training condition, nor baseline scores on any of the outcome measures, all p's > .136. However, girls completed more assessment sessions than boys, χ2 (4) = 9.83 p = .043. The REC-T scores at baseline were significantly smaller than zero, t (171) = -7.34, p <.001, indicating that at baseline participants generally showed a positive interpretation bias. Interpretation bias was not correlated with anxiety or depressive symptoms, r = .11, p = .147, and r = .13, p = .093, respectively. Table 2 shows descriptive statistics for both training groups for all outcome measures. Statistics of the original and final models for all hypotheses can be found in Table 3, and Table 4 shows the relevant parameter estimates. 10.1371/journal.pone.0194274.t002 Table 2 Outcome measures per training condition. Condition Outcome measure a T1 b pre-training assessment T2 post-training assessment FU1 3 months follow-up FU2 6 months Follow-up FU3 12 months follow-up M SD M SD M SD M SD M SD CBM-I (n = 134) REC-T -0.32 0.58 -0.93 0.77 - - - - - - SCARED 20.31 12.13 17.57 12.73 18.15 12.01 16.73 12.37 16.30 12.35 CDI 8.68 5.54 7.70 6.52 7.34 5.76 6.68 6.42 6.10 6.13 Positive mood 200.52 62.04 198.57 71.46 - - - - - - Negative mood 41.00 48.36 38.42 45.90 - - - - - - RSES 30.14 4.88 31.19 5.27 31.21 5.05 32.04 5.14 31.84 5.27 PTQ 35.18 12.27 34.14 12.61 32.78 11.18 33.55 14.02 32.16 13.50 PMT-K 7.66 3.59 7.05 3.66 7.20 3.35 6.73 3.79 5.98 3.62 SDQ 10.69 5.12 10.04 5.35 9.35 5.51 9.21 5.74 8.25 5.13 SDQ-parent 6.44 5.06 6.34 5.32 5.61 4.6 5.05 4.33 5.09 4.18 Placebo (n = 39) REC-T -0.34 0.56 -0.60 0.60 - - - - - - SCARED 18.15 10.85 16.00 10.04 17.67 9.36 15.60 8.12 14.39 7.55 CDI 8.87 6.42 8.14 5.63 5.72 4.21 8.10 6.36 7.61 6.60 Positive mood 201.53 59.11 201.29 65.03 - - - - - - Negative mood 47.11 44.19 37.63 44.78 - - - - - - RSES 29.62 4.85 29.89 5.69 31.16 5.21 29.55 4.91 30.37 5.62 PTQ 36.26 12.91 32.33 12.12 32.44 9.96 32.35 13.14 29.28 12.21 PMT-K 7.97 3.32 7.28 3.28 7.94 3.84 8.10 3.45 7.11 4.34 SDQ 9.82 5.43 9.11 4.32 8.37 3.34 9.05 5.92 7.39 4.07 SDQ-parent 7.32 5.07 6.42 4.76 6.37 4.38 6.46 5.39 7.16 5.34 a REC-T = Recognition Task; SCARED = Screen for Child Anxiety Related Emotional Disorders; CDI = Children’s Depression Inventory; RSES = Rosenberg Self-Esteem Scale; PTQ = Perseverative Thinking Questionnaire; PMT-K = Performance Motivation Test for children; SDQ(-P) = Strengths and Difficulties Questionnaire (Parent) b Note that for positive and negative mood, T1 and T2 refer to pre- and post-stressor mood respectively, both assessed at the post-training assessment session. 10.1371/journal.pone.0194274.t003 Table 3 Statistics of the original and final models for all hypotheses. Outcome measure a Model b Model fit Time Condition Condition * Time Condition * Time * Moderator c     AIC BIC F df F df F df F df REC-T Condition * Time 662.37 689.00 27.95 *** 1, 165.15 2.69 1, 166.90 4.40 * 1, 165.15 - - Bias index probes Condition * Time 13009.71 13258.79 15.68 *** 7, 111.48 16.23 *** 1, 143.61 1.23 7, 111.48 - - Condition + Time 13003.89 13219.44 22.53 *** 7, 108.59 14.13 *** 1, 115.99 - - - - Negative probes Condition * Time 13604.12 13855.77 21.10 *** 7, 106.79 0.94 1, 159.57 1.01 7, 106.79 - -   Time 13596.64 13809.58 31.98 *** 7, 106.64 - - - - - - Positive probes Condition * Time 13661.39 13913.10 49.51 *** 7, 109.89 7.29 1, 164.33 2.99 ** 7, 109.89 - - SCARED Condition * Time 4169.85 4278.97 5.92 *** 4, 112.69 0.31 1, 165.17 0.30 4, 112.69 - -   Time 4161.45 4248.75 9.87 *** 4, 111.99 - - - - - -   Condition * Time * Bias change 3994.09 4145.51 4.86 ** 4, 108.81 0.07 1, 152.12 0.61 4, 108.81 0.41 4, 111.28   Condition * Time * Bias 4140.06 4292.52 6.20 *** 4, 111.63 0.30 1, 165.79 0.31 4, 111.63 0.14 4, 116.08   Condition * Time * Life events 4163.99 4316.75 5.17 ** 4, 120.80 0.71 1, 172.59 0.29 4, 120.80 0.29 4, 120.80   Condition * Time * Sessions 4175.90 4328.67 5.80 *** 4, 148.77 0.63 1, 192.33 0.31 4, 148.77 1.23 4, 147.64 CDI Condition * Time 3339.44 3448.47 1.65 4, 91.67 0.28 1, 160.35 0.98 4, 91.67 - -   Time 3333.20 3420.43 2.57 * 4, 91.52 - - - - - -   Condition * Time * Bias change 3195.44 3346.73 1.11 4, 86.70 0.17 1, 146.41 0.91 4, 86.70 0.70 4, 88.82   Condition * Time * Bias 3316.46 3468.80 1.78 4, 92.25 0.29 1, 157.75 0.87 4, 92.25 0.31 4, 97.63   Condition * Time * Life events 3319.01 3471.66 2.10 4, 102.16 0.13 1, 164.63 1.28 4, 102.16 1.05 4, 102.16   Condition * Time * Sessions 3347.13 3499.78 1.87 4, 137.44 0.09 1, 174.91 2.40 † 4, 137.44 1.38 4, 139.42 Positive mood Condition * Time 3357.13 3383.42 0.36 1, 156.03 0.01 1, 159.98 0.01 1, 156.03 - -   Time 3353.14 3371.92 0.55 1, 156.03 - - - - - - Negative mood Condition * Time 3137.53 3163.82 5.08 * 1, 157.41 0.13 1, 160.34 1.58 1, 157.41 - -   Time 3135.17 3153.95 3.46 † 1, 157.34 - - - - - - RSES Condition * Time 3305.06 3414.48 1.91 4, 102.41 1.44 1, 158.48 0.77 4, 102.41 - -   Time 3398.80 3386.33 3.72 ** 4, 105.65 - - - - - - PTQ Condition * Time 4232.85 4341.92 6.36 *** 4, 95.27 0.03 1, 163.50 0.94 4, 95.27 - -   Time 4226.58 4313.84 6.17 *** 4, 94.86 - - - - - - PMTK Condition * Time 2773.03 2882.11 5.69 *** 4, 100.40 0.91 1, 159.23 0.45 4, 100.40 - -   Time 2765.36 2852.62 9.04 *** 4, 100.54 - - - - - - SDQ Condition * Time 3252.50 3361.71 4.37 ** 4, 101.22 0.70 1, 165.87 0.17 4, 101.22 - -   Time 3243.96 3331.33 5.86 *** 4, 100.91 - - - - - - SDQ-P Condition * Time 3148.66 3258.91 2.28 † 4, 115.53 1.40 1, 160.67 0.19 4, 115.53 - -   Time 3140.77 3228.97 3.18* 4, 115.10 - - - - - - † p < .10 * p ** p *** p Note that most p-values between p a REC-T = Recognition Task; SCARED = Screen for Child Anxiety Related Emotional Disorders; CDI = Children’s Depression Inventory; RSES = Rosenberg Self-Esteem Scale; PTQ = Perseverative Thinking Questionnaire; PMT-K = Performance Motivation Test for children; SDQ = Strengths and Difficulties Questionnaire b Condition = CBM-I versus Placebo; Time = two levels for REC-T, eight levels for probes, and five levels for all other outcome measures. Bold print = final model, based on AIC and BIC and significance of parameters. Lower AIC and BIC values represent a better model fit. Note that moderation models were tested after testing general training effects on primary outcome measures (SCARED and CDI). c Moderator refers to the specific potential moderator included in the model (Bias change, Bias, Life events, or Sessions) 10.1371/journal.pone.0194274.t004 Table 4 Parameters estimates of significant effects. Training effects   CBM-I a T2 FU1 FU2 FU2 T2 CBM-I     B SE B SE B SE B SE B SE B SE REC-T b Condition * Time - - -0.26 † 0.15 - - - - - - -0.35 * 0.17 Bias index probes Condition + Time c -102.19 *** 27.19 - - - - - - - - - - SCARED Time - - -2.33 *** 0.54 -2.41 * 0.95 -4.22 *** 0.95 -4.98 *** 0.97 - - CDI Time - - -0.63 * 0.29 -1.19 * 0.46 -1.37 ** 0.50 -1.35 * 0.54 - - RSES Time - - 0.82 ** 0.30 1.06 * 0.47 1.33 ** 0.42 1.63 ** 0.47 - - PTQ Time - - -1.51 * 0.59 -2.40 * 1.01 -3.42 ** 1.10 -4.64 *** 1.02 - - PMTK Time - - -0.61 ** 0.17 -0.61 * 0.30 -1.08 ** 0.32 -1.75 *** 0.31 - - SDQ Time - - -0.45 † 0.26 -1.00 * 0.42 -1.12 * 0.43 -1.90 *** 0.40 - - SDQ-P Time - - 0.20 0.23 -0.88 * * 0.28 -1.08 ** 0.34 -0.83 * 0.34 - - † p .10 * p < .05 ** p < .01 *** p < .001 Note that most p-values between p a Reference categories for parameters estimates were the placebo condition and the pre-training assessment (T1). T2 = post-training assessment; FU1 = 3 months follow-up; FU2 = 6 months follow-up; FU3 = 12 months follow-up. b REC-T = Recognition Task; SCARED = Screen for Child Anxiety Related Emotional Disorders; CDI = Children’s Depression Inventory; RSES = Rosenberg Self-Esteem Scale; PTQ = Perseverative Thinking Questionnaire; PMT-K = Performance Motivation Test for children; SDQ(-P) = Strengths and Difficulties Questionnaire (Parent) c Time effects are not included in this Table, since this model included the eight training sessions as time points. Bias index was significantly reduced at all sessions compared to the first session (all p’s < .001, parameter estimates between B = -489.60, SE = 55.51, and B = -275.29, SE = 57.90), except for the fourth session, p = .224. BODY.RESULTS.PRIMARY OUTCOME MEASURES: Our first and primary hypothesis, that CBM-I would result in reduced anxiety and depressive symptoms compared to Placebo, was not confirmed, as no significant Condition x Time interactions were observed for SCARED and CDI scores. For anxiety a significant main effect of Time was found, p < .001, and for depressive symptoms a marginal Time effect, p = .043, both indicating a general decrease in symptoms from T1 to T2, that remained significant or marginally significant at all follow-up assessments. BODY.RESULTS.SECONDARY COGNITIVE OUTCOME MEASURES: Our second hypothesis, that CBM-I would result in a reduction of negative interpretation bias compared to placebo, was confirmed by a marginal Condition x Time interaction for interpretation bias assessed with the REC-T, p = .037. That is, a larger reduction in negative interpretation bias (i.e. a relative increase in positive interpretations) was found in the CBM-I group compared to Placebo. For interpretation bias assessed with RTs to probe scenarios (bias index = RTs to positive probes—RTs to negative probes), the expected Condition x Time interaction was not significant, but significant main effects of Condition and Time were found, both p's <.001, indicating a general increase in positive interpretation bias and a more positive bias in the CBM-I group compared to placebo. Testing separate models for negative and positive probes revealed only a significant effect of Time for negative probes, p <.001, indicating reduced RTs over time in both groups. For positive probes, the expected Condition x Time interaction was observed, p = .007, indicating a significantly larger reduction in RTs to positive probes in the CBM-I group compared to the Placebo group. This indicates that participants who were following the CBM-I training became quicker in responding to positive probes, suggesting quickening of positive interpretations. Fig 2 shows the development of RTs throughout training sessions. 10.1371/journal.pone.0194274.g002Fig 2Mean reactions times (RT) to negative probe scenarios (NP) and positive probe scenarios (PP) during training sessions for the two training groups. BODY.RESULTS.MODERATION OF TRAINING EFFECTS: Our third hypothesis, that training effects on anxiety and depressive symptoms would be larger for participants who showed a larger reduction in negative interpretation bias, was not confirmed, as no three-way interactions between Condition, Time and change in interpretation bias were observed. Our fourth hypothesis, that training effects on anxiety and depressive symptoms would be moderated by baseline interpretation bias, stressful life events, or number of training sessions completed, was also not supported, as no three-way interactions between Condition and Time, and these potential moderators were observed. BODY.RESULTS.SECONDARY EMOTIONAL OUTCOME MEASURES: Finally we explored training effects on secondary emotional outcomes, that is, stress-reactivity immediately post-training, and self-esteem, perseverative negative thinking, test-anxiety, and social-emotional and behavioural problems post-training and at follow-up. Contrary to our expectations, no changes in mood were observed in response to the stress task, nor was mood affected by training condition. For all other secondary outcome measures, significant Time effects were found, indicating reductions in symptoms, with some variability in the specific comparisons between time points that were significant. No differential training effects were observed between the CBM-I and Placebo group for these measures. BODY.DISCUSSION: The aim of the current study was to investigate the short- and long-term effects of multiple sessions of online CBM-I on interpretation bias, anxiety, depression, and emotional resilience in unselected adolescents. Our primary hypothesis was that CBM-I would result in reduced symptoms of anxiety and depression compared to placebo. Furthermore, we hypothesized that CBM-I would reduce negative interpretation bias (i.e. enhance positive interpretation bias), and would have enhancing effects on emotional resilience. As hypothesized, interpretation bias as assessed with the recognition task became more positive by CBM-I training compared to CBM-I placebo, although this effect just fell short of significance after Bonferroni-Holm correction. This trend converges with accumulating evidence on the efficacy of CBM-I in changing interpretive style in both adults and adolescents [8], [12], [13], [24]. Note that a neutral control condition was used instead of a negative control condition, and the former has been found to yield smaller or even no effects in previous research (e.g. [46]). Interpretation bias displayed in response to probe scenarios became more positive in both groups, but across sessions the CBM-I group displayed the most positive bias. Changes in interpretation bias were expected to be accompanied by increased resilience and reduced anxiety and depressive symptoms, but such corresponding effects were not found, neither on the short-term nor on the long-term. Both the CBM-I and Placebo group showed a general decrease in anxiety and depression symptoms over time, but did not differ from each other. Whether this reflects a natural decline, expectancy effects or an unintended positive effect of the placebo training (cf. [23], [47]) is unclear. However, a similar overall decline of anxiety symptoms in adolescents has also been found in a previous study that used a non-intervention instead of a placebo control condition [25]. Thus it seems most parsimonious to attribute the overall decline of symptoms to a natural course. Including the following four moderators: change in interpretation bias, baseline interpretation bias, stressful life events, or number of training-sessions completed, did not change these results. For the secondary emotional outcome measures, the same pattern of general improvement was observed. In previous CBM-I research, findings on emotional outcomes have also been more mixed [8]. It has been argued that changing interpretive style on a training-related task might be relatively easy, but more time may be necessary for transfer to daily life and emotional symptoms [20]. That is, a change in interpretation bias might only affect emotions in interaction with daily situations. However, the lack of long-term effects in our study (up to one year later), suggests that time alone is not enough to obtain generalization to emotional outcomes. As negative biases have also developed during a long time period and in response to life experiences, more time in between training sessions or including booster sessions might be necessary [48]. The timing of training and assessments is an important issue for further research. Our study was one of the first to investigate long-term effects of CBM-I in adolescents. The only previous study on CBM-I as a preventive intervention in adolescents similarly found limited effects [24] also at two-year follow up [25], but in a long-term study in adults more promising effects were observed [22]. Although a comparable training paradigm was used, two important differences should be noted here. First, the training in the study of Khalili-Torghabeh et al. [22] was performed in the laboratory rather than online, and CBM effects are generally stronger in such laboratories than online (meta-analysis [7]). This might be related to the lack of experimental control in online studies, which seems especially important in an intervention where task compliance and timing is essential, as is the case with CBM. In contrast, online CBT might suffer less from such distracting environments or technical issues, and small to medium effects on anxiety and depression in young people have been found in a recent meta-analysis [49]. Second, contrary to their pre-selected samples with heightened symptoms, we used unselected adolescents. In our sample, with a relatively low level of symptoms that further decreased over time, it might have been harder to detect any training related changes. We hypothesized that CBM-I would also increase emotional resilience in healthy adolescents, but did not find such effects. Contrary to our expectations, no correlation was observed between interpretation bias and symptoms of anxiety and depression or secondary emotional measures. This might question the relevance of interpretation bias in unselected samples (compared to (sub-)clinical samples), and thereby undermine the basis of CBM-I as an intervention to increase emotional resilience in such a population. However, the variability in symptom levels in the current sample was high, and previous research has shown that interpretation bias scores on the recognition task are associated with anxiety in adolescents [15]. Therefore, the lack of a correlation in the current study, might be related to limitations of our assessment method. The recognition task was administered in group format, and performance might have suffered from a lack of concentration and motivation. Therefore, the observed scores might not be a fully accurate reflection of existing interpretation biases. A more general limitation of the recognition task, is its strong resemblance to the CBM-I training task, rendering it vulnerable to practice and demand effects. To investigate transfer effects, it would be necessary to add other tasks that differ more from the training paradigm, like a homophone or face classification task [50]. Previous attempts to demonstrate generalization to other types of interpretation bias assessments have been mainly unsuccessful (e.g. [51], [52]. Note that, although also task-specific, the development of reaction times to probes during training confirmed the change in interpretation bias found on the recognition task. Another limitation of the current study concerns the high drop-out rates, which are not uncommon in longitudinal and/or online research (cf. [24], [53]). Many participants did not complete the intended amount of training and assessments, and the current results (intention-to-treat approach) might thus not reflect the full potential of multi-session training. Although drop-out was unrelated to emotional functioning at baseline, girls completed relatively more assessment sessions. Therefore, long-term results should be interpreted carefully when referring to adolescent boys. Drop-out at follow-up assessments also reduced power to detect long-term effects, but as we also found no short-term effects, it is unclear whether effects would have been found in a larger sample. Note that we applied a relatively conservative data-analytic approach, including the simultaneous examination of all time points and stringent correction for multiple testing. Although this seems an adequate approach to reduce the risk of Type I errors and to assess robust effects of the intervention, it also reduced power to observe small effects for specific comparisons, particularly in combination with our unbalanced randomization (smaller placebo group). Although steps were taken to increase engagement and compliance in the current study (by including feedback, progress bar, financial compensation, e-mail, text messages and phone call reminders), more motivating features might be necessary to improve adherence. For example, training might become more appealing when adding gaming elements or a social network environment [54]. This seems especially important in adolescent samples and in healthy samples who may miss an intrinsic motivation to change. Whether intended motivating features indeed increase engagement and adherence, needs to be monitored carefully. Improving adherence is not only relevant for reducing attrition (and increasing representativeness) in intervention studies, but particularly for potential implementation of training paradigms that prove to be effective. Preventive programs should be acceptable for the targeted population, and apart from attractive tasks, this might also require providing a clear rationale [55], which is not current practice in CBM training studies [56]. Whether more explicit instructions (cf. [57]) and psychoeducation will improve efficacy is thus another important question for future research. To summarize, the CBM-I training was marginally effective in increasing a positive interpretation bias in unselected adolescents, as indexed by both reaction times during probe trials during training and a separate assessment task. However, these changes were not paralleled by a change of any of the emotional measures, neither at the short- nor at the long-term. Consistent with previous findings among adolescents (e.g., [25]), symptoms of anxiety and depression generally decreased over time. Yet, this decline was not especially pronounced in the active condition. Given the limitations of online research (especially in unselected samples), including the high drop-out rates, it would be premature to conclude that CBM-I has no potential, but in its current form, it seems of little use for universal prevention. BODY.SUPPORTING INFORMATION: S1 AppendixAdditional manipulations and measures.(DOCX)Click here for additional data file. S1 FileCONSORT checklist.(DOCX)Click here for additional data file. S1 ProtocolTrial protocol.(PDF)Click here for additional data file. S2 ProtocolAdditional documentation trial protocol.(DOCX)Click here for additional data file. S3 ProtocolInformation letters.(PDF)Click here for additional data file. S4 ProtocolTrial protocol translation.(DOCX)Click here for additional data file.

TITLE: A phase IIa dose-finding and safety study of first-line pertuzumab in combination with trastuzumab, capecitabine and cisplatin in patients with HER2-positive advanced gastric cancer ABSTRACT.BACKGROUND:: Pertuzumab plus trastuzumab provides a more comprehensive blockade of HER2 signalling than trastuzumab alone. Therefore, we conducted a phase IIa study of the pharmacokinetics and safety of pertuzumab plus trastuzumab and chemotherapy in advanced gastric cancer (aGC). ABSTRACT.METHODS:: Patients received pertuzumab 840 mg for cycle 1 and 420 mg q3w for cycles 2–6 (Arm A) or pertuzumab 840 mg q3w for six cycles (Arm B). Trastuzumab, cisplatin and capecitabine were also given for six cycles, then trastuzumab q3w until disease progression or unmanageable toxicity. The co-primary endpoints were day 43 pertuzumab serum trough concentration (Cmin) and safety. ABSTRACT.RESULTS:: Thirty patients were randomised. Mean pertuzumab Cmin at day 43 was 40.0 μg ml−1 (s.d.: 17.3) in Arm A and 62.7 μg ml−1 (29.1) in Arm B. Mean day 43 Cmin in Arm A was ∼37% lower than that seen in metastatic breast cancer. The safety profiles were similar between arms and treatment was well tolerated. Partial responses were achieved by 86% and 55% of patients in Arms A and B, respectively. ABSTRACT.CONCLUSIONS:: On the basis of the pharmacokinetic and safety data, the 840 mg q3w pertuzumab dose has been selected for a phase III study of pertuzumab, trastuzumab and chemotherapy in HER2-positive aGC. BODY: Globally, gastric cancer is the fourth most commonly occurring cancer (Ferlay et al, 2010), with variable overall survival rates; the 5-year survival rate ranges from 10–30% in most of the world to 50–70% in Korea and Japan (Howlader et al, 2011; Matsuda and Saika, 2013; Jung et al, 2013a; De Angelis et al, 2014). Although surgical resection of localised gastric tumours offers a potentially curative therapy, most patients present with inoperable, advanced or metastatic disease requiring palliative treatment (Dicken et al, 2005), with the exception of some countries in East Asia, such as Korea and Japan, where national screening programmes are conducted (Dicken et al, 2005; Jung et al, 2013b). Combining multiple chemotherapy agents has improved overall survival rates compared with single agents or best supportive care (Wagner et al, 2006), and the current standard chemotherapy regimen for patients with advanced gastric cancer (aGC) contains a fluoropyrimidine plus a platinum compound (Jackson et al, 2009). However, the prognosis of patients with aGC remains poor and improved treatment options are required for this disease. Human epidermal growth factor receptor 2 (HER2) overexpression/amplification is an established negative prognostic biomarker in breast cancer and occurs in ∼20% of cases (Slamon et al, 1987, 1989), whereas 17–22% of gastric cancer cases have been reported to be HER2-positive (Tanner et al, 2005; Lordick et al, 2007; Zhang et al, 2009; Bang et al, 2010). In the phase III ToGA trial, the addition of the HER2-targeted humanised monoclonal antibody trastuzumab to first-line fluoropyrimidine plus cisplatin chemotherapy significantly increased overall survival in patients with HER2-positive advanced gastric or gastro–oesophageal junction cancer, with further survival benefit in patients whose tumours had high levels of HER2 expression (immunohistochemistry (IHC) 3+ or IHC 2+ and fluorescence in situ hybridisation-positive; Bang et al, 2010). The grade 3–4 adverse event (AE) profiles were similar between the trastuzumab plus chemotherapy arm and the chemotherapy-only arm, with the exception of increased rates of diarrhoea in patients receiving trastuzumab (Bang et al, 2010). On the basis of the data from the ToGA trial, trastuzumab was approved in combination with capecitabine plus cisplatin or 5-fluorouracil plus cisplatin for the first-line treatment of patients with HER2-positive metastatic adenocarcinoma of the stomach or gastro–oesophageal junction (Herceptin Summary of Product Characteristics, 2013). Lapatinib, an oral tyrosine kinase inhibitor of epidermal growth factor receptor and HER2, has also been tested in HER2-positive gastric cancer. However, lapatinib did not improve overall survival when combined with chemotherapy for first-line (Hecht et al, 2013) or second-line treatment (Bang et al, 2013) of HER2-positive aGC. Pertuzumab is a humanised monoclonal antibody that targets a different epitope of HER2 from trastuzumab (Cho et al, 2003; Franklin et al, 2004). Whereas trastuzumab binds to subdomain IV of HER2 (Cho et al, 2003) to inhibit ligand-independent signalling (Junttila et al, 2009), pertuzumab binds to subdomain II (Franklin et al, 2004), the dimerisation domain, to inhibit HER2 heterodimerisation and, consequently, ligand-dependent signalling (Agus et al, 2002). Pre-clinical studies have suggested that trastuzumab and pertuzumab have complementary mechanisms of action and that the combination of the two antibodies provides a more efficient blockade of HER2 signalling than either antibody alone (Nahta et al, 2004; Scheuer et al, 2009). In the CLEOPATRA study of patients with HER2-positive metastatic breast cancer (MBC), the combination of pertuzumab, trastuzumab and docetaxel significantly improved progression-free (Baselga et al, 2012) and overall survival (Swain et al, 2013) compared with placebo, trastuzumab and docetaxel. Furthermore, pre-clinical studies of a HER2-positive human gastric cancer xenograft model showed enhanced anti-tumour activity when pertuzumab and trastuzumab were combined, compared with that seen with either antibody alone (Yamashita-Kashima et al, 2011). Therefore, the combination of pertuzumab with trastuzumab and chemotherapy has the potential to improve survival outcomes in patients with HER2-positive aGC compared with trastuzumab and chemotherapy alone. In patients with HER2-positive aGC, the observed steady-state exposure of trastuzumab was lower than that seen with an identical dose in MBC, and patients with trastuzumab concentrations in the lowest quartile had a shorter median overall survival (7.7 (range, 6.3–10.6) months) than patients in the upper three quartiles (15.7 (range, 14.1–18.9) months; Yang et al, 2013). Furthermore, in a separate analysis, patients with the lowest trastuzumab serum trough concentrations had the highest rate of disease progression and shortest overall survival (Cosson et al, 2014). Therefore, we aimed to study the pharmacokinetics (PK) of pertuzumab in combination with trastuzumab and chemotherapy to identify the pertuzumab dose that produces a steady-state trough serum concentration (Cmin) of ⩾20 μg ml−1 in at least 90% of patients with HER2-positive gastric cancer. This PK target was derived from dose-response xenograft studies of pertuzumab administered as a single agent showing effective tumour-growth inhibition with serum trough concentrations of 5–25 μg ml−1 (Malik et al, 2003), and is also the PK target that was used to determine the pertuzumab dose in breast cancer. In patients with HER2-positive breast cancer, the PK target was achieved with a pertuzumab loading dose of 840 mg followed by 420 mg q3w, a dosing regimen that has shown promising activity in early and advanced breast cancer treatment settings (Baselga et al, 2010; Cortés et al, 2012; Gianni et al, 2012; Schneeweiss et al, 2013; Swain et al, 2013). Importantly, phase I/II trials of both weight-based and fixed pertuzumab doses in multiple tumour types have not revealed a maximum tolerated dose (Agus et al, 2005; Attard et al, 2007; Albanell et al, 2008). Here we present the results of JOSHUA, a phase IIa study of the PK and safety of two different doses of pertuzumab in combination with trastuzumab and chemotherapy, to identify the optimal pertuzumab dose for clinical studies in HER2-positive gastric cancer. BODY.MATERIALS AND METHODS.STUDY DESIGN AND TREATMENT: JOSHUA was a randomised, multicentre, open-label phase IIa trial (NCT01461057) evaluating two different doses of pertuzumab in patients with HER2-positive advanced gastric or gastro–oesophageal junction cancer. Treatment was given in 3-weekly cycles. Patients were randomised to receive an initial pertuzumab dose of 840 mg for cycle 1, followed by a dose of 420 mg for cycles 2–6 (Arm A) or pertuzumab 840 mg for cycles 1–6 (Arm B). Patients in both treatment arms received cisplatin (80 mg m−2) and capecitabine (1000 mg m−2 BID for 14 days) for cycles 1–6; in the absence of disease progression or unmanageable toxicity, patients could receive capecitabine for more than six cycles, at the discretion of the investigator. All patients received trastuzumab at 8 mg kg−1 for cycle 1, followed by 6 mg kg−1 for subsequent cycles, until investigator-assessed disease progression or unmanageable toxicity. The study was approved by the institutional review board of each participating centre or the competent authority and ethics committee. The study was conducted in full accordance with the International Conference on Harmonisation Good Clinical Practice guidelines and the Declaration of Helsinki. All patients provided written informed consent. The co-primary endpoints of the study were Cmin at day 43 (cycle 2 trough concentration) and safety. The aim of the PK endpoint was to identify the pertuzumab dose that produced a steady-state Cmin of ⩾20 μg ml−1 in at least 90% of patients. The aim of the safety endpoint was to assess the incidence of all AEs. An exploratory objective of the study was to assess the anti-tumour activity of pertuzumab in combination with trastuzumab and chemotherapy in this disease setting. BODY.MATERIALS AND METHODS.PATIENT POPULATION: Patients ⩾18 years old were eligible if they had histologically confirmed, inoperable, locally advanced or metastatic HER2-positive adenocarcinoma of the stomach or gastro–oesophageal junction. HER2 positivity was defined as IHC 3+ or IHC 2+ and in situ hybridisation (ISH)-positive (ISH positivity was defined as a HER2 : CEP17 signal ratio of ⩾2.0) by central testing. Eligible patients had measurable disease or non-measurable, evaluable disease according to Response Evaluation Criteria In Solid Tumors (RECIST) version 1.1 (Eisenhauer et al, 2009). Patients were required to have an Eastern Cooperative Oncology Group performance status of 0 or 1, a baseline left ventricular ejection fraction (LVEF) of ⩾55% and a life expectancy of ⩾3 months. Patients were eligible if they had not received prior treatment for advanced or metastatic disease; prior (neo)adjuvant therapy was allowed if completed ⩾6 months before study enrolment, whereas platinum-based (neo)adjuvant therapy was prohibited. Patients were not eligible for the study if they had a serious cardiac illness or medical condition. BODY.MATERIALS AND METHODS.ANALYSIS OF PK: For pertuzumab PK assessment, pre-dose and post-dose serum samples were collected at cycles 1, 2, 3, 4 and 6. Pre-dose samples were collected up to 6 h before the administration of pertuzumab and post-dose samples were collected up to 30 min after the pertuzumab infusion. The cycle 2 trough (cycle 3 pre-dose) sample had to be collected on study day 43 (21 days after the cycle 2 dose), regardless of whether the cycle 3 dose was given, delayed or not given. In addition, weekly serum samples were collected in cycles 1 and 2. A validated bridging enzyme-linked immunosorption assay was used to measure the concentration of pertuzumab in serum samples. The assay used a monoclonal anti-idiotype antibody to capture pertuzumab and had a minimum quantifiable concentration in human serum of 150 ng ml−1. BODY.MATERIALS AND METHODS.ASSESSMENTS: For tumour response assessments, all measurable and non-measurable, evaluable disease had to be documented at screening and reassessed at the end of cycles 3 and 6 according to RECIST v1.1, after which patients were followed for tumour response according to local standards. Response was assessed by the investigator based on physical examinations, computed tomography scans or magnetic resonance imaging. Other methods were allowed if they were amenable to evaluation per RECIST. LVEF assessments by echocardiogram or multiple-gated acquisition scan were performed at baseline and then every three cycles until disease progression or treatment discontinuation. LVEF assessments were performed at the treatment discontinuation visit, every 6 months for the first year following treatment discontinuation, then annually for up to 3 years following treatment discontinuation. Patients who discontinued study treatment permanently due to LVEF decline continued to have LVEF assessments repeated as clinically indicated, with a maximum interval of 3 months between assessments, until LVEF returned to >50%, or 1 year after the treatment discontinuation visit, whichever occurred first. Thereafter, LVEF assessments were performed annually for up to 3 years after the treatment discontinuation visit. AEs were graded according to The National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 4.0 (National Cancer Institute, Bethesda, MD, USA). Real-time safety monitoring occurred on an ongoing basis for all patients and expedited reporting was performed for serious AEs and AEs of special interest. AEs and serious AEs were recorded throughout the study and up to 6 months after the last dose of study treatment, with the exception of cardiac AEs, which were followed for 12 months after the last dose of study treatment, and symptomatic LVEF, which was required to be reported up to 3 years after the last dose of study treatment. Study drug-related serious AEs continued to be recorded regardless of the time elapsed since the last dose of study treatment. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: A sample size of 15 patients per arm was considered sufficient to determine the dose needed to achieve the target Cmin with an acceptable degree of precision (coefficient of variation <15%). This was determined based on trastuzumab PK data from the ToGA trial and the assumption that pertuzumab behaves similarly to trastuzumab in aGC. However, due to variability, outliers and the limited number of data points due to patient withdrawals, it was not possible to accurately assess the primary PK endpoint on the basis of the observed percentage of patients who achieved the target Cmin. Therefore, a post hoc bootstrap analysis was conducted to estimate the percentage of patients at or above the target Cmin and the confidence interval (CI) of this estimate (analysis performed in R Statistics Software version 2.10.1, R Foundation for Statistical Computing, Vienna, Austria). For each pertuzumab dose group, a sample of 15 patients was generated on the basis of the mean and s.d. of the observed data assuming a log-normal distribution, and the percentage of patients with a pertuzumab Cmin of ⩾20 μg ml−1 was computed. The process was repeated 1000 times to generate an estimated percentage of patients with a pertuzumab Cmin of ⩾20 μg ml−1 and a 95% CI. BODY.RESULTS.PATIENT AND DISEASE CHARACTERISTICS: Between December 2011 and April 2012, 30 patients enroled into the study and 15 were randomised into each arm. The baseline demographics and disease characteristics of patients were generally well balanced between arms, although there were some imbalances in terms of age and sex, with older patients and more men in Arm A (Table 1). The majority of patients had metastatic gastric cancer. The data cutoff occurred on 31 December 2012, at which point the mean follow-up time was 312 days and 260 days in Arms A and B, respectively. Fourteen patients in Arm A and 11 patients in Arm B had received all six cycles of pertuzumab (Table 2). Seven patients in Arm A and three patients in Arm B were still receiving study treatment while the remaining patients had discontinued: eight patients in Arm A (due to disease progression) and 12 patients in Arm B (eight due to disease progression, one due to an AE (worsening renal failure), one at the physician's decision (due to pneumonia), one death (fungal pneumonia) and one withdrawal by the patient). BODY.RESULTS.PK OF PERTUZUMAB: Mean pertuzumab serum concentration–time profiles are shown in Figure 1. Pertuzumab concentrations were similar between arms at cycle 1 (when the dose was the same for each arm). However, from cycle 2 onwards, pertuzumab concentrations were higher in Arm B than in Arm A, consistent with the higher dose in Arm B. In Arm A, the mean pertuzumab Cmin increased ∼2-fold from day 22 to day 106, whereas in Arm B the increase in mean pertuzumab Cmin over this time was slightly higher, at ∼2.9-fold. Overall, pertuzumab concentrations were dose proportional. Fifteen patients in Arm A and 13 patients in Arm B had data available to assess pertuzumab Cmin at day 43. One patient in Arm B had abnormally low pertuzumab concentrations, with a day 22 Cmin below the assay's lower limit of quantification and a day 43 Cmin of 0.610 μg ml−1. After excluding this patient from the analysis, the day 43 mean (s.d.) pertuzumab Cmin in Arms A and B was 40.0 μg ml−1 (17.3) and 62.7 μg ml−1 (29.1), respectively, and the geometric mean Cmin in Arms A and B was 36.8 μg ml−1 and 56.4 μg ml−1, respectively. Summary box-scatter plots in Figure 2 show the day 43 pertuzumab Cmin values observed in this study and in the pertuzumab arm of the CLEOPATRA trial, which studied HER2-positive MBC. Mean pertuzumab Cmin with the 840/420 mg dose in HER2-positive aGC (Arm A) was ∼37% lower than that observed with the same dose in HER2-positive MBC (in CLEOPATRA). Mean pertuzumab Cmin with the 840 mg q3w dose in HER2-positive aGC (Arm B) was similar to that observed with the 840/420 mg dose in HER2-positive MBC. As day 43 pertuzumab Cmin samples from Arm B were only available for 12 patients, the ability to select the most appropriate pertuzumab dose based on the observed data was limited. Therefore, a bootstrap analysis was conducted to estimate the percentage of patients who achieved the target pertuzumab Cmin of ⩾20 μg ml−1 at day 43 and the CI of this estimate. This analysis showed that 91.6% (95% CI 78.3, 99.2) and 98.3% (95% CI 91.4, 99.97) of patients in Arms A and B, respectively, achieved the target Cmin. BODY.RESULTS.SAFETY: At the time of data cutoff, patients in Arm A had received a median of six (range, 4–6) doses of pertuzumab and patients in Arm B had received a median of six (range, 1–6) doses of pertuzumab. Overall, the AE profiles were similar between arms. Diarrhoea was the most common AE and was mostly grade 1–2; onset was in cycle 1, with the frequency decreasing during subsequent cycles. Figure 3 shows the incidence of diarrhoea at each treatment cycle. No patient discontinued therapy due to diarrhoea. There were 24 reports (16 in Arm A and 8 in Arm B) of grade ⩾2 diarrhoea in 18 patients (11 in Arm A and 7 in Arm B). Onset of the first incidence of grade ⩾2 diarrhoea in these patients occurred on median (range) study day 12 (1–237) (study day 13 (6–237) in Arm A, study day 8 (1–16) in Arm B). There were 13 instances (8 in Arm A (in seven patients) and 5 in Arm B (in three patients)) of capecitabine dose reduction due to an AE that occurred either during the duration of grade ⩾2 diarrhoea or on day 1 of the subsequent treatment cycle, following resolution of grade ⩾2 diarrhoea. For these 10 patients, initial capecitabine dose reduction occurred on median (range) study day 35.5 (22–181) (study day 42 (23–181) in Arm A, study day 22 (22–56) in Arm B). The total number of grade ⩾3 AEs was 78 in Arm A and 60 in Arm B and the most frequent grade ⩾3 AEs are shown in Table 3. Seventy-three percent and 67% of patients in Arms A and B, respectively, experienced at least one serious AE and the total number of all serious AEs was 35 in Arm A and 19 in Arm B. Serious AEs that occurred in ⩾2 patients overall were diarrhoea, febrile neutropenia, acute renal failure, asthenia, fatigue, gastric obstruction, hyponatraemia, mucosal inflammation, neutropenia, pneumonia, pulmonary embolism and vomiting. One serious AE in Arm B (fungal pneumonia) resulted in death. This was considered by the investigator to be related to the chemotherapy agents. No patient experienced symptomatic heart failure. Two patients, both in Arm A, experienced asymptomatic LVEF decline. After delaying study treatment, their LVEF values recovered and these patients continued receiving study treatment. BODY.RESULTS.OVERALL RESPONSE: An exploratory endpoint of the study was to assess the anti-tumour activity of pertuzumab with trastuzumab, cisplatin and capecitabine in patients with HER2-positive aGC (Table 4). At the end of cycle 3, 10 (67%) and 7 (58%) patients in Arms A and B, respectively, had achieved a partial response, whereas an additional 4 patients in each arm (27% and 33%, respectively) had stable disease. One patient in each arm had progressive disease. At the end of cycle 6, a partial response was observed in 12 (86%) and 6 (55%) patients in Arms A and B, respectively, whereas 2 (14%) and 3 (27%) patients, respectively, had stable disease. Two patients in Arm B had progressive disease. BODY.DISCUSSION: The primary objective of this study was to identify the pertuzumab dose that produces a steady-state concentration of ⩾20 μg ml−1 in at least 90% of patients with HER2-positive aGC. The PK data show that, as expected, the 840 mg q3w dose resulted in higher pertuzumab concentrations overall than the 840/420 mg dose. The data demonstrate that both doses resulted in day 43 trough concentrations above the target of 20 μg ml−1 in at least 90% of patients; however, the lower bound of the 95% CI in Arm A in the bootstrap analysis was 78.3%, suggesting a greater risk of not achieving the PK target in at least 90% of patients with the 840/420 mg pertuzumab dose and that the 840 mg q3w pertuzumab dose is more likely to maintain trough concentrations above the target in at least 90% of patients. Moreover, the 840 mg q3w pertuzumab dose produced trough concentrations in patients with HER2-positive aGC similar to those observed in HER2-positive MBC in CLEOPATRA, whereas the 840/420 mg dose produced lower trough concentrations. Exposure-response analysis of data from the ToGA trial in aGC showed that patients with the lowest trastuzumab serum trough concentrations had the highest rate of disease progression and shortest overall survival (Yang et al, 2013; Cosson et al, 2014). Therefore, the 840 mg q3w pertuzumab dose is expected to provide greater treatment benefit than the 840/420 mg dose in patients with HER2-positive aGC. Many population PK models have found patient demographic factors, such as age, body weight and gender, and also receptor number and disease-related factors, such as number of metastatic sites, circulating tumour receptors and tumour burden, to have a statistically significant impact on PK (Bruno et al, 2005; Gupta et al, 2012; Zhu et al, 2014). However, exploratory analysis did not show any apparent correlation between PK and these covariates in the small sample size of our study. Furthermore, no PK differences were observed between Asian and non-Asian patients, consistent with previous analyses (Dirks and Meibohm, 2010; Chiba et al, 2014; data not shown). As such, the reasons for the lower pertuzumab concentrations in aGC compared with MBC are currently unknown. At this interim safety assessment, the combination of pertuzumab, trastuzumab and chemotherapy was well tolerated. The AE profiles were similar between arms and consistent with those observed with trastuzumab and chemotherapy in the ToGA trial (Bang et al, 2010) with the exception of higher rates of diarrhoea. Events of diarrhoea in JOSHUA were mostly grade 1–2 and occurred early during treatment, with their frequency decreasing during subsequent cycles, particularly following cycle 6. Diarrhoea was manageable and no patient discontinued study treatment due to this AE. Although more patients discontinued treatment in Arm B than Arm A (12 vs 8 patients), it is not clear whether this was due to the higher pertuzumab dose, as AEs leading to discontinuation were not uniform. As a pertuzumab dose of 1050 mg q3w has been shown to be safe and tolerable in phase I/II studies in a variety of solid tumour types (Gordon et al, 2006; Attard et al, 2007; de Bono et al, 2007; Albanell et al, 2008; Gianni et al, 2010), the similarity of the safety profiles of the two doses in JOSHUA is not unexpected. Preliminary data from the exploratory efficacy analysis show that patients with HER2-positive aGC treated with the combination of pertuzumab, trastuzumab, capecitabine and cisplatin achieved high rates of partial response (86% in Arm A and 55% in Arm B) or stable disease (14% in Arm A and 27% in Arm B) at the end of cycle 6. The overall response rates observed in JOSHUA compare favourably with that seen in patients with HER2-positive aGC treated with trastuzumab, capecitabine/fluorouracil and cisplatin in the ToGA trial (47% Bang et al, 2010). Due to the small sample size and lack of a comparator arm in JOSHUA, no firm conclusions can be drawn about the activity of the study drug regimen or the difference between response rates in the two dose groups. However, results from JOSHUA support further investigation of dual blockade anti-HER2 therapy with pertuzumab plus trastuzumab in HER2-positive aGC. In summary, a pertuzumab dose of 840 mg q3w in patients with HER2-positive aGC produces trough concentrations comparable to those seen in HER2-positive MBC without increasing the incidence of AEs. Therefore, the 840 mg q3w pertuzumab dose has been selected for an ongoing phase III study of first-line pertuzumab, trastuzumab and chemotherapy in HER2-positive metastatic gastric and gastro–oesophageal junction cancer (JACOB, NCT01774786; Hoff et al, 2013).

TITLE: Effects of Substituting Soybean Meal for Sunflower Cake in the Diet on the Growth and Carcass Traits of Crossbred Boer Goat Kids ABSTRACT: The present study was conducted to determine the best level of substitution of soybean meal by sunflower cake in diets for kids through the evaluation of quantitative carcass traits. Thirty-two Boer kids X 1/2 NDB (no defined breed), males, non-castrated, with 4 months of age and initial body weight of 15±3.2 kg, were randomly assigned to individual pens. The treatments contained four substitution levels of soybean meal by sunflower cake (0, 33, 66 and 100% DM). At the end of the experimental period, the animals were slaughtered. There was no influence of the treatments on any of the mean values of the evaluated measures (p>0.05): 21.78 kg (body weight at slaughter), 8.65 kg (hot carcass weight), 8.59 kg (cold carcass weight), 40.27% (hot carcass yield), 39.20% (cold carcass yield), 7.73 cm2(rib eye area), 46.74 cm (carcass outer length), 45.68 cm (carcass internal length), 36.92 cm (leg length), 26.04 cm (leg perimeter), 48.66 cm (hind perimeter), 58.62 cm (thoracic perimeter), 0.20 (carcass compactness index), 68.48% (total muscle of the leg), 2.79% (total leg fat), 55.19% (subcutaneous leg fat), 28.82% (total bone), 81.66 g (femur weight), 14.88 cm (femur length), 0.38 (leg muscularity index), 2.53 (muscle:bone ratio) and 33.42 (muscle:fat ratio). The substitution of soybean meal by sunflower cake may be recommended up to a level of 100% without alterations to quantitative carcass traits. BODY.INTRODUCTION: Carcass weight is a factor that has a great influence on the market value of an animal, as in some countries there are marked preferences and price differentials according to carcass weight (Oliveira et al., 2008). Besides that, one must take into consideration the carcass components, since what is sought in the animal is its maximum carcass yield and minimum carcass non-components (Cezar and Sousa, 2007). The consumer shows a preference for cuts with a higher proportion of muscle and subcutaneous fat in an adequate quantity as to ensure tenderness and juiciness, so products brought to the market should present these characteristics. In animal production, a food supply of good nutritional value is important for adequate animal performance and carcass yield, but the high cost of food is a factor that must be taken into consideration. For this reason, cheaper types of food alternative to the ones commonly used (corn and soybeans) have been studied which would not compete with human food. As an option, by-products of oil extraction for biodiesel production (meals and cakes) have been used for this purpose. In Brazil, due to its diversity of climate and ecosystems, there are several species of oilseeds with potential for biodiesel production. The processing of seeds generate considerable amount of by-products that need to be evaluated as alternative sources for animal feed (Dias et al., 2010). Among the various alternatives available, sunflower cake is an option for animal feed because it is a good source of protein and energy (Oliveira et al., 2007) which may increase the yield of goat carcasses, given that the goat breeding systems carried out most extensively in northeastern Brazil are characterized by low animal performance. This study was conducted to evaluate the best level of substitution of soybean meal by sunflower cake in the diet of 1/2 Boer kids through the study of carcass characteristics. BODY.MATERIAL AND METHODS: The experiment was conducted at the School of Veterinary Medicine and Animal Science of the Federal University of Bahia, located in Salvador, Brazil, over a period of 68 days from October to December 2008, with seven days for animal adaptation to diets and daily management and 61 days for data collection. Thirty-two 1/2 Boer kids X 1/2 NDB (no defined breed) were used, all non-castrated males, at an age of four months and an initial body weight of 15±3.2 kg. Prior to the beginning of the experiment, the animals were weighted, marked, de-wormed and vaccinated against clostridia, endo and ectoparasites, and then assigned to individual 1.0 m2 pens with a suspended wooden slatted floor, and provided with food and water in a covered area. The kids were distributed into four treatment groups, constituting diets with 0, 33, 66 and 100% substitution of soybean meal by sunflower cake in the dry matter. Diets were formulated following the NRC guidelines (1981) to meet the nutritional requirements of growing kids, with target gains of 150 g per day. The concentrates were composed of ground corn, soybean meal, sunflower cake and minerals, and were used to supplement the forage, ground Tifton hay (3–4 cm), in a 50:50 proportion of forage: concentrate. The chemical composition of the diet ingredients (Table 1) and the experimental diets (Table 2) were evaluated according to AOAC (1990) for the estimation of dry matter (DM), ash, crude protein (CP) and ether extract (EE). The tests for the evaluation of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined according to Van Soest et al. (1991). Non-fibrous carbohydrates were calculated according to Mertens (1997). Diets were offered twice daily, at 9 am and 4 pm. The leftovers were weighted daily and the amount of food provided was adjusted to allow for 10% of the offered food remaining. Water was supplied ad libitum daily. The metabolizable energy (ME) was calculated following the NRC recommendations (2001), using the equations: DE = TDN×0.04409 (Mcal/kg) and ME = DE×0.082. Analyses were performed at the Laboratory of Animal Nutrition of the School of Veterinary Medicine and Animal Science of the Federal University of Bahia in Brazil. At the end of the experiment, animals with final body weight of 22±2.30 kg were fasted for 12 h and then weighed to determine the body weight at slaughter (BWS) and, later, the animals were stunned (electro) followed by bleeding, dehiding and evisceration. After the removal of the head and limbs, the carcasses were weighted to determine the hot carcass weight and the hot carcass yield was determined by the formula: HCW = (HCW/BWS)×100. The carcasses remained in cold storage (4°C) for 24 h and were then weighed to obtain the cold carcass weight (CCW). The biometric measurements were evaluated according to Yañez et al. (2004) as follows: carcass internal length (distance between the front edges of the pubic bone and the edge of the first ribs at its midpoint), carcass outer length (distance between the joint and cervicothoracic intercoccigeal first joint), leg length (distance between the greater trochanter and the edge of the tarsal-metatarsal joint), hind perimeter (perimeter in the croup, based on the trochanters of the femurs), leg perimeter (perimeter drawn on the middle part of the leg above the femoral-tibio-patellar articulation) and thoracic perimeter (perimeter measured behind the palette). The carcasses were longitudinally divided into two half carcasses, and the left half carcass was sectioned into five anatomical regions: neck (1st to 7th cervical vertebrae), shoulder (bone basis, scapula, upper arm and carpus), ribs (1st to 13th thoracic vertebrae), loin (longissimus lumborum muscle and dissected vertebral bones) and leg (section between the last lumbar and first sacral vertebrae). The cuts were individually weighed to determine the percentages that represented about half the weight of the remaining reconstituted carcass according Cezar and Souza (2007). The carcasses were cut between the 12th and 13th ribs for determination of the rib eye area (REA), which was made using a clean transparency sheet and a pen. REA cm2 was obtained after scanning the images. The carcass compactness index (cold carcass weight divided by carcass internal length) was calculated according Yañez et al. (2004). The muscularity, muscle: bone and muscle: fat ratios were obtained after dissection and weighing of the muscles lining the femur (biceps femoris, semitendinosus, adductor, semimembranosus and quadriceps) and the completely clean femur, using the formula described by Purchas et al. (1991): muscularity=WM5/FLFL in which WM5 is the weight (g) of the five muscles and FL is the femur length (cm). The proportion of muscle to bone was obtained by dividing the weight of the five above mentioned muscles by the weight of the femur. The experiment was a completely randomized design with four treatments and eight repetitions. The data were submitted to an analysis of variance (GLM procedure, SAS 2002) and the initial body weight was added as covariable for all variables analyzed. Orthogonal polynomial contrasts were used to examine responses (linear and quadratic) to an increase in the substitution levels of soybean meal by sunflower cake in the diets (REG procedure, SAS, 2002) with significance level of 5%. BODY.RESULTS AND DISCUSSION: The substitution of soybean meal by sunflower cake in the diet of goats did not affect (p>0.05) body weight at slaughter (Table 3). It is probable that the chemical composition of sunflower cake was responsible for the similarity among weights and treatments, because with increasing of substitution of the soybean meal by sunflower cake, it was observed that non-fibrous carbohydrate intake decreased and intake of neutral detergent fiber and ether extract increased (Aly Agy et al., 2011). This allowed the animals to present similar metabolizable energy intake, resulting in a similar body weight. Analyzing the average weight gain and feed conversion sunflower cake was found to decrease the average daily weight gain by 0,002 kg for each 1% inclusion of sunflower cake, while the feed conversion showed an increase, ranging from 5.36 to 7.58 kg dry matter by kg of weight gain (Aly Agy et al., 2011). Although these results were influenced by the substitution of soybean meal with sunflower cake, they did not promote a significant reduction in body weight at slaughter. Weights and carcass yields as well as rib eye area showed no differences (p>0.05) with the substitution of soybean meal by sunflower cake. As the addition of sunflower cake in the diet resulted in no differences in body weight between treatments, the variables of weight and rib eye area followed the same pattern, as according to Pereira Filho et al. (2008), animals of similar body weights imply similar carcass weight and rib eye area measures. The values of hot and cold carcass weight and rib eye area were close to those reported by Pereira Filho et al. (2008) for 1/2 Boer kids, slaughtered between 20 and 25 kg. According to Dias et al. (2008), an increase in the NDF content of the diet, such as that found in this study (Table 1), should result in a reduction in carcass yield, since the fiber content in the diet has a direct impact on the increased volume of the gastrointestinal tract, in addition to limiting consumption due to more time spent on fiber fermentation. The non- significant effect (p>0.05) for the carcasses in this work can be explained by the low physical effectiveness of the fiber, since the sunflower cake was provided ground which decreased fiber retention time in the gastrointestinal tract. This alternative feed may therefore be an option to circumvent the limitations caused by the consumption of a food supply high in NDF. The measurements showed no differences (p>0.05) with the substitution of soybean meal by sunflower cake (Table 4). These results support evidence by the anatomical harmony law proposed by Boccard and Dumont (1960), in which the body regions of the carcass are proportional in animals of similar weight and fat content. Similarly, the carcass compactness index was not affected by the addition of sunflower cake in the diet. The carcass compactness index is a measure related to cold carcass weight and carcass internal length. The substitution of soybean meal by sunflower cake did not influence these two variables, so the carcass compactness index followed the same pattern. The commercial cuts and yields were not influenced (p>0.05) by the substitution of soybean meal by sunflower cake (Table 5). These variables are related to the carcass weight, which remained constant with the increased substitution of soybean meal by sunflower cake. According to Boccard and Dumont (1960), in carcasses with similar weight and fat content, the body regions are in similar proportions. With an increase in carcass weight, cuts which develop earlier such as the leg and shoulder have decreased yield, and in goat carcasses from 30 kg, the yield of rib starts to exceed the yield of leg (Colomer-Rocher, 1992). This is undesirable behavior, since the leg is considered a first category cut because it is a region with higher muscle deposition and with little connective tissue content, which ensures softness and commercial valorization. Tissue composition, leg muscularity and the muscle: bone and muscle:fat ratios (Table 6) were not influenced (p>0.05), probably because the substitution of soybean meal by sunflower cake provided the same energy intake for animals of different treatments, resulting in similar weights, which was also the case for animals of similar ages. According to Madruga (1999) subcutaneous fat in goats is characteristically very thin, and the abdominal cavity is the main fat depot, where 50% to 60% of the total fat is located between the abdomen and viscera, and the major part of it is removed at evisceration. Some authors (Banskalieva et al., 2000; Grande et al., 2003) have already reported this particular goat carcass trait as desirable as it results in leaner carcasses compared with other animal species. The index values of leg muscularity found in this study were greater than the ones found by Dias et al. (2008) who worked with goats fed wheat bran; an increased amount of muscle was found on the goats fed with sunflower cake as well. The total percentage of muscle found in this work was also higher than that found by other authors (Table 6). This was probably because the animals in this study were slaughtered at a younger age, and were thus still in the process of muscle deposition. The muscularity index proposed by Purchas et al. (1991) considers the weight of the muscles surrounding a given length of a bone and the bone itself. According to Abdullah et al. (1993), muscularity has a better correlation with the scores of carcass conformation than the muscle: bone ratio. This may be explained by the fact that muscularity is obtained by the ratio between the weight of the five muscles and the length of the femur and not by the ratio of two weight measures. Similarly, the muscle:fat and muscle:bone ratios were not influenced by levels of sunflower cake in the diet, since the body weight at slaughter of the animals in different treatments were similar. These two variables are related to the weight and the age of the animal, since these are variables dependent on the development of the tissues in question. The muscle:bone ratio was lower than reported by Pereira Filho et al. (2008); however, the muscle:fat ratio was superior. The animals in this study probably achieved a weight of 20 kg earlier because these same authors state that bone tissue showed negative growth, indicating that increasing age and weight of the animal led to decreasing bone growth, even completely stopping, while the muscle tissue continued to develop, thus increasing the muscle: bone ratio. According to Rosa et al. (2002), leg fat will grow later, regardless of the feeding method, while the muscle growth is isometric, indicating that a higher muscle: fat relationship in this cut will be obtained in carcasses of younger animals. The variables in the muscle: bone and muscle:fat ratios are important in determining the proportions of the tissues for commercial cuts, and higher values of these variables mean a greater amount of muscle tissue and a higher commercial value. Based on the evaluation of the carcass traits, the substitution of soybean meal by sunflower cake in the diet of 1/2 Boer goats may be recommended up to a level of 100% without alterations to quantitative carcass traits.

TITLE: The status of serum γ-interferonand antiviral antibodies in patients with type I and type 2 diabetes: A comparative study ABSTRACT.OBJECTIVES:: There is an association between viral infection and development of diabetes mellitus. This study aimed to investigate the role of rubella virus, cytomegalovirus and coxsackievirus in patients with type 1 (T1D) and type 2 (T2D) diabetes mellitus in respect to the glycemic control and immune response presented by serum γ-interferon leveland antiviral antibodies. ABSTRACT.MATERIALS AND METHODS:: A total number of 160 (70 male and 90 female) T1D and 75 T2D (25 male and 50 female) patients allocated randomly from Martyr Layla Qasm center for diabetes mellitus in Erbil, Iraq, were enrolled in the study. Serum IgG antibody (I.U./mL) against rubella virus, cytomegalovirus coxsackievirus as well as serum interferon-g were determined. ABSTRACT.RESULTS:: Type-1 diabetic patients with positive anti-coxsakievirus antibody presented with significantly shorter duration of illness (4.822 ± 2.442 year) and poorer glycemic control (HbA1c %: 9.895 ± 1.272) This observation was not noticed with other viral infection as well as in T2D. Significant alterations in serum interferon-g (8.051 ± 13.371 pg/ml) were observed in T1D and related to coxasackievirus infection (13 patients had a level higher than 10.975 pg/ml; the upper limit of 95% C.I of control, and 34 had a level less than 4.457 pg/ml; the lower limit of 95% C.I of control). ABSTRACT.CONCLUSIONS:: Subjects with type 1 diabetes and Coxsackie infections seem to have a different immunological and clinical profile. This needs further study. BODY.INTRODUCTION: Viral infections such as mumps, rubella, enteroviruses, cytomegalovirus, rotavirus, and parvovirus have all been associated with human type-1 diabetes (T1D).[12] An association between diabetes and virus infection was first made in 1864 in a patient who developed the disease following mumps infection. It is now known that the ssRNA enveloped mumps virus is capable of infecting islet and pancreatic cells in vitro and in vivo, respectively, and mediating direct beta cell cytolysis.[3–5] Similarly, rubella virus was first associated with human T1D in 1969. Additionally, cytomegalovirus (CMV) infection was linked to the development of T1D in 1979. The mechanisms by which viruses implicated in pathogenesis of T1D include: first, direct infection of beta cells which resulted in beta cell lysis and release of self-antigens which are picked up by antigen presenting cells (APCs) that in turn activate self-reactive lymphocytes that mediate beta cell destruction, leading to the expression of hyperglycemia.[13] Second, viral infection of APCs may cause an increased expression of cytokines that activate self reactive lymphocytes, or directly mediate beta cell cytolysis.[3] Third, viral antigens with homology to self-epitopes cross react, leading to the activation of self-reactive lymphocytes that mediate beta cell destruction i.e. 'molecular mimicry'.[6] Finally, in experimental animal models, viral infections may cause a transient lymphopenia that disturbs the equilibrium between selfreactive lymphocytes and regulatory T lymphocytes, tipping the immune balance toward an autoimmune environment.[7] There are increasing reports of association between hepatitis C and type-2 diabetes (T2D),[89] but there is no evidence of association between rubella, cytomegalovirus or coxsacki B viral infection and T2D. This study is aimed to compare the sero-positive T2D and T1D patients toward rubella virus, cytomegalovirus and coxsackievirus in respect to the glycemic control and g-interferon in a small sample of patients lived in the Kurdistan, north of Iraq. BODY.MATERIALS AND METHODS: This cross-sectional study was conducted in Martyr Layla Qasm center for diabetes mellitus in Erbil, Iraq during the period of 1st of August 2008 to 30 December 2009. The study was approved by the local scientific committee of college of Pharmacy, Hawler Medical University. A consent form was obtained from each participant prior to the study. A total number of 160 (70 male and 90 female) T1D and 75 T2D (25 male and 50 female) patients allocated randomly (using randomized tables) from patients attended the diabetic center over the period of sixteen months were enrolled in the study. Fasting venous blood samples were obtained from participants and the sera were separated for determination of glucose, glycosylated hemoglobin (HbA1c %). ELISA-based determination of serum IgG antibody (I.U./mL) against rubella virus, cytomegalovirus coxsacki virus were used. The concentration of antibodies at the cut-off absorbance were: 15 I.U./mL (absorbance 2 at λ 450nm), 1.2 I.U./mL (absorbance 1.2 at λ 450nm) and 100 I.U./mL (absorbance 1.5 at λ 405nm) against rubella virus, cytomegalovirus and coxsacki virus respectively. The serum antibody concentration was calculated according to the following equation.[10]: Also the serum immunoglobulin M(mg/dl) is determined by ELISA Interferone-γ was determined in serum using enzyme linked immunosorbent assay (ELISA) technique. In brief, serum samples were added into the wells, incubated with shaking at 37°C for 2 h, then washed and biotinylated antibody and streptavidin-HRP conjugate were added in consequence. After 30 min incubation, the wells were washed and the substrate was added, incubated with shaking at room temperature for 20 min followed by adding stopping solution and then the absorbance was read at wavelength 450 nm. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: The results are expressed as number, percent and mean ± SD. The data had normal distribution and were analyzed using two tailed unpaired Student's "t" test, and 95% confidence intervals (95% C.I.) test taking P≤ 0.05 as the lowest limit of significance. BODY.RESULTS: Table 1 shows that the age of T1D patients presented with antibody against coxsackievirus is significantly less than corresponding age of T1D patients with negative anti-coxsackie virus antibody. Such observation is not detected in patients with T2D who had anticoxackie virus antibody [Table 2]. Type -1 diabetic patients with positive anti-coxsakievirus antibody presented with significant short duration of illness (4.822 ± 2.442 years, P< 0.01) while those with anti-rubella or anti cytomegalovirus antibody did not show significant difference from corresponding patients without positive antibody [Table 1]. Such observation again not demonstrated in patients type-2 diabetes [Table 2]. Although the fasting serum glucose of T1D patients was higher than corresponding T2D which amounted approximately 1.5 fold but the difference in fasting serum glucose in each type of diabetes did not show significant differences regarding the presence of antibody against any studied virus [Tables 1 and 2]. The HbA1c % as indicator of glycemic control was significantly higher (which indicated poor glycemic control) in T1D patients with positive antibody against coxsackie virus (9.895 ± 1.272%) [Table 1]. In T2D patients with positive anti-coxsackie virus antibody, the HbA1c % is non significantly lower than corresponding patients without antibody [Table 2]. The serum level of interferon-g shows variations in both T1D and T2D patients which reflected in non significant differences [Tables 1 and 2]. Further statistical analysis using 95% C.I. revealed that all T1D patients who have positive antibody against virus, having either significant low or high antibody level [Table 1]. More than 70% of T1D patients with positive anti-coxsackie or anti-cytomegalovirus antibody have significant low serum interferon-γ[Table 1]. [Table 2] shows that T2D patients with positive anti-viral antibody required less doses of insulin compared with those with negative anti-viral antibody. There are no significant differences between serum level of IgM between patients with T1D and T2D (181.1 ± 58.8 vs 183.8 ± 54.1 mg/dl respectively) and these values are less than reference value of healthy subjects in the laboratory (219 ± 44 mg/dl). Table 1 Characteristics of type-1 diabetes (T1D) patients Table 2 Characteristics of type-2 diabetes (T2D) patients BODY.DISCUSSION: The results reported here show that certain Coxsackievirus is one of the many factors that involved in the glycemic control and in the immune response (presented with serum interferon-γ in T1D and not in T2D). It is well known that T1D occurs in patients with congenital rubella.[11] However, most of these patients have the HLA and immune markers characteristic of type 1 diabetes. In addition, Coxsackievirus B, cytomegalovirus, adenovirus, and mumps have been implicated in inducing certain cases of the disease. It is generally believed that the environmental agents trigger disease development in genetically susceptible individuals.[12–14] In this study, the viruses, though they may not have been directly shown to be implicated in inducing T1D have been probably linked to an alteration in the glycemic control as evidenced by the short duration of illness and high percent of HbA1c (%). This observation is not found in T2D and this supports the previous suggestion that viruses may trigger the disease in susceptible individuals. The significant short duration of illness in T1D is in agreement with the Horwitz et al. study who reported experimentally that a Coxsackievirus B4 infection accelerates diabetes development in transgenic mice.[15] Recently, it has been observed that interferon-γ production is critical for the mechanism by which a coxasackivirus B4 infection accelerates the progression to overt diabetes in transgenic mice and this explain the significant alteration in serum interferon-γ of T1D patient.[16] Further evidence about the interaction of interferon-g with Coxasackievirus infection was reported by the one who found that Coxsakievirus B infection triggers the production of interferon-γ.[17] This study adds a further observation that the changes in interferon-γ are associated with significant poor glycemic control. In T2D patients, only 4 cases out of 75 patients have positive anti-coxasackievirus antibody and this factor among many factors explains the non significant changes in duration of illness and glycemic control in T2D. This study points out the role of Coxasackievirus in poor glycemic control in T1D while its role in T2D is negligible. In fact T2D patients with Coxasackievirus infection required less recommended dose to control their glycemia than corresponding patients without viral infection. One of the limitations of the study is determination of the proinflamatory markers that indicate an association between viral infection and diabetes. The other limitation of the study is related to the many factors involved in using high doses of insulin in patients with positive antiviral antibodies; therefore logestic regression could be a useful model to avoid this problem, but the sample size is small. It may be concluded that the clinical profile of Coxsackievirus antibody associated type 1 diabetes is an area where future research should be carried out, and this may have implications for the better management of these patients.

TITLE: A Double-Blind Study on Acupuncture Sensations with Japanese Style of Acupuncture: Comparison between Penetrating and Placebo Needles ABSTRACT: To investigate the acupuncture sensations elicited by the Japanese style of acupuncture, penetrating acupuncture and skin-touch placebo needles were randomly administered at various insertion depths (5 and 10 mm for the penetrating needles and 1 and 2 mm for the placebo needles) at LI4 to 50 healthy subjects. Among the 12 acupuncture sensations in the Massachusetts General Hospital Acupuncture Sensation Scale (MASS), "heaviness" was the strongest and most frequently reported sensation with the 10 mm needles, but not with the 5 mm needles. There were no significant differences in number of sensations elicited, MASS index, range of spreading, and intensity of needle pain for 5 mm penetration versus 1 mm skin press and 10 mm penetration versus 2 mm skin press. The MASS index with 2 mm skin-touch needles was significantly larger than that with 1 mm skin-touch and 5 mm penetrating needles. The factor structures in the 12 acupuncture sensations between penetrating and skin-touch needles were different. The acupuncture sensations obtained in this study under satisfactorily performed double-blind (practitioner–patient) conditions suggest that a slight difference in insertion depth and skin press causes significant differences in quantity and quality of acupuncture sensations. BODY.1. INTRODUCTION: De qi, a characteristic constellation of sensations felt by patients during acupuncture needling, has long been regarded as an important factor related to clinical effects [1]. For the last several decades, much effort has been made to investigate the quantity and quality of acupuncture sensations in detail. Acupuncture sensation questionnaires such as the Acupuncture Sensation Scale (ASS) [2], the Subjective ASS (SASS) [3], the Massachusetts General Hospital ASS (MASS) [1], and the Southampton Needle Sensation Questionnaire (SNSQ) [4] have been developed as tools to objectively scrutinise acupuncture sensations. Using these questionnaires, acupuncture sensations elicited by manual acupuncture, electroacupuncture [3, 5], and various other manipulations [6] have been studied. Also, comparisons of acupuncture sensations among acupoints [2, 3, 7] and between acupuncture-experienced and acupuncture-inexperienced subjects [8, 9] have been made. The Standards for Reporting Interventions in Clinical Trials of Acupuncture currently recommend reporting de qi in clinical studies [10, 11]. Acupuncture sensations are an essential factor in inducing an analgesic effect [3] and improving local blood flow in the skin and muscles [12, 13]. The relationship between acupuncture sensations and analgesic effects has been clearly demonstrated in clinical trials of acupuncture treatment on epicondylalgia [14] and osteoarthritis of the knee [15, 16]. In recent randomised controlled trials, acupuncture sensation questionnaires were utilised to examine the relationship between acupuncture sensations and efficacy of acupuncture treatment [3, 17–25]. In some of these studies, acupuncture sensations reported on the MASS with placebo/sham acupuncture needles were analysed [17, 22–25] in order to better understand acupuncture-specific sensations, as well as the mechanism of acupuncture treatment. However, there have been several limitations to these studies. First, the studies were single-blinded, with the practitioner being aware of which needle was being applied. Second, practitioners did not keep the insertion depth, and in the case of placebo needles, skin press, constant throughout the trials. Also, the methods of acupuncture used in most of the above studies were of the Chinese style, limiting their validity in other styles, such as the Japanese style of acupuncture. Almost all of the studies that investigated acupuncture sensations were performed using 0.2–0.38 mm diameter needles [2, 3, 6, 7, 9, 12–15, 19–21], which are relatively large diameter needles not usually used in Japanese acupuncture. Unlike Chinese acupuncture, Japanese acupuncture typically uses thin needles, 0.16–0.18 mm in diameter [26], with a shallow insertion of <5 mm in depth [27] and a tapping-in method to penetrate the skin using a guide tube [28]. Although there have been clinical studies that employed the Japanese style of acupuncture to investigate de qi, patients reported only whether they felt de qi, as well as a dull sensation, and a skin-penetration sensation [29–32]. To the best of our knowledge, no report has used acupuncture sensation questionnaires to analyse acupuncture sensations with the Japanese style of acupuncture. In this study, we investigated acupuncture sensations elicited with needles in the Japanese style of acupuncture. The study was double-blinded (practitioner–patient) and insertion depth and skin press were kept constant. To measure the sensations, we translated the MASS into Japanese (Japanese MASS) and reported criterion-related validity, construct validity, and reliability [33]. At present, MASS is the most plausible candidate for a standard tool since it is the most frequently applied scale in clinical studies in English-speaking areas. Further, it has already been translated into Chinese, and this version has been validated for clinical use [34]. The aim of this study was to investigate and compare acupuncture sensations elicited with penetrating needles inserted 5 and 10 mm deep and with nonpenetrating, skin-touch placebo needles pressed 1 and 2 mm deep. BODY.2. METHODS.2.1. STUDY DESIGN AND PARTICIPANTS: The study design was a randomised, double-blinded (practitioner–patient), and crossover clinical trial comprising four conditions. Overall, 50 healthy subjects (32 males, 18 females, aged 22.3 ± 7.5 years) who had normal Japanese language ability and no nervous system disorders were recruited. All subjects had acupuncture experience and knowledge about acupuncture, including acupuncture sensations or de qi. One acupuncturist with 12 years of acupuncture treatment experience (male, aged 35 years) applied the treatment. The acupuncture treatment was performed in a laboratory at Tokyo Ariake University, Tokyo, Japan. The laboratory was maintained at 24°C–26°C. The study was approved by the Ethics Committee of the Tokyo Ariake University of Medical and Health Sciences (approval number 198). The study objectives and protocol were explained to each participant using a written form, and all participants provided written consent. BODY.2. METHODS.2.2. INTERVENTIONS: We used penetrating acupuncture needles and skin-touch placebo needles designed for double-blinding (practitioner–patient) [28–31, 35–37] (Figure 1). For each subject, we prepared 5 mm penetrating needles to insert 5 mm from the body surface, 10 mm penetrating needles to insert 10 mm from the body surface, 1 mm skin-touch needles to press into the skin 1 mm with a protruding blunt tip, and 2 mm skin-touch needles to press into the skin 2 mm with a protruding blunt tip. The appearances of the four needles were indistinguishable. The diameter of the needles was 0.18 mm. Based on our previous study using ordinary acupuncture needles [33], the needles were applied at LI4 (large intestine meridian) [38], the most frequently used point in acupuncture research [2, 3, 7–9, 22, 32, 33], on the right or left hand with a rotating technique until the needle handle made contact with the top of the opaque tube, which was followed by the tapping-in method [28]. After needle insertion, the acupuncturist rotated the needle by 180 degrees clockwise and anticlockwise alternately at 1 Hz for 30 s [5, 8, 9, 39, 40] and then removed it immediately. BODY.2. METHODS.2.3. PROCEDURE: Fifty sets of four needles (a 5 mm penetrating needle, a 10 mm penetrating needle, a 1 mm skin-touch needle, and a 2 mm skin-touch needle) were sealed in bags for sterilisation, and then they were sterilised. Before applying the needles, the needle tips were set just above the bottom of a guide tube for penetrating needles and just above the lower stuffing for skin-touch placebo needles. Before the trial, all 50 subjects and the acupuncturist were informed that penetrating and/or skin-touch needles would be used. The subjects lay on their backs with their arms resting along their sides. The acupuncturist disinfected each subject's skin between the first and second metacarpal bones on the back of both hands with ethanol. Then he picked a needle randomly from the set of four and applied the needle at LI4. After the first needle, one of the three remaining needles was applied at LI4 on the other hand. In the second trial after a one-week interval, he applied the remaining two needles at LI4 on both hands in the same way as in the first trial. To exclude bias, we randomly assigned subjects to right or left LI4 for the first needling using a web-generated table of random numbers (http://randomization.com). In the second trial, right and left LI4 were applied in the reverse order to the first trial. As a result, the four types of the needles were applied to both hands in random order (chi-square test, p = 0.27). Immediately after removal of each needle, subjects completed the following questionnaires: (1) the Japanese MASS, on which subjects rated the intensity of each acupuncture sensation corresponding to the 12 descriptors on a numerical rating scale (NRS) from 0 (none) to 10 (strongest imaginable); if the subjects felt other sensations besides the 12 descriptors, they reported the additional sensation and rated it on the NRS; (2) a human figure to shade the range of spreading of acupuncture sensation to be evaluated as 0 (none), 1 (localized), 2 (digit/wrist), 3 (lower forearm), 4 (upper forearm), and 5 (beyond) according to the MGH Acupuncture Sensation Spreading Scale [1]; and (3) the visual analogue scale (VAS) to evaluate needle pain, on which subjects rated the intensity of pain from 0 (no pain) to 100 (most severe pain, unbearable). To evaluate the degree of subject and practitioner blinding, the subjects and acupuncturist were asked to record whether they thought the needle was "penetrating," "skin-touch," or "unidentifiable." They then rated their confidence in their guess on a 100 mm VAS for confidence from 0 (no confidence) to 100 (complete confidence) [28, 30, 36, 37]. The acupuncturist and subjects also reported adverse events during each needling, if any. BODY.2. METHODS.2.4. STATISTICAL ANALYSIS: The Friedman test and Dunn's multiple comparison test among and between groups were conducted for the numbers and intensities of the 12 descriptors in the MASS, MASS indices (the magnitude of overall acupuncture sensations) [1], range of spreading, intensities of needle pain, and subjects' confidence. Frequencies of the 12 descriptors were compared among and between the four types of needles with Cochran's Q test and Dunn's multiple comparison test. For subjects' guesses, Kappa coefficients for 5 mm penetrating needles versus 1 and 2 mm skin-touch needles and for 10 mm penetrating needles versus 1 and 2 mm skin-touch needles were calculated. Further, Bang's blinding index (BI) for each type of needles was calculated by the numbers of guesses. The Mann–Whitney U test was used to identify differences in intensities for the 12 MASS descriptors and degrees of spreading between subjects who guessed a needle they received as "penetrating" and subjects who guessed a needle they received as "skin-touch." To evaluate factor structures of acupuncture sensations, we performed an exploratory factor analysis according to the Japanese MASS ratings with penetrating and skin-touch needles. Furthermore, to estimate which acupuncture sensations in the MASS were related to subjects' guesses at needle authenticity, we added "penetrating" in subjects' guesses as a variable and performed an exploratory factor analysis. A principal component extraction method was used in the factor analyses. Factors with eigenvalues greater than 1.0 were extracted and a varimax rotation involving Kaiser normalisation below 25 iterations for convergence was performed. Statistical analyses were performed using SPSS Version 24 (IBM Japan, Ltd., Tokyo, Japan). BODY.3. RESULTS.3.1. NUMBERS OF ACUPUNCTURE SENSATIONS ELICITED, MASS INDEX, SPREADING OF ACUPUNCTURE SENSATION, AND INTENSITY OF NEEDLE PAIN WITH EACH TYPE OF NEEDLES: The upper rows in Table 1 show the numbers of acupuncture sensations elicited with each type of needles, the MASS index, range of spreading, and the intensity of needle pain. The upper rows in Table 2 show the comparisons among the four needles for each index. There were no significant differences in the four indices between 5 mm penetration and 1 mm skin press and between 10 mm penetration and 2 mm skin press. The rates of occurrence and intensities of each of the 12 acupuncture sensations are shown on the left and right in Figure 2, respectively. More than 50% of subjects felt "deep pressure" and "tingling" with all four types of needles. "Heaviness" was statistically characterised by both occurrence frequency and intensity with 10 mm penetration, but not with 5 mm penetration, 1 mm skin press, or 2 mm skin press. The 2 mm skin-touch needles tended to induce greater "tingling," "sharp pain," and "aching" compared with the 1 mm needles. BODY.3. RESULTS.3.2. RELATIONSHIP BETWEEN SUBJECTS GUESS AND INTENSITY OF ACUPUNCTURE SENSATIONS: The intensities of "heaviness" and "aching" with penetrating needles guessed as "penetrating" were significantly higher than those with penetrating needles guessed as "skin-touch" ("heaviness": p = 0.001, "aching": p = 0.015). As for skin-touch needles, the intensities of "heaviness," "aching," "dull pain," and "soreness" for needles guessed as "penetrating" were significantly higher than those guessed as "skin-touch" ("heaviness": p = 0.002, "aching": p = 0.015, "dull pain": p = 0.027, and "soreness": p = 0.030). No significant difference in the intensity of "sharp pain" was revealed between correct and incorrect guesses by subjects (penetrating needles: p = 0.087, skin-touch needles: p = 0.310). BODY.3. RESULTS.3.3. FACTOR ANALYSIS: Tables 3 and 4 show the results of factor analysis in the 12 acupuncture sensations with penetrating and skin-touch needles, respectively. Factors 1, 2, and 3 of penetrating needles were inferred as "deep dull-heavy sensation," "no pain sensation," and "superficial pain sensation." Factors 1, 2, and 3 for skin-touch needles were inferred as "superficial pain sensation," "deep dull-heavy sensation," and "thermal sensation." Factor 4 of skin-touch needles, which was a prominent difference from penetrating needles, was inferred as "soreness." To explore the association between MASS sensations and subjects' guesses at needle authenticity, we added "penetrating" in subjects' guesses as a variable and performed an exploratory factor analysis. Table 5 shows the results of factor analysis that include "penetrating" in subjects' guesses as an additional variable in the 12 acupuncture sensations. "Penetrating" in subjects' guesses was categorised as the third factor with "heaviness," "aching," and "dull pain." BODY.3. RESULTS.3.4. BLINDING ASSESSMENTS OF NEEDLES: The lower rows in Tables 1 and 2 show BIs for subjects' and practitioner's guesses, confidence in guesses, and the Kappa coefficients between needles applied (penetrating or skin-touch needles) and subjects' or practitioner's guesses (penetrating or skin-touch). For the practitioner, the BIs of each type of needles were close to zero and his confidences were very low, which indicates that his guesses were random. For the subjects, although the blinding statuses of the 10 mm penetrating needles and the 1 mm skin-touch needles were assessed as "unblinded," there were no significant differences among the four types of needles in their confidences and no significant difference in subjects' confidence between correct and incorrect guesses for both penetrating (p = 0.155) and skin-touch (p = 0.819) needles, which indicates that the subjects could not identify the nature of the needle. Except for subject blinding for 10 mm penetrating needles versus 1 mm skin-touch needles, the Kappa coefficients indicated that the degree of agreement was poor for both the subjects and the practitioner. BODY.3. RESULTS.3.5. ADVERSE EVENTS: Dot haemorrhage occurred in three cases with the 10 mm penetrating needle. There were no critical adverse events. BODY.4. DISCUSSION: In this study, we applied double-blind 5 and 10 mm penetrating needles and 1 and 2 mm skin-touch needles to investigate acupuncture sensations with the Japanese style of needling using Japanese MASS. We found that "heaviness" was a distinctive feature for 10 mm needle penetration. The total number of sensations elicited, the MASS index, range of spreading, and the intensity of needle pain with the 5 and 10 mm penetrating needles were similar to those with the 1 and 2 mm skin-touch needles, respectively. However, the factor structures of acupuncture sensations were different between penetrating and skin-touch placebo needles, implying that acupuncture penetration and insertion depth may be associated with different sensation patterns. Literature suggests that the depth of needle insertion, that is, shallow (minimal/superficial) or deep insertion, affects acupuncture sensation [41–44] and efficacy [45–48]. This indicates that the depth of needle insertion is an influential factor in determining the sensation and efficacy of acupuncture and that insertion depth should be controlled when investigating acupuncture sensations. In this study, we applied Takakura needles for double blinding and for maintaining a fixed insertion/touch depth [35]. The needles for double blinding can blind patients and practitioners to exclude any bias from psychological factors. Previous studies suggested that acupuncture sensations can be elicited by phantom acupuncture or sham laser acupuncture, which implies that patient-practitioner interaction may alter acupuncture sensations [49, 50]. Therefore, we believe that the acupuncture sensations obtained in this study were unbiased in terms of technique. "Heaviness" was revealed as a distinctive sensation with 10 mm penetration insertion (Figure 2). The results were similar to those of our previous study using Japanese MASS, in which 10 mm penetration with ordinary needles of 0.18 mm diameter was applied [33]. Literature suggests that "heaviness," "aching," "dull pain," and "soreness" are all regarded as important acupuncture sensations with manual acupuncture [3, 7, 17, 51]. We found that the sensations of "aching," "dull pain," and "soreness" from 2 mm skin-touch needles were not different in frequency and/or intensity from 10 mm penetrating needles (Figure 2). Furthermore, although spreading of acupuncture sensations has been considered as one of the specific characteristics of de qi [18, 52–54], the spreading with 2 mm skin-touch needles was similar to that with 10 mm penetrating needles (Table 1). Also, despite the finding that stimulating the fascia and muscles was reported to elicit "dull," "heavy," and "spreading" sensations frequently [43], our results indicated that only "heaviness" was exclusive to deep needle insertion. Similar occurrences of "deep pressure" and "tingling" with the four types of needles meant that these sensations were also not exclusive to 10 mm penetrating needles. With 5 mm penetrating needles, "tingling" and "deep pressure" were as frequently reported as with 1 and 2 mm skin-touch needles, and "heaviness" was much less elicited compared with 10 mm penetration (Figure 2). Despite needle insertion, "heaviness" was not a specific sensation with 5 mm penetration. Acupuncture sensations with 5 mm penetrating needles were similar to those with 1 and 2 mm skin-touch needles, which endorses the potential feasibility of blinding patients using these needles. The differences in acupuncture sensation between 10 mm penetration (deep insertion) and 5 mm penetration (shallow insertion) indicate that a slight difference in insertion depth should not be ignored when acupuncture sensations are evaluated. The majority of indications for acupuncture sensations with 10 mm penetration were higher than those with 5 mm penetration in this study, suggesting that acupuncture sensations with shallow insertions tend to be weaker than those with deep insertions [41, 42, 44]. If the needle grasp felt by acupuncturists as de qi is due to mechanical coupling of the needle with connective tissue [55], a needle depth of 10 mm should contact larger connective tissues than that of 5 mm. This may explain why the 5 mm penetration did not elicit "heaviness," which was exclusive to deep insertion, if the de qi felt by acupuncturists is assumed to be simultaneously felt by patients. The tip of the 2 mm skin-touch needle is estimated to reach the subcutaneous tissues according to an MRI study performed at LI4 [56]. The stimulation of such superficial tissues with a 2 mm skin press may produce strong sensations. The 2 mm skin-touch needles could be a reliable placebo control for 10 mm penetrating needles because of the similarities in number of sensations elicited (except for "heaviness"), MASS index, range of spreading, and intensity of needle pain between these needles. Interestingly, we found that acupuncture sensations elicited with 1 mm skin-touch needles were quite similar to those with 5 mm penetrating needles as determined by similarities in number of sensations elicited, MASS index, range of spreading, intensity of needle pain, and the occurrence and intensity of every descriptor of the acupuncture sensations. These results suggest that 1 mm skin-touch needles could be a reliable placebo control for 5 mm penetrating needles. We found that the intensity of acupuncture sensations was entirely different, especially aching, tingling, and sharp pain, between 1 and 2 mm skin-touch needles. This may provide insight into some inconsistent results in other studies using other superficial placebo needles. Several results on acupuncture sensations with single-blind skin-touch needles have been reported. For instance, White et al. investigated acupuncture sensations in patients suffering from hip or knee pain between Streitberger placebo needles and penetrating acupuncture needles and concluded that the Streitberger placebo needle is a convincing control based on similarities in intensity for "dull," "radiating," "stinging," and "electric" sensations [20]. Liang et al. reported that pain scores with sham needle applications using the Park device were larger than those with real penetrating needles [40]. In contrast, it was reported that the intensities of "aching" with verum needles, which is considered to be equivalent to pain [17, 22], were higher than those with the Streitberger placebo needles. Further, it was reported that the intensities of acupuncture sensations with single-blind placebo needles were considerably dependent on needling by practitioners [20, 57]. Taken together, these studies call for a better way to control the depth/pressure of the nonpenetrating placebo needle in acupuncture research. Using factor analyses of the 12 acupuncture sensations on the Japanese MASS, the construct validity of MASS was confirmed using double-blinded, controlled-depth needles. Considering that all subjects had experienced acupuncture in addition to the double blinding of this study, an extraction of the three factors latent in the 12 descriptors, including a factor of sensations unrelated to pain such as "warmth," "numbness," and "fullness/distension," was more valid and reliable than the extraction of the two factors revealed in our previous, unblinded study, in which half of the subjects had not experienced acupuncture. Furthermore, this is the first factor analysis of acupuncture sensations with skin-touch placebo needles under practitioner blinding. The factor structure of acupuncture sensations with real penetrating needles was different from that with skin-touch placebo needles. For instance, although "soreness," which was categorised as a "dull-heavy" sensation, was reported to be one of the particular sensations experienced with real penetrating needles [17], the soreness elicited with skin-touch placebo needles in this study was not involved in the "dull-heavy" factor. It is worth noting that patient blinding was almost successfully conducted from the Kappa coefficients and subjects' confidence. Also, successful blinding for the practitioner was confirmed with 53.5% unidentified needles in addition to the Kappa coefficients and blinding indices. These results indicate that the methods used in this study have removed much of the bias present in previous studies. Consistent with previous observations that "sharp pain" is frequently elicited by acupuncture stimulation in the epidermis, dermis, and shallow subcutaneous tissue [43], "sharp pain" was the most predominant sensation with 2 mm skin-touch needles in this study. In a previous study, for subjects who believed that the phantom acupuncture they received was real acupuncture, the intensities of "dull pain" and "heaviness" were similar to those with real needles, but those of "sharp pain" and "tingling" were less than those with real needles [49]. In the current study, however, no significant difference in the intensity of "sharp pain" was revealed between correct and incorrect guesses by subjects. "Sharp pain" was not a distinguishing cue for the subjects. To be more precise, the decisive cues for subjects for identifying the needles as "penetrating" seemed to be "aching," "dull pain," and "heaviness" from the factor analysis, including subjects' guesses as additional variables. This was the first study to investigate acupuncture sensations with penetrating needles and skin-touch placebo needles while keeping the depth of insertion and skin press constant. The results obtained in this study suggest that a slight difference in the depth of needle insertion and skin press significantly influences the quantity and quality of acupuncture sensations, which indicates a future direction of studies on acupuncture sensations. Double-blind needles [35] must play a significant role in future clinical acupuncture trials. There are several limitations in this study. Only one acupuncturist participated in this study. We thus cannot test interpractitioner credibility in this experiment. Also, we only recruited healthy subjects. Acupuncture sensations in patients are reported to be different from healthy subjects [53, 58]. Future studies are needed to evaluate the sensations of different types of needles in patient populations. BODY.5. CONCLUSION: Potential factors in the 12 acupuncture sensations on the Japanese MASS were different between double-blinded penetrating and skin-touch placebo needles. The strongest and most frequent sensation observed was "heaviness" for 10 mm penetration. The total number of sensations elicited, the MASS index, range of spreading, and the intensity of needle pain with 1 and 2 mm skin-touch needles were similar to those with 5 and 10 mm penetrating needles, respectively. A slight difference in the depth of insertion and skin press caused significant differences in the intensity and quality of acupuncture sensations.

TITLE: Quality of care for remote orthopaedic consultations using telemedicine: a randomised controlled trial ABSTRACT.BACKGROUND: Decentralised services using outreach clinics or modern technology are methods to reduce both patient transports and costs to the healthcare system. Telemedicine consultations via videoconference are one such modality. Before new technologies are implemented, it is important to investigate both the quality of care given and the economic impact from the use of this new technology. The aim of this clinical trial was to study the quality of planned remote orthopaedic consultations by help of videoconference. ABSTRACT.METHOD: We performed a randomised controlled trial (RCT) with two parallel groups: video-assisted remote consultations at a regional medical centre (RMC) as an intervention versus standard consultation in the orthopaedic outpatient clinic at the University Hospital of North Norway (UNN) as a control. The participants were patients referred to or scheduled for a consultation at the orthopaedic outpatient clinic. The orthopaedic surgeons evaluated each consultation they performed by completing a questionnaire. The primary outcome measurement was the difference in the sum score calculated from this questionnaire, which was evaluated by the non-inferiority of the intervention group. The study design was based on the intention to treat principle. Ancillary analyses regarding complications, the number of consultations per patient, operations, patients who were referred again and the duration of consultations were performed. ABSTRACT.RESULTS: Four-hundred patients were web-based randomised. Of these, 199 (98 %) underwent remote consultation and 190 (95 %) underwent standard consultation. The primary outcome, the sum score of the specialist evaluation, was significantly lower (i.e. 'better') at UNN compared to RMC (1.72 versus 1.82, p = 0.0030). The 90 % confidence interval (CI) for the difference in score (0.05, 0.17) was within the non-inferiority margin. The orthopaedic surgeons involved evaluated 98 % of the video-assisted consultations as 'good' or 'very good'. In the ancillary analyses, there was no significant difference between the two groups. ABSTRACT.CONCLUSIONS: This study supports the argument that it is safe to offer video-assisted consultations for selected orthopaedic patients. We did not find any serious events related to the mode of consultation. Further assessments of the economic aspects and patient satisfaction are needed before we can recommend its wider application. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00616837 ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12913-016-1717-7) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: Patients need secondary care consultations after referrals from their general practitioners (GPs), or they need follow-up consultations for earlier treatment or for chronic disease. According to the health authorities in Norway, it is a public responsibility to provide necessary health and care services to the entire population regardless of place of residence. Decentralised services using outreach clinics or modern technology are methods to reduce both patient transports and costs to the healthcare system [1]. The University Hospital of North Norway (UNN) is the tertiary referral hospital for the North Norway regional health trust, covering approximately 470,000 inhabitants (2012) and an area of 112,975 km2. UNN is also the local hospital for Troms and northern Nordland County, covering 187,000 inhabitants (2012) and an area of 31,500 km2. In 2014, the trust's expenses for patient travel accounted for 3.2 % of the hospital's total budget, not including expenses for ambulance transport by car, boat or air [2]. As one of the outpatient clinics with the highest number of patients, many of whom need assistance by accompanying persons when travelling or who are not able to use public transport, decentralising orthopaedic outpatient consultations is of special interest. Telemedicine equipment is improving rapidly with regard to quality, cost and user-friendliness; these, together with the distribution of high-speed telecommunication networks, may make it tempting to implement this new technology without further investigation. However, before new methods in healthcare delivery are implemented, it is important to investigate the quality and safety of the care given as well as the economic impact of such innovation to discover any pitfalls and reduce unwanted events. An earlier non-randomised study demonstrated good accuracy by telemedicine-assisted consultation for trauma management compared to standard consultations [3]. A randomised controlled trial (RCT) found telemedicine capable of providing a satisfactory standard of care in the management of minor injuries [4]. Another RCT evaluated patients coming to an emergency department and found telemedicine to be a satisfactory treatment technique [5]. Others suggest that telemedicine is an alternative to conventional visits for orthopaedic patients in an outpatient setting [6–9]. In one study, real-time videoconference was found to suitably provide orthopaedic care to rural areas; however, further investigations, including a cost–benefit analysis, were recommended [10]. Also, telehealth via real-time videoconference was reported to be effective by connecting an Antarctic station and Japan to treat orthopaedic cases [11]. However, there are few randomised studies regarding telemedicine and orthopaedic patients, none of which were conducted in Norway [12–16]. Some of the earlier studies demonstrated the importance of transmitting X-ray images of adequate quality as a factor to improve telemedicine for remote orthopaedic consultation; this was performed with a separate document camera [8, 17]. The X-ray system at UNN is fully digitalised: digital images taken at one location are electronically available at other locations within the hospital trust. From this background, the aim of this RCT was to study the quality of remote telemedicine consultations in an outpatient clinic as compared to ordinary consultations. The study is reported according to the consort 2010 guidelines [18]. Telemedicine in this study means the use of real-time videoconference and digitalised X-rays. Our study hypothesis was as follows: The introduction of telemedicine service in the form of real-time videoconference for the selected orthopaedic patients will cause no reduction of the professional quality of the patient treatment administered by the doctor involved in the consultations; it will also increase patient satisfaction and lower costs. The study hypothesis examines the non-inferiority of telemedicine consultation versus conventional outpatient consultation. Our choice of a non-inferiority trial design was based on the expectation that a slightly lower-quality score of the evaluation by the physician of the video-assisted consultations would be compensated by increased patient satisfaction and/or reduced travel expenses. In this paper, we present the method of the study and the analyses of the professional quality of the patients' treatment. BODY.METHODS: This RCT featured two parallel groups that were allocated into remote consultations at a regional medical centre (RMC) (3.5 h by car from Tromsø) as an intervention and into standard consultations in the orthopaedic outpatient clinic at UNN as a control. BODY.METHODS.TECHNICAL EQUIPMENT: At RMC, a screen (ViewSonic, Modl nr VS10946-Ie) with a codec and camera situated on top (Tandberg 990MXP) was installed. The orthopaedic surgeon at UNN controlled the camera, which could be used to zoom in on the patients (to look at a post-operative wound) or follow them when walking, for example. At UNN, in a standard outpatient clinic room, another camera, codec (Tandberg 1500MXP) and similar screen were installed. These were connected to a standard PC to show the X-rays to the patient if he or she wanted. The Norwegian Health Network transmitted data over a secure broadband connection (10 Mbps full duplex). BODY.METHODS.PARTICIPANTS: All of the patients were recruited from the four northernmost municipalities in Troms County in Northern Norway: Kåfjord, Skjervøy, Nordreisa and Kvænangen. The 6,500 km2 area is sparsely populated with approximately 12,000 inhabitants (in 2013), 50 % of which live in five small towns. The patients, who all were referred to or scheduled a visit at the orthopaedic outpatient clinic at UNN Tromsø, were evaluated according to the inclusion and exclusion criteria defined by the orthopaedists running the study (Table 1).Table 1Patients' inclusion and exclusion criteriaInclusion criteriaExclusion criteriaNew referred to orthopaedic outpatient clinic UNN, Tromsø (e.g. knee osteoarthritis, hallux valgus)Follow up after orthopaedic surgery (e.g. arthroplasty of the hip)Follow up after orthopaedic trauma (operated or not)Follow up of chronic orthopaedic disordersWritten consentExpectancy of advanced physical examination/tests (e.g. shoulder- and "young knee" problem)Unable to give informed consent (e.g. Dementia, soldiers, prisoners)Need of interpreterTo be seen by a specific orthopaedic surgeonNeed of contemporary procedures (e.g. CAT-scan, ultrasound)Contemporary other outpatient clinical consultation BODY.METHODS.INTERVENTIONS: The remote consultations were performed through real-time videoconference, where a trained nurse was with the patient at the remote location and the orthopaedic surgeon was located at UNN. The preselected orthopaedic surgeons (three consultants, two experienced registrars) carried out their daily work at the orthopaedic department and conducted the consultations as part of their daily routine. They were randomly selected according to who were available at the consultation time. The orthopaedic surgeon ran the consultation after some initial training and technical assistance. Before beginning the study, two nurses from the RMC were trained at the orthopaedic outpatient clinic. They attended casting courses and were trained in clinical examination techniques. The trained nurses received the patient at the remote site, assisted during the consultation and performed physical tasks, for example, changed a cast or removed stitches. No physician was with the patient at the remote site. A digital X-ray lab served by a radiograph was available at the RMC. Digital X-rays were, if appropriate, available at the time of the consultation. Radiologists at UNN later described the X-rays and included them in the hospital's standard X-ray records. The standard consultations took place at the hospital outpatient clinic. In each consultation, the usual mandatory registration and documentation in the patient's medical records was done by the orthopaedic surgeon, including the conclusion of the consultation, agreement between surgeon and patient regarding any follow-up appointments, prescriptions, referrals for operation, further investigation, physiotherapist training and/or an application for orthopaedic aid if needed. BODY.METHODS.OUTCOMES: Following each consultation, the orthopaedic surgeon immediately evaluated the professional quality of the telemedicine and the standard consultation. The evaluation comprised answering a questionnaire with five five-level questions (very good, good, neither good nor bad, bad, very bad), each measuring five categories of experience: cooperation, information, examination/evaluation, treatment and overall evaluation of the consultation. (Questions presented in Table 3). The questions regarding information and treatment included the additional option 'not applicable'. All of the questions were equally weighted, and a sum score was calculated. The primary outcome measurement was the difference between standard and video-assisted consultations in the sum score. Additional analyses were done to support the evaluation of the professional quality of the consultation. The orthopaedic surgeon recorded the duration of the consultation as well as agreement on further action (follow-up consultation/discharge/referrals). The patients received a questionnaire three and 12 months after the last consultation to report events or complications, including any need for additional contact with health services as well as patient-reported outcome measures (EQ-5D-3L and EQ-VAS). Two postal reminders were sent, and an additional telephone call was placed to non-responders. The patients' hospital medical records were screened for additional information relevant to the study. These included complications linked to the referred condition (reported or not by the patient); if referred for operation, whether operated as referred or not; total number of consultations for the actual disorder in the study and if they had been referred again for the same condition over the subsequent two years. The orthopaedic surgeon questionnaire after the video-assisted consultations included five additional five-level questions (very good, good, neither good nor bad, bad, very bad) regarding cooperation with other health workers, technical issues, previous experience with video-assisted consultations and expectations regarding a video-assisted consultation compared to a standard consultation before and after the conducted consultation. The secondary endpoints were comprised of patient satisfaction and economic analyses, assessed via questionnaires given to the patients and specialists after each consultation and mailed to the patients three and 12 months after the last consultation in the study. The health economic outcomes and patient satisfaction will be reported in separate papers. Baseline data were collected via a questionnaire that the patients completed immediately after the first consultation. This included demographic variables (age, gender, occupation, education), indicators used for measuring patient-reported outcomes, cause of consultation and experience with different specialist outpatient clinics. English translations of the questionnaires used in the study can be viewed in the Additional files 1, 2, 3 and 4. BODY.METHODS.SAMPLE SIZE: The sample size calculation was based on the quality sum score assessed by the consulting physicians; the results indicated that we needed at least 191 patients in each group to achieve 90 % power to detect non-inferiority using a one-sided two-sample t-test, a standard deviation equal to 1.0 and a 5 % significance level. The margin of non-inferiority was set at 0.30, as a difference in sum score between the groups ≤ 0.3 was rated as not clinically relevant using a questionnaire with five-level questions (1–5). BODY.METHODS.RANDOMISATION: Randomisation of patients was performed via a password-protected, web-based randomisation database created by the Unit for Applied Clinical Research, Faculty of Medicine, Norwegian University of Science and Technology, Trondheim. It was a blocked randomisation of unknown size and stratified by municipality and age (≤18 and ≥ 65 in one group and 19–64 years of age in the other). Blinding was not applicable. BODY.METHODS.IMPLEMENTATION: Some of the patients were referred directly to participate in the study by their General Practitioner (GP) or specialists at the hospital, but most of the eligible patients were contacted for inclusion after review (by a secretary or the corresponding author) of the hospitals' waiting lists or evaluation of newly referred patients. Up to two invitation letters were sent by mail. The orthopaedic surgeon running the study did the final evaluation to ensure that each patient met the inclusion criteria; the same surgeon also performed the randomisation. The study patients were thereafter given a consultation appointment according to a planned schedule. BODY.METHODS.STATISTICAL METHODS: The baseline characteristics were presented as means (standard deviation) or numbers (percentages). Generalised estimating equations (GEE) were used to assess the differences between the intervention and the control group and to assess the non-inferiority of the intervention group. The exchangeable covariance structure was specified in the GEE models in order to control for two or more consultations for some of the participants. In additional models, we recoded the items regarding the evaluation of the consultation to very good (yes/no) and used GEE assuming a binomial distribution with a logit link function. The study design was based on the intention to treat principle, but the analyses of the primary outcome – the sum score – were not strictly by intention to treat principle, since 3.2 % of the randomised patients did not meet for a consultation (5.0 % in the control and 1.5 % in the intervention group). The ancillary results were presented as means (standard deviation) or numbers (percentages) and analysed using two-sample t-tests or chi-square tests, as appropriate. Statistical analyses were performed using STATA version 13.1 (StataCorp LP Texas, USA). BODY.RESULTS: Eligible patients from the four municipalities were recruited between November 2007 and August 2012 and were seen at the outpatient clinic at the first available slot after randomisation, or for follow-up patients, when scheduled. The last consultation in the study was conducted in October 2012. A review of the patient files was performed between May 2013 and October 2014. The baseline characteristics are shown in Table 2; they did not reveal any significant differences between the groups. Figure 1 shows the flow chart, including the data collection points. A total of 559 consultations (257 at UNN and 302 at RMC) from 389 patients (190 at UNN and 199 at RMC) were included. The specialists' evaluation questionnaires were completed for all of the consultations (100 %); one consultation in each group missed all of the questions, forming the sum score (0.5 %). A total of 547 (98 %) of the patients completed the questionnaire (249 at UNN and 298 at RMC). One patient in each group did not attend their follow-up appointments due to other more serious disorders. A total of 125 (66 %) of the UNN-allocated patients versus 136 (68 %) of the RMC participants returned the 3-month questionnaire, and 143 (75 %) and 144 (73 %) returned the 12-month questionnaire. All 389 participating patients' electronic medical records were reviewed as planned. Four patients from UNN and two from RMC died of other disorders within two years after their last consultation.Table 2Descriptive baseline characteristics from 1st consultation according to location a UNN, standard consultation (n = 190)RMC, video conference consultation (n = 199)Males75 (39)82 (41)Age, years46.7 ± 24.948.8 ± 24.0Age 1-18 years46 (24)43 (22) 19-64 years86 (45)91 (46) 65-90 years58 (31)65(33)The patient residential municipality Kvænangen25 (13)26 (13) Nordreisa82 (43)90 (45) Skjervøy47 (25)45 (23) Kåfjord36 (19)38 (19)Cause of consultation New referral69 (36)81 (41) Control after elective surgery25 (13)22 (11) Control after trauma surgery33 (17)35 (18) Control after trauma, no surgery55 (29)50 (25) Chronic disease8 (4)11 (6)EQ-5D-3 L index (n = 165 + 178)b 0.70 ± 0.250.68 ± 0.26EQ VAS 1–100 (n = 140 + 150)b 75 ± 1873 ± 19Patient assessment of own health in general; 5-leveled scale (n = 180 + 191)b 2.00 ± 0.832.05 ± 0.83Employment status (n = 177 + 190)b Full time worker45 (25)56 (30) Part time worker23 (13)20 (11) Homemaker12 (7)19 (10) Unemployed2 (1)2 (1) Retired/disability benefit55 (31)61 (32) Student/pupil40 (23)32 (17)Education (n = 158 + 176)b Primary school85 (54)92 (52) Secondary school39 (25)54 (31) University34 (21)30(17)Number of outpatient consultations last 6 months before 1st consult. (n = 180 + 188)b Only the actual consultation109 (61)128 (68) 2 to 3 times64 (36)52 (28) 4 times or more7 (4)8 (4) UNN University Hospital of North Norway, RMC Regional Medical Centre a Values are mean ± SD or number (percent) b Number of item responses in UNN and RMC respectivelyFig. 1Flow diagram of the enrollment, allocation, follow- up and data collections points BODY.RESULTS.OUTCOMES AND ESTIMATION: The reasons for discharge from the study were as follows: patient did not need further follow-up (n = 216, RMC 113 [57 %]/UNN 103 [55 %]); patient was referred for surgery (n = 55, RMC 22 [11 %]/UNN 33 [17 %]); patient was referred to another outpatient clinic (n = 8, RMC 3 [2 %]/UNN 5 [3 %]); patient required further follow-up at the orthopaedic department for chronic conditions (n = 74, RMC 41 [21 %]/UNN 33 [17 %]); patient required follow-up with his or her own GP (n = 6, RMC 2 [1 %]/UNN 4 [2 %]); patient needed a consultation specific to the orthopaedic outpatient clinic at UNN (n = 27, RMC 16 [8 %]/UNN 11 [6 %]); patient was referred for admission to the ward (RMC 1 [0.5 %]/UNN 0 [0 %]) (p = 0.424). The reasons that 27 patients needed follow-up consultations specific to UNN (standard consultation) were as follows: the physician was not satisfied with the examination at the remote location (n = 3); patient needed removal of osteosynthesis implants (n = 13); patient needed diagnostic anaesthetic injection tests (n = 3); patient needed a CAT scan (n = 5); other causes (n = 3). Except for 'not satisfactorily examined at the remote location', these causes were equally distributed between both groups. The primary outcome – the sum score of the orthopaedic surgeon's evaluation – was significantly lower, in other words, 'better', at UNN compared to RMC (1.72 versus 1.82, p = 0.0030). However, the 90 % CI for the difference in score (0.05, 0.17) was within the non-inferiority margin (Fig. 2). Subgroup analyses restricted to the first consultation of newly referred patients (n = 150) and the first follow-up consultation of those who were not newly referred (n = 238) showed similar results with slightly wider CIs (−0.02, 0.18) and (0.03, 0.20), respectively. When the five different questions forming the sum score were assessed separately, the questions regarding how the orthopaedic surgeon evaluated the examination/evaluation of the patient and the overall evaluation of the consultation demonstrated significantly higher scores in the RMC group (Table 3).Fig. 2Observed treatment differences for video-assisted consultation (RMC) minus standard consultation (UNN) for sum-score of the specialist evaluation of the consultation. Blue dashed line = 0.3 non-inferiority margin, CI = Confidence intervalTable 3Orthopaedic surgeon's evaluation of the consultation per allocationa UNN,RMC, video p-valueb p-valuec standard consultationconference consultationHow well did you perceive the patient cooperated during the consultation? (254 + 299)d p = 0.58 p = 0.75 Very good95 (37)105 (35) Good157 (62)190 (64) Neither good nor bad2 (1)3 (1) Bad0 (0)1 (0) Very bad0 (0)0 (0)How well could you evaluate/examine the patient? (243 + 290)d P < 0.001 P < 0.001 Very good98 (40)57 (20) Good144 (59)225 (78) Neither good nor bad1 (0)7 (2) Bad0 (0)1 (0) Very bad0 (0)0 (0)How well could you treat the patient? (246 + 292)d p = 0.068 p = 0.039 Very good23 (16)12 (7) Good119 (83)155 (91) Neither good nor bad1 (1)2 (1) Bad0 (0)1 (1) Very bad1 (1)0 (0) Other (not applicable)102122How well could you inform the patient? (254 + 298)d p = 0.106 p = 0.28 Very good54 (22)50 (17) Good191 (77)233 (79) Neither good nor bad4 (2)12 (4) Bad0 (0)0 (0) Very bad0 (0)0 (0) Other (too young)53Overall how well could you assess/treat/checking the patient? (254 + 293) d p = 0.0047 p = 0.040 Very good56 (22)43 (15) Good198 (78)242 (83) Neither good nor bad0 (0)7 (2) Bad0 (0)1 (0) Very bad0 (0)0 (0)Sum score, mean(SD)1.72 ± 0.381.82 ± 0.38 p = 0.0030NA UNN University Hospital of North Norway, RMC Regional Medical Centre a Values are number (percent) or mean ± SD b Test for equality between UNN and RMC using generalised estimating equations (GEE) c Test for equality between UNN and RMC using GEE with a logit link function and a binary response very god (yes/no) d Number of item response in UNN and RMC respectively There were a few missing values in the five questions forming the sum score from 6 up to 26 (1.1–4.7 %). A sensitivity analysis, in which the missing values were replaced with the highest score in the intervention group and the lowest score in the control group, still demonstrated a difference in sum score that was within the non-inferiority margin (90 % CI 0.14–0.27). BODY.RESULTS.ANCILLARY ANALYSES: Additional analyses are shown in Table 4. The mean consultation duration was not significantly different between the groups (p = 0.60). In the subgroup analyses restricted to patients who required casting, we observed a significantly longer mean consultation time in the RMC group (29.0 min) compared to the UNN group (22.6 min, p = 0.0063). Casting was performed in 11 % of the consultations. All of the patients at the RMC underwent their planned operation. In the UNN group, two patients were not operated on due to the occurrence of other serious disorders, four patients improved during the waiting time and did not need the planned surgery and one did not appear for an unknown reason. There were no significant differences in the number of operated patients between the two groups (p = 0.432). Of the 190 patients allocated to UNN, 147 had one consultation, 27 had two, 11 had three, three had four, one had five and one patient had six consultations before discharge from the study. Of the 199 patients allocated to the RMC, 135 had one consultation, 39 had two, 15 had three, seven had four, two had five and one had six consultations. There was a tendency toward more consultations in the RMC group, but this was not statistically significant (p = 0.057). Also, the subgroup analyses of the number of consultations per patient according to the cause of the consultation did not demonstrate any significant differences. The patients who had their appointment at the RMC were not more likely to be referred again within two years for the same disorder (p = 0.858). Furthermore, no significant difference was observed in the subgroup of 'discharged patients' (i.e. in those who were not referred for operation, a standard consultation or any follow-up appointment for chronic disorders with the orthopaedic department within six months). The patient-reported outcome measure at three and 12 months and the change from the baseline to 12 months did not demonstrate any difference between the two groups. This will be analysed in a separate paper.Table 4Ancillary results according to locationa UNN, standard consultation (n = 190)RMC, video conference consultation (n = 199) P- value** Consultation durations, minutesb 20.9 ± 7.4720.5 ± 8.90.603Operation Referred to surgery33 (17 %)22 (11 %)0.074 Operated26 (14 %)22(11 %)0.431Referred again within 2 years Overall (n = 190 + 199)19 (10 %)21 (11 %)0.858 Among "discharged patient" (n = 145 + 159)c 12 (8 %)18 (11 %)0.373Number of consultations per included Overall (n = 190 + 199)1.35 ± 0.781.52 ± 0.910.057 New referredd (n = 69 + 81)1.06 ± 0.291.17 ± 0.440.067 Control patientse (n = 121 + 118)1.52 ± 0.911.75 ± 1.010.071Complication Overall (n = 190 + 199)g 40 (21 %)33 (17 %)0.259 Patient reported at 3 month,(n = 109 + 119)f 15 (14 %)16 (13 %)0.095 Patient reported at 12 month, (n = 132 + 133)f 23 (17 %)14 (11 %)0.105 UNN University Hospital of North Norway, RMC Regional Medical Centre a Values are mean ± SD and number (percent) b 553 consultations, missing data: 4 of 257 in UNN and 3 of 302 in RMC group c Patient with no appointment at orthopedic department within 6 month for the actual disorder, presented according to location. (Patient neither referred to operation nor to a required standard consultation or follow-up for chronic disorder) d One patient in each group did not meet to follow up consultation e Cause of consultation – control after elective surgery, trauma or chronic diseases f Denominator/number differs due to non-item response, presented according to location g Evaluation of the patient's records and patient reported at 3 and 12 months, presented according to location ** P-value calculated with t-test or chi square test when appropriate BODY.RESULTS.THE TELEMEDICINE CONSULTATION: For the video-assisted consultations, the orthopaedic surgeon evaluated the cooperation with other health workers as 'very good' (99 %) and 'good' (1 %) and the technical performance as 'very good' (14 %), 'good' (78 %), 'neither good nor poor' (7 %) and 'poor' (<1 %). There was no change in the orthopaedic surgeons' evaluation of a video-assisted consultation compared to a standard consultation before and after the actual consultation, which were evaluated as equal (98–99 %). All of the video-assisted consultations were conducted as planned. Due to technical trouble, 17 consultations were delayed – two subsequent consultations for 75 and 60 min, the rest for 17 min (mean). BODY.DISCUSSION: The main finding in our study is that the orthopaedic surgeon evaluated the video-assisted consultations as not being inferior to the standard consultations. The sum score was significantly lower in the control group compared to the intervention group, but the difference was within the non-inferiority margin. The difference in sum score was 0.1 on a scale from 1 to 5, which is lower than the assumed accepted difference of clinical relevance. A total of 98 % of the remote consultations versus 99 % of the standard consultations were evaluated as 'good' or 'very good' for all of the questions in the questionnaire, except for the question regard information to the patient which for 96 % of the consultations at RMC were evaluated as 'good' or 'very good'. X-rays are an important part of an orthopaedic consultation. In our study, X-rays were performed immediately prior to the consultations in 88 % (UNN) and 87 % (RMC) of the cases. This might contribute to the orthopaedic surgeons' positive evaluations. At an orthopaedic consultation, it is important to reach a conclusion for a further treatment plan based on the patient's history, the clinical examination/evaluation and any additional tests or investigations (mainly X-rays). Therefore, it is expected that a consultation without the possibility of physically examining the patient directly will be evaluated as less optimal than a standard consultation. This could explant the significant difference in evaluation of question regarding evaluation/examination of the patient. The overall question of how well the orthopaedic surgeon could assess/treat/check the patient is also influenced by the latter. Due to the lack of a standard validated questionnaire for the orthopaedic surgeons' evaluation of the consultations, we created one. The five questions relevance were evaluated by eight, not in the study engaged, orthopaedic surgeons, item content validity index, CVI = 0.976, calculated and reported as recommended by Polit and Beck [19]. All of the questions were related to assessment, which could be affected by the different consultation situations. Others have used similar questions. For example, Brennan et al., who evaluated emergency physicians' ability to use telemedicine to evaluate and treat patients with pre-selected chief complaints in an emergency department, reported a mean of 3.8 (1 = not very satisfied, 5 = very satisfied) in the physicians' comfort level in making diagnoses and performing treatment in the telemedicine group. They did not report any mean in the control group or p-values, but they concluded that telemedicine was a satisfactory technique for the chosen group of patients [5]. A similar result was reported by Wan et al. They evaluated the feasibility of remote consultation for pain management, orthopaedics and general surgery using telemedicine. They had a mean score of 3.6 for the physicians' satisfaction with seeing the patient via videoconference [20]. Aarnio et al. found that 23 out of 29 (six missing) orthopaedic surgeons responded with 'good' or 'very good' as their level of overall satisfaction with teleconsultations, and 20 evaluated the physical examination with aid as 'good' or 'very good' [8]. In another study regarding remote surgical consultations by videoconference, Aarnio et al. demonstrated that 92 % of the consulting surgeons fully agreed that their decisions were as good as they would have been in a usual outpatient clinic consultation [21]. In this study, we did not find any serious events related to the mode of consultation. This finding is strengthened by the fact that our institution is the only hospital in this region, thereby allowing us to discover serious events that the participants do not report, as long as these resulted in contact with the hospital. The patient-reported complications included a wide variety of causes, many of which were not related to the treatment or patient evaluation at the consultations. The complications, evaluated based on the patients' reports, and total complications, which also include complications revealed from the patients' medical records, were not different between the two groups. Because of the lack of a standard questionnaire for measuring orthopaedic surgeons' satisfaction of consultations, we performed additional analyses to support the evaluation of the quality of care of the consultation. We did not find any significant difference between the two groups concerning referral to operation, regardless of whether the planned operations were performed or not. This was also the case when the analysis was restricted to the new referred patients (data not presented), which is in conjunction with the findings of another follow-up study on videoconferencing with orthopaedic outpatients [9]. Another important finding in our study is that the mean consultation duration was not significantly different between the groups. This is in contrast to what others have reported, where the duration of telemedicine consultations was significantly longer than that of standard consultations [4]. Our data does not give a clear explanation for this finding, although our consultations' duration of 20 min generally was longer [8, 22]. The scheduled duration for each consultation (including consultation, documentation and study registration) was 30 min, which may have influenced the overall amount of time. Another factor could be that all of the consultations in our study were scheduled. Urgent consultations, which represented the largest proportion of consultations in other studies, were not included [4, 10, 23]. One could expect that if the patients were not satisfied with the outcome of the consultation, they would be more likely to be referred again if they still had problems or pain. We did not find any difference between the groups regarding re-referrals, or when analysing subgroups according to different causes of inclusion or how they were discharged from the study. These findings support that, in our study, videoconference consultations are not inferior to standard care. To our knowledge, others have not reported this. Even if there was a tendency toward a higher number of consultations per patient in the video-assisted group, the difference between the two groups was not significant. After the first consultation, 32 % of the patients in the RMC group were discharged compared to 36 % in the UNN group (p = 0.389). Wallace et al. reported that patients in the virtual outreach group were offered follow-up appointments to a larger degree compared to patients receiving standard consultations, especially orthopaedic and ear, nose and throat (ENT) patients [24]. Another study reported that a significantly higher proportion of patients assessed by an emergency medicine specialist using telemedicine were offered a follow-up consultation compared to patients assessed by an on-site emergency medicine specialist [4]. One possible explanation for this difference could be our thorough evaluation of the participants' orthopaedic condition before their inclusion in the study. For example, we did not include the first visit for emergency patients and excluded patients with an expected need for advanced clinical examination or treatment. Two of the three patients who were not satisfactorily evaluated at RMC had a combination of back and hip pain. Another study has also reported inadequate assessment of patient histories that present with back problems at telemedicine consultations [6]. Our telemedicine approach might be improved if it was an option to have another trained health worker together with the patient at the remote site than the trained nurses used in our study. For example, in a further study on video assisted remote consultations for orthopaedic patients it could be tested whether the possibility to have a physiotherapist together with the patient could increase the potential for examining/testing the patients, and thus both increase the quality of the telemedicine consultations and expand its use to a wider range of patients. BODY.CONCLUSIONS: This study found that it was safe to offer video-assisted remote consultations for selected orthopaedic patients. The strengths of this study are that is was conducted in a real-life clinical setting. We did not find any serious events related to the mode of consultation. Further assessments of the economic aspects and patient satisfaction are needed before we recommend a wider application.

TITLE: Comparison of vinorelbine with cisplatin in concomitant chemoradiotherapy in head and neck carcinoma ABSTRACT.AIM:: Head and neck cancer is one of the most commonly occurring malignancies in the world. In India, the most commonly occurring head and neck cancers are those of the oral cavity and the pharynx. The majority of these cancers present with stage III/IV disease. Surgery and radiation therapy are the main treatment modalities. Concomitant chemoradiation is being investigated with the goal of improved local control that translates into improved survival. In this background, we have started this prospective randomized trial to ascertain the dose, schedule and sequence of therapy and to note whether Vinorelbine as radiosensitizer is equally effective as Cisplatin, comparing compliance, local control and toxicity. ABSTRACT.PATIENTS AND METHODS:: Forty patients of advanced head and neck cancer were randomized into two arms. Arm A received weekly injection Cisplatin 40mg/m2 along with radiation. Arm B received weekly injection of Vinorelbine 6mg/m2 along with radiation. Radiotherapy was delivered at a dose of 6,600-7,000 Gy in conventional fractionation in a telecobalt machine. ABSTRACT.RESULTS:: The complete response (CR) rate was higher in arm B (90%) than in arm A (70%). Major toxicities included neutropenia, anemia, mucositis and nausea. ABSTRACT.CONCLUSION:: Concomitant chemoradiation with Vinorelbine produced more CR than chemoradiation with Cisplatin in advanced head and neck cancer. Toxicities were more in the Cisplatin arm, but they were manageable. Although a majority of the study was performed using Cisplatin as the radiosensitizer, Vinorelbine can be recommended as radiosensitizer in advanced head and neck malignancy. BODY.INTRODUCTION: Head and neck malignancy is the one of the most commonly occurring malignancy in India. The overall male to female ratio is nearly 4:1. It usually occurs in the 5th decade and above. The prognosis of head and neck cancer depends on the primary site, grade and anatomical extent of the disease. Early-stage head and neck cancers can be cured with surgery and/or radiotherapy but, for advanced stages, the local failure rate sometimes approached as high as 50%. To improve the results, combined modality treatment with chemotherapy has been investigated. The three approaches to the use of primary chemotherapy are neoadjuvant chemotherapy,[1–3] adjuvant chemotherapy and concomitant chemoradiotherapy. Concomitant chemoradiation is being investigated with the goals of improved local control translating into improved survival, reduction of distant metastasis and preservation of organ function. The purpose of administering chemotherapy and radiotherapy is to take advantage of the radiosensitizing capability of many of the active drugs for this disease and effect a substantial-enough increase in locoregional control, which would translate to increased survival.[4] Patients who received concomitant chemoradiotherapy had marginally improved rates of locoregional control and disease-free survival. This was observed primarily in patients with oropharyngeal cancer[5] as compared to other cancers. The drugs most commonly employed as part of a radiation combined approach are Cisplatin, 5FU and hydoxyurea. Cisplatin has widespread use in combined modality treatment in lung cancers[6] and head and neck cancers.[7] Recently, Vinorelbine[8–11] was used as a radiosensitizer. A majority of the studies was performed using Cisplatin[1213] as a radiosensitizer, although some studies also support use of Vinorelbine as a radiosensitizer. BODY.PATIENTS AND METHODS: This study was carried out in the radiotherapy department of I.P.G.M.E.R, Kolkata, from September 2004 to July 2005. Forty patients of head and neck cancer were randomized into two arms, with 20 patients in each arm. Patients of head and neck carcinoma having stage II-IV disease with squamous cell histology were included in this trial. These patients had no prior surgery, chemotherapy or radiotherapy. The performance status was >70% (according to Karnofsky's scale). Hematological parameters were within the normal range, like hemoglobin >11 mg%, absolute neutrophil count >1,900, platelet count >1 lakh/mm3, serum bilirubin <1 mg%, liver enzymes within 1.5-times of the normal limit and serum creatinine <1.5 mg%. Patients were excluded from the study if they had already received some form of anticancer therapy, if there was presence of metastatic disease, if they had participated in a clinical trial in the last 30 days, if there was simultaneous participation in a clinical trial or if they had any uncontrollable systemic illness like diabetes, tuberculosis and hypertension. BODY.PATIENTS AND METHODS.TREATMENT PROTOCOL: Patients who fulfilled the above eligibility criteria were required to sign the informed consent form and were then randomized to assign either of the treatment arms. Arm A: External beam radiotherapy (EBRT) along with weekly injection Cisplatin 40 mg/m2 IV. Arm B: EBRT along with weekly Vinorelbine 6 mg/m2 IV. The dose of EBRT was 66-70 Gy, with conventional fractionation, using a telecobalt machine with cord sparing after 4,400 cGy. Response was assessed by local examination and indirect laryngoscopy 1 month after completion of radiotherapy. Regular follow-up was carried out at monthly intervals. Local control was recorded using the terminology complete response (CR), partial response (PR) and progressive disease (PD) (as per WHO definition). Toxicity assessment was carried out weekly during treatment and thereafter monthly up to 3 months for acute toxicities using Radiation Therapy Oncology Group criteria. Subsequently, patients were being followed-up monthly up to 6 months and then at 3-monthly intervals for any sign of recurrence and treatment-related morbidity. BODY.RESULTS.PATIENT CHARACTERISTICS: From September 2004 to July 2005, 41 patients were enrolled. One patient in arm B dropped out due to mucositis. Patient characteristics are listed in Table 1. The majority of the patients are in the range of 50–70 years. Patients were predominantly male (95%). They had a good performance status. The larynx and laryngopharynx were the dominant sites (47.5%). Histologically, all were squamous cell carcinoma, the majority of which was well-differentiated (62.5%). Stage III disease was predominant (67.5%). Patients were equally distributed among the two treatment arms. Table 1 Patient characteristics Arm A Arm B Age  Median 56.50 62.50  Range 43–70 31–73 Gender  Male 19 19  Female 01 01 Addiction  Smoker 15 18  Nonsmoker 05 02 Site  Laryngopharynx 13 06  Glottis 02 04  Hard palate 00 00  Pyriform fossa 03 03  Tongue 01 04  Tonsil 01 01  Cheek 00 01  Retromol trigone 00 01 Stage  II 00 02  III 16 11  IV 04 07 Histology  Well differentiated 05 08  Mod differentiated 14 11  Poor differentiated 01 01 BODY.RESULTS.RESPONSE TO TREATMENT: All the patients who completed the treatment were assessed in terms of CR, PR, stable disease and PD. Ninety percent of the patients in arm B achieved CR. This result is better than the weekly Cisplatin arm, which has 70% CR (as shown in Table 2). Table 2 Response to treatment Age Arm A (RT+Cisp) Arm B (RT+Vinorelbine) CR 14 18 PR 04 02 SD 02 00 PD 00 00 When arm B was compared with arm A in terms of CR, it was not statistically significant. BODY.RESULTS.ACUTE TOXICITY: All the toxicities were higher in the Cisplatin-containing arm. All the toxicities were higher in arm A when Cisplatin was used as a radiosensitizer compared with the Vinorelbine arm. Mucositis was almost similar in both arm B and arm A. When arm B was compared with arm A, myelosuppression was higher in arm A (statistically significant, P-value 0.05). Skin reaction was also lower in the Vinorelbine arm when compared with the other arm. Nausea was significantly higher in arm A (RT+cisplatin) when compared with arm B [Table 3.] Table 3 Toxicity Toxicity Arm A (RT+Cisplatin) Arm B (RT+Vinorelbine) Mucositis 20 19 Skin reaction 14 07 Nausea 16 04 Myelosuppression 13 06 BODY.RESULTS.LATE TOXICITY: As the follow-up is short, no definite comment of late toxicity is possible at this stage. All the patients are alive and no serious complication has occurred till date. BODY.DISCUSSION: Therapeutic approach in head and neck cancer is widely discussed and is a debatable one also, with the optimum treatment modality, the intention of treatment and managing toxicities occupying the mind of the physician with the survival effect defining the effectivity of treatment modality. The management of primary cancer is considered separately for each anatomic site. If external beam radiation therapy is selected, it may be given with either conventional once-daily fractionation to 66-70 Gy in 2 Gy/fraction, 5 days a week in a continuous course or with an altered fractionation schedule. EBRT may also be delivered with intensity-modulated radiation therapy (IMRT)[14] to reduce the dose to the normal tissues.[15] The disadvantages of IMRT are that it is much more time consuming to plan and treat the patient, the dose distribution is often less homogeneous so that "hot spots" may increase the risk of late complication and the risk of marginal miss may be increased. Whether an altered fractionation schedule is better than a conventional one depends on the altered fractionation technique that is selected. Altered fractionation schedules shown to result in improved locoregional control rates are the University of Florida hyperfractionation technique and HD Anderson concomitant boost technique. The Randomized Radiation Therapy Oncology Group 90-03 found that acute toxicity is increased with altered fractionation whereas late toxicity is comparable with that of conventional fractionation. Management of the neck is closely tied to management of the primary site. The rationality of combining chemotherapy with radiation in doses mentioned was: to improve the locoregional control rate and increase the response in this fairly advanced diseaseassessment of tolerability of patients with a concurrent approach, determining the dose to normal tissues tolerability to avoid toxic effectsdecrease the distant metastasis rates by acting on systemic micrometastasis present at the diagnosis in more than 50% of the cases. Calais et al,[16] recently reported that disease-free survival and 3-year rate of locoregional control were significantly improved with concomitant chemotherapy, although patients in the combined radiation therapy–chemotherapy arm experienced higher rates of grade 3 or 4 mucositis, feeding tube placement and severe cervical fibrosis. Although a majority of studies were performed by using Cisplatin as the radiosensitizing drug, some studies also support the use of Vinorelbine as a radiosensitizer. After 1-year follow-up, CR is higher in the Vinorelbine plus radiation arm followed by the Cisplatin plus radiation arm, which needs further evaluation. Although toxicities like mucosal, hematologic and dermatologic were higher in they concomitant arm, they were manageable. All toxicities were significantly higher when Cisplatin was used as a radiosensitizer. Compliance was also greater with Vinorelbine as toxicities were less when compared with Cisplatin. Our study had a limited number of patients and the duration of follow-up is also short. Further evaluation of treatment protocol with large number of patients and also with prolonged follow-up may have a positive impact on survival as the response rate is already showing improvement in a concomitant protocol.

TITLE: The effect of different types of insoles or shoe modifications on medial loading of the knee in persons with medial knee osteoarthritis: a randomised trial ABSTRACT.ABSTRACT: Many conservative treatments exist for medial knee osteoarthritis (OA) which aims to reduce the external knee adduction moment (EKAM). The objective of this study was to determine the difference between different shoes and lateral wedge insoles on EKAM, knee adduction angular impulse (KAAI), external knee flexion moment, pain, and comfort when walking in individuals with medial knee OA. Seventy individuals with medial knee OA underwent three‐dimensional walking gait analysis in five conditions (barefoot, control shoe, typical wedge, supported wedge, and mobility shoe) with pain and comfort recorded concurrently. The change in EKAM, KAAI, external knee flexion moment, pain, and comfort were assessed using multiple linear regressions and pairwise comparisons. Compared with the control shoe, lateral wedge insoles and barefoot walking significantly reduced early stance EKAM and KAAI. The mobility shoe showed no effect. A significant reduction in latter stance EKAM was seen in the lateral wedge insoles compared to the other conditions, with only the barefoot condition reducing the external knee flexion moment. However, the mobility shoe showed significant immediate knee pain reduction and improved comfort scores. Different lateral wedge insoles show comparable reductions in medial knee loading and in our study, the mobility shoe did not affect medial loading. © 2015 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 33:1646–1654, 2015. BODY: Knee osteoarthritis (OA) is the most common form of OA and is the leading cause of pain and disability in older adults.1 At the current time, there is no cure for knee OA and therefore non‐surgical conservative management is at the forefront of the treatment for the disease. In the UK, National Institute of Clinical Excellence (NICE) guidelines recommend conservative management techniques such as footwear and insoles to be part of the management of the condition.2 The medial compartment of the knee joint is more often affected than the lateral compartment.3 Dynamic joint loading has been implicated both in the development of knee pain associated with OA4 and the progression of the disease.5 During walking the ground reaction force passes medial to the knee in the frontal plane, this creates a moment that adducts the tibia relative to the femur, with the peak load on the medial compartment almost 2.5 times more than that on the lateral compartment.6 The external knee adduction moment (EKAM), captured from three‐dimensional motion analysis and inverse dynamics, is a valid and reliable proxy representing dynamic load distribution and is the primary mechanism, along with the external knee flexion moment, for the majority of compressive load in the joint.6, 7, 8 The EKAM typically has an early stance peak (first) and a late stance peak (second) with the first peak always higher than healthy controls regardless of severity, whereas the second peak is only higher in the more severe individuals.9 Therefore, given the target population for conservative management (mild and moderate knee OA), the primary parameters of interest are the first peak in the EKAM and also the knee adduction angular impulse (KAAI), which is the area under the adduction curve.10 These two parameters have been demonstrated to be related to severity11 to structural features of OA and to progression.12, 13 Therefore, reducing the EKAM during walking and other activities could be effective in delaying progression in medial knee OA. Many unloading strategies are available including proximal and distal gait adaptations, direct orthotic management at the knee such as valgus knee braces,14, 15 or indirectly at the foot and ankle interface such as shoes/footwear and foot orthoses such as lateral wedge insoles.16, 17, 18 The latter are popular as they are typically inexpensive with good adherence to treatment. Different types of shoes and orthotics have been shown to reduce the EKAM in knee OA trials16, 18, 19 but these have not been directly compared in terms of their effects on medial knee loading and clinical responses. Further, in recent studies directly measured medial compressive loads have been shown to be affected by the magnitude of the external knee flexion moment20 in that a reduction in EKAM may not correspond with a true reduction in medial loads if a corresponding increase in knee flexion moment was seen. The literature on the different effects of lateral wedge insoles and shoe modifications on the knee flexion moment has also not been fully described. There is also not one type of lateral wedge insole, but rather several types such as heel only, full length, and full length insoles with medial support, also with different angulations of lateral incline. In this study we chose to investigate a full length lateral wedge insole (typical) as these have been found to be better than heel only wedges21 and also one with a medial arch support (supported), as this was previously found to be better functionally for the foot and ankle and more comfortable.22 We have demonstrated in a previous paper the effects on early stance peak EKAM and external knee flexion moment of these two different types of lateral wedge insole.18 In addition, other footwear based approaches to lowering medial load have been proposed. One such shoe treatment which aims to mimic barefoot walking during gait,16 which is perceived as the best walking style for reducing medial loading, has been developed and recommended as efficacious for medial knee OA. These shoes have not been directly compared with traditional lateral wedge insoles in terms of their effects on medial knee load. Understanding which treatment reduces medial loading and reduces pain may provide guidance in terms of which, if any, of these treatments is most likely to be efficacious for medial knee OA. The objectives of this study were to determine which of several different conservative treatments (barefoot, shoes, and insoles) most lowers the EKAM during walking, to determine if any concurrent changes occur in the external knee flexion moment and to compare the degree of immediate knee pain reduction and comfort during usage. BODY.METHODS: The study is a Level 1 evidence randomised clinical trial (ISCRTN 83706683) whereby ethical approval was obtained from the North West Research Ethics Committee (09/H1013/51). BODY.METHODS.PARTICIPANTS: Participants with knee pain were recruited from the following sources: orthopaedic/physiotherapy clinics and advertisements in local media. The eligibility criteria for participation in the study were aged 45 years and above, medial tibiofemoral OA with radiographs demonstrating Kellgren and Lawrence grade 2 or 3 in the affected painful knee with medial greater than lateral joint space narrowing, and at least mild pain during walking on a flat surface during the last week assessed by the KOOS pain subscale (P5).23 Radiographs were generally acquired as part of the patient's routine care and were read by an experienced academically based musculoskeletal radiologist according to the OARSI atlas.24 When no radiographs were available, we accepted evidence from recent arthroscopies or knee MRI's as providing sufficient information to evaluate eligibility. Patients were excluded if they presented with pain more localised to the patellofemoral joint on examination than medial joint (wedge inserts are not appropriate for disease in this compartment and lowering the EKAM may make them worse), had tricompartmental knee OA or grade 1 or grade 4 tibiofemoral OA on the Kellgren and Lawrence scale. Other exclusions included a history of high tibial osteotomy or other realignment surgery, total knee replacement on the affected side, or any foot and ankle problems, such as painful hallux valgus; plantar fasciitis; peripheral neuropathy or any foot and ankle pain, that contraindicated the use of the load modifying footwear interventions. In addition, participants were excluded if they had severe coexisting medical morbidities or used orthoses prescribed by a podiatrist or orthotist. Eligible participants were invited to attend the gait laboratory where informed consent was obtained. BODY.METHODS.INTERVENTIONS: The analyses were conducted in the context of a single visit randomised trial. We tested five conditions: barefoot, a flat soled shoe (Ecco Zen) (control), two different lateral wedge insoles each which have been shown to reduce EKAM in patients with medial knee OA18, 25 and a mobility shoe16 meant to mimic barefoot walking. Both lateral wedge insoles consisted of a 5 degree lateral wedge. The major difference between the lateral wedge insoles was that one had medial support (referred to hereafter as the "supported" wedge18 whereas the other had no medial support (the "typical" wedge).25 During the trial, these lateral wedges were inserted into the flat‐soled control shoe with participants having a minimum of 5 min familiarisation period to the condition. The mobility shoe was a flexible grooved shoe16 (see Fig. 1). Figure 1Lateral wedge insoles and mobility shoes.Copyright © 2015 Wiley Periodicals, Inc. BODY.METHODS.PROTOCOL: All participants underwent gait analysis in all of the conditions. The order of presentation of the different conditions was randomised prior to participants' enrolment using computer‐generated permutations (using http://www.randomization.com/). Patients walked at their self‐selected speed in all conditions. Upon completing each treatment, participants were asked to compare the knee pain experienced under that treatment while walking to pain when wearing their own shoes by scoring this pain on a 5‐point Likert scale ranging from −2 (indicating much better pain compared to the participants' normal shoes) to +2, (indicating much worse pain compared to the participant's normal shoes). Additionally, we asked participants to rate each condition's comfort, in comparison to their normal everyday shoes. This was measured on a 10 cm VAS, with scores ranging from −5 (much less comfortable than the participants' normal shoes), to +5 (much more comfortable than the participant's normal shoes). All outcomes were measured in all five study conditions (control shoe, typical lateral wedge, supported lateral wedge, mobility shoe, and barefoot.) A 16 camera Qualisys OQUS3 motion analysis system operating at 100 Hz and four AMTI force plates operating at 200 Hz were used to measure kinematics and kinetics during the trials. Each participant completed a minimum of three successful trials at a self‐selected walking speed. The CAST marker set technique26 was employed whereby rigid clusters of four non‐orthogonal markers were positioned over the lateral shank, lateral thigh, and sacrum to track the movements of the limbs. Retroreflective markers were glued securely to the control shoes with the foot modelled as a rigid segment. A reference trial was conducted where retroreflective markers were placed on bony landmarks specifying their location in relation to the clusters and to approximate joint centre. Ankle and knee joint centres were calculated as midpoints between the malleoli and femoral epicondyles respectively. The hip joint centre was calculated using the regression model of Bell et al.27 based on the anterior and posterior superior iliac spine markers. Using an inverse dynamic approach Visual 3D (C‐Motion, Rockville, Maryland) we calculated the EKAM and sagittal plane external knee flexion moment (KFM) during stance phase for all of the trials and conditions. A custom Matlab (Matlab, USA) programme was used to extract the peak EKAM and KFM during early stance (up to 50% of stance phase) and the peak latter stance EKAM (from 50% of stance phase) and to calculate the knee adduction angular impulse (KAAI),9 which is the area under the adduction moment curve during the entire stance phase of gait. EKAMs and KFMs were normalised to participant's mass (Nm/kg) with the KAAI normalised to participant's mass and stance time (Nm/kg s). BODY.METHODS. : Multiple linear regression was used to test for differences in continuous outcomes of interest, between the 5 different experimental conditions. We created four models, one for each of the gait outcomes of interest (EKAM [first and second peak] KAAI, and KFM). In each model, the predictor variable was the orthotic intervention, coded as "dummy variables"—giving 5 predictor variables in total, one for each condition). The control shoe condition was used as the reference group. The model also accounted for the repeated‐measures design of the study by including the participant ID as a panel variable. We used a Hausman specification test to check for the validity of using a random‐effects model, in preference to a fixed‐effects model of the same specification. The test did not show statistical significance and consequently, a random‐effects model was used. We checked for model fit by investigating residuals against fitted plots. Since model residuals appeared heteroskedastic, robust standard errors (using sandwich estimators) were used to improve estimates of standard errors. Post‐hoc pairwise contrasts were produced, using linear combinations of the beta coefficients from the model to test for differences in all possible comparisons of the orthotics conditions, with ten pairwise tests for each of the three outcomes considered (EKAM, KAAI, and maximum external flexion moment). To counter issues of multiple testing, confidence intervals and p‐values from these pairwise tests were adjusted using the Benjamini‐Hochberg procedure,28, 29 using a false discovery rate (FDR) of 0.05 (see supplementary material). Because patient perceived change in knee pain was not normally distributed, we used Wilcoxon Sign Rank tests to investigate whether the distribution of patient‐perceived pain change ranks were equal to zero, in each orthotic condition separately. Finally, for each condition, we measured if the patient‐perceived change in comfort was different from zero. To test this, we used a random‐effects linear regression model, with the participants' comfort ratings as the outcome variable. The predictor variable again was the orthotic intervention condition, coded as "dummy variables", as in the EKAM/KAAI regression. We then combined the model intercept with the beta coefficients of each condition in turn. This tests if the mean comfort rating in each is equal to zero. Additionally, as both walking speed and knee flexion moment were considered potential confounding variables, we repeated the above models, with walking speed and external knee flexion moment added as additional covariates. All statistical analysis was performed using the statistical software package Stata (version 13.1; Stata Corporation, College Station, TX), with an alpha level of 0.05 (two‐sided) for the assessment of statistical significance. BODY.RESULTS: The flow of participants into the study is shown in Figure 2. The characteristics of the 70 participants were: a mean age of 60.3 years (SD 9.6), mean BMI of 30.5 kg/m2 (SD 4.9), and 27 (38.6%) were female. Data on Kellgren‐Lawrence (K‐L) grades were available for 62 of the 70 study participants, and of these, the mean K‐L grade was 2.6 (SD 0.5). We reviewed recent knee arthroscopy reports or MRIs for 8 participants who did not have x‐rays prior to the study to ensure that these subjects also had medial > lateral cartilage loss and other features of OA. Figure 2Consort Figure: those eligible/enrolled/randomised/studied.Copyright © 2015 Wiley Periodicals, Inc. When we examined the effects of the conditions on measures of medial loading (Tables 1 and 2, Fig. 3), we found that barefoot walking had the greatest effect on early stance peak EKAM, lowering it by −7.6% (p < 0.001 vs. control shoe). Both lateral wedges reduced the early stance peak EKAM by −5.9 and −5.6% (p = 0.001 vs. control shoe) for typical and supported respectively as we have previously reported.18 However, the mobility shoe did not produce a significant reduction in the early stance peak EKAM compared with the control shoe (−1.6%, p = 0.38). Table 1 EKAM, KAAI, Knee Flexor moment, Comfort Rating (VAS), and Walking Speed by Condition Condition EKAM 1st Peak, Nm/kg Mean (SD) EKAM 2nd Peak, Nm/kg Mean (SD) KAAI, Nm/kg*s Mean (SD) Knee Flexor Moment (KFM), Nm/kg Mean (SD) Comfort Rating (‐5 to +5), Mean (SD) Walking Speed, m/s Mean (SD) Control shoe 0.39 (0.16) 0.33 (0.14) 0.16 (0.07) 0.61 (0.24) ‐0.24 (2.29) 1.08 (0.33) Typical lateral wedge 0.37 (0.15) 0.30 (0.13) 0.14 (0.07) 0.61 (0.23) 0.84 (2.42) 1.08 (0.33) Supported lateral wedge 0.37 (0.15) 0.31 (0.14) 0.15 (0.07) 0.62 (0.23) 1.35 (2.13) 1.09 (0.33) Mobility shoe 0.39 (0.16) 0.32 (0.14) 0.15 (0.07) 0.60 (0.24) 2.40 (2.22) 1.11 (0.34) Barefoot 0.36 (0.15) 0.33 (0.14) 0.15 (0.07) 0.57 (0.22) 0.48 (2.35) 1.04 (0.33) © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. Table 2 Effects of Study Footwear on Moments and Walking speed compared with control shoe EKAM 1st Peak EKAM 2nd Peak KAAI Knee Flexor Moment (KFM) Walking Speed (m/s) Condition mean change (95% CI), p % change mean change (95% CI), p % change mean change (95% CI), p % change mean change (95% CI), p % change mean change (95% CI), p % change Typical lateral wedge ‐0.023 (‐0.035 to ‐0.012), <0.001*** ‐5.85 ‐0.028 (‐0.036 to ‐0.02), <0.001*** ‐8.49 ‐0.012 (‐0.016 to ‐0.009), <0.001*** ‐7.95 ‐0.002 (‐0.022 to 0.018), 0.818 ‐0.39 0.003 (‐0.007 to 0.013), 0.558 0.28 Supported lateral wedge ‐0.022 (‐0.035 to ‐0.009), 0.001** ‐5.63 ‐0.018 (‐0.026 to ‐0.01), <0.001*** ‐5.52 ‐0.009 (‐0.013 to ‐0.005), <0.001*** ‐5.52 0.013 (‐0.004 to 0.03), 0.133 2.17 0.009 (‐0.002 to 0.019), 0.105 0.79 Mobility shoe ‐0.006 (‐0.021 to 0.008), 0.384 ‐1.61 ‐0.005 (‐0.015 to 0.005), 0.294 ‐1.59 ‐0.004 (‐0.009 to 0.001), 0.090 ‐2.74 ‐0.006 (‐0.029 to 0.017), 0.611 ‐0.99 0.029 (0.018 to 0.039), <0.001*** 2.65 Barefoot ‐0.03 (‐0.044 to ‐0.016), <0.001*** ‐7.62 0.001 (‐0.011 to 0.013), 0.856 0.34 ‐0.007 (‐0.013 to ‐0.001), 0.023* ‐4.30 ‐0.035 (‐0.057 to ‐0.013), 0.002** ‐5.74 ‐0.042 (‐0.056 to ‐0.028), <0.001*** ‐3.89 Asterisks denote magnitude of p‐value as follows:*p <0.05; **p<0.01; ***p<0.001. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. Figure 3EKAM time series plots for the different conditions (n = 70).Copyright © 2015 Wiley Periodicals, Inc. For the second peak in EKAM during late stance, both of the lateral wedge insoles significantly reduced the magnitude of this peak in comparison to the control condition. There was no difference in the mobility or barefoot conditions in comparison to the control condition. For the knee adduction angular impulse (KAAI), the barefoot condition and the two lateral wedge conditions were significantly different in comparison to the control condition (barefoot −4.3%, p = 0.023; typical wedge −7.95%, p < 0.001; supported wedge −5.5%, p < 0.001). Pairwise comparisons (see supplementary material eTables S1–3) showed that there were no significant differences in the effects on the early stance peak in EKAM between each of the two lateral wedge conditions and barefoot walking. However, the early stance peak in EKAM in the barefoot condition was reduced significantly more than the mobility shoe (mean difference −0.024 Nm/kg, p = 0.004). For the second peak in EKAM, both of the lateral wedge insoles had significantly greater reductions than the barefoot (typical wedge mean difference −0.029 Nm/kg, p < 0.01; supported wedge mean difference −0.019 Nm/kg, p = 0.004) and mobility (typical wedge mean difference −0.023 Nm/kg, p < 0.01; supported wedge mean difference −0.013 Nm/kg, p = 0.024) conditions. A larger second peak reduction in the typical wedge resulted in a significant reduction in KAAI in comparison to the mobility shoe (mean difference 0.008 Nm/kg s, p = 0.011). In comparison with the control shoe and all other conditions, the barefoot condition had significant reductions in the maximum external knee flexion moment (KFM) (etable 3) during early stance. No other changes in external knee flexion moment were seen. Compared with the control shoe, walking speed increased by 0.03m/s with the mobility shoe (95%CI 0.02–0.04, p < 0.001) and slowed by 0.04 m/s with barefoot walking (95%CI −0.05 to −0.03, p < 0.001), but with adjustment for walking speed, this did not affect the overall findings or their significance. Additional adjustment for external knee flexion moment changes also did not affect the differences seen between conditions in medial load measures. In contrast with the findings with regard to medial loading, immediate reductions in knee pain were seen in two conditions: the supported (but not the typical) wedge (as reported previously)17 and the mobility shoe (both p < 0.001) (see Fig. 4). A significant worsening of knee pain was reported by patients for the control shoe (not the subject's own shoe) (p = 0.015) and barefoot walking (p = 0.054). Figure 4Participant rating of knee pain during use of each condition compared with knee pain using their own shoe.Copyright © 2015 Wiley Periodicals, Inc. In terms of comfort, the control shoe was rated as less comfortable than the participant's everyday shoes (see Table 3). Even though the wedges were placed inside these control shoes, both lateral wedges were rated as more comfortable than everyday shoes as were the mobility shoes. Table 3 Participants report of shoe/condition comfort Mean Comfort Rating compared with participants’ own shoe (10cm VAS, ‐5 to +5) Condition mean (95% CI), p Control ‐0.243 (‐0.777 to 0.291), 0.373 Typical lateral wedge 0.844 (0.31 to 1.379), 0.002** Supported lateral wedge 1.349 (0.814 to 1.883), <0.001*** Mobility shoe 2.403 (1.869 to 2.937), <0.001*** Barefoot 0.464 (‐0.074 to 1.002), 0.091 Asterisks denote magnitude of p‐value as follows:*p <0.05; **p<0.01; ***p<0.001. Negative value represents report that condition is less comfortable than current shoe whereas positive value represents report that condition is more comfortable. Wedges were placed inside control shoe . © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. BODY.DISCUSSION: To examine the effects of shoes and orthotics suggested as effective in unloading the medial knee compartment, we carried out a randomised trial, comparing these treatments. We found that barefoot walking and lateral wedge insoles all significantly reduced medial loading in the first part of stance phase with both of the lateral wedge insoles reducing medial loading during latter periods of stance. The two types of lateral wedge insoles showed roughly comparable effects on the knee adduction moment and impulse with only the barefoot walking significantly altering the sagittal moment. Although the mobility shoe did not reduce medial knee loading, participants reported that it diminished knee pain more than the typical wedge, control shoe, and barefoot, and was rated as more comfortable than the other treatments. While lateral wedge inserts have not been shown to decrease knee pain in knee osteoarthritis,30 they do reduce medial loading. Since excess loading in the medial compartment contributes to knee pain and disease progression and since knee OA treatments that alter this are likely to be popular and inexpensive, further exploration of their possible effects is desirable. In that vein, our work suggests two important findings. First, they suggest that lateral wedge insoles reduce medial knee loading more than a control shoe throughout the whole of stance phase and significantly better than both barefoot walking and the mobility shoe during latter stance where the supported insole reduces immediate knee pain better than the typical device with increased comfort. Secondly, whist the mobility shoes did not reduce medial loading significantly, they were rated highly by participants for reductions in knee pain and comfort. Barefoot walking was found to have the greatest reduction in EKAM in comparison to the control shoe and is in agreement with a previous study by Shakoor and Block.31 However, they found a greater reduction (−11.9%) in the peak EKAM (KAAI was not assessed in that study). Differences between our study and that of Shakoor and Block could have accounted for the difference in the magnitude of effect. We focused on the early stance first peak EKAM and not the peak EKAM (which is sometimes different) and we used one control shoe whereas they compared barefoot to a person's individual shoes. We found importantly that barefoot walking reduced medial loading during latter stance in comparison to the control shoe, but had increased medial loading in the latter period of stance in comparison to the lateral wedge insoles. This reduced latter stance reduction in EKAM in the lateral wedge insoles, whilst not directly related to severity or progression, would contribute to a greater reduction in the overall loading during stance phase (KAAI) which has been related to cartilage loss.13 Different shoes may differ in their effect on medial knee loading and our control shoe may have been more effective than some personal shoes in reducing knee medial loads. An exploration of types of personal shoes and their effects on knee loading was beyond the scope of this trial but this is an important next step to determine what role different footwear has on medial knee loading. In agreement with Jones et al.,22 there was no change in the reduction of EKAM or KAAI with the two different lateral wedge insoles. This is in contrast to the work by Nakijima et al.32 who reported that a lateral wedge insole with an arch support reduced medial knee loading more than a standard lateral wedge. One reason for this difference is that the lateral wedge insole with the medial support used in this study is an off‐the‐shelf device and not custom made as in the study by Nakajima et al. It is noteworthy that the medial support incorporated into our lateral insole was not hard and could readily be compressed with weight‐bearing and this may underlie the similar effects of both insoles we studied. Both insoles were deemed to be comfortable (Table 3) but the supported lateral wedge received a greater overall comfort score and significantly reduced pain immediately in comparison to the typical wedge. The mechanism for these reductions in EKAM and KAAI are perceived to be related to the change in the centre of pressure location for the lateral wedge insoles33 which leads to a greater reduction in moment arm. The barefoot walking had a slightly slower speed but this was not associated with changes in EKAM or KAAI. Therefore, the mechanism for this is potentially due to altered foot mechanics but this was not the remit of this paper and further research is needed. External knee flexion moments also contribute to medial knee loading and effects of shoe inserts or shoes on flexion moments could affect medial loading independently of EKAM or KAAI. We found no significant effects of shoe inserts or shoes on external knee flexion moment.20 Only barefoot walking reduced flexion moments and this may have been a consequence of a slower overall walking speed but this needs to be further explored. Further, recent work by Trepczynski and colleagues8 using instrumented knee prostheses suggests that the external knee flexion moment contributes importantly to medial knee loading only during activities when the knee is overly‐flexed, such as stair climbing and squatting or kneeling. Our participants were only required to walk on level ground and our findings on flexion moments suggest that with this activity, most of the variance in medial loading is readily explained by the EKAM and KAAI. Our results on the effects of the mobility shoe contrast with earlier studies in that we found a reduction of just greater than 1% in medial knee loading during early stance. One possible reason could have been the choice of control shoe. As noted earlier Shakoor and cowokers16 tested mobility shoes against the individuals' own shoes. Those authors comment that the choice of shoe worn by the patient has an effect on reduction in medial knee loads compared with the mobility shoe. It is also possible that medial loading reductions occur over time with the mobility shoe.34 While the mobility shoe did not show expected reductions in medial loading, the participant's immediate knee pain scores were significantly improved in comparison to the control shoe with a favourable comfort rating. This suggests that patient adherence would be high and if medial loading were reduced significantly over time, this could be an effective intervention. The reductions in pain seen in the mobility shoe and the lateral wedge insoles disagree with some longitudinal studies and the full reason behind why there were these changes in not known. However, one of the potential reasons could be due to an increased comfort in both the supported insole and the mobility shoe which reflected better perceived pain scores. As with any study there are limitations other than the ones described earlier. The pain and comfort responses were assessed immediately and it is possible that these may change over time. However, previous work35 has suggested that immediate pain response and longer term pain response with wedges are highly correlated. In conclusion, different lateral wedge insoles show comparable reductions in medial knee loading with the supported insole reducing pain more. In our study, the mobility shoe did not affect medial loading. While we confirmed findings of other studies in demonstrating a clearcut reduction in early stance medial loading when walking barefoot, barefoot walking increased medial loading during the latter period of stance and may not be the best for medial loading reduction. BODY.AUTHORS' CONTRIBUTIONS: All authors contributed to the study design, collection, analysis and editing and approval of the final manuscript. BODY.SUPPORTING INFORMATION: Additional supporting information may be found in the online version of this article. Supporting Information Table S1: EKAM 1st Peak Post‐Hoc Pairwise Comparisons. Supporting Information Table S2: EKAM 2nd Peak Post‐Hoc Pairwise Comparisons. Supporting Information Table S3: KAAI Post‐Hoc Pairwise Comparisons. Supporting Information Table S4: Max Flexor Moment Post‐Hoc Pairwise Comparisons.Click here for additional data file.

TITLE: High-Intensity Interval Training as an Efficacious Alternative to Moderate-Intensity Continuous Training for Adults with Prediabetes ABSTRACT: Aims. High-intensity interval training (HIIT) leads to improvements in various markers of cardiometabolic health but adherence to HIIT following a supervised laboratory intervention has yet to be tested. We compared self-report and objective measures of physical activity after one month of independent exercise in individuals with prediabetes who were randomized to HIIT (n = 15) or traditional moderate-intensity continuous training (MICT, n = 17). Method. After completing 10 sessions of supervised training participants were asked to perform HIIT or MICT three times per week for four weeks. Results. Individuals in HIIT (89 ± 11%) adhered to their prescribed protocol to a greater extent than individuals in MICT (71 ± 31%) as determined by training logs completed over one-month follow-up (P = 0.05, Cohen's d = 0.75). Minutes spent in vigorous physical activity per week measured by accelerometer were higher in HIIT (24 ± 18) as compared to MICT (11 ± 10) at one-month follow-up (P = 0.049, Cohen's d = 0.92). Cardiorespiratory fitness and systolic blood pressure assessed at one-month follow-up were equally improved (P's < 0.05). Conclusions. This study provides preliminary evidence that individuals with prediabetes can adhere to HIIT over the short-term and do so at a level that is greater than MICT. BODY.1. INTRODUCTION: It is estimated that ~35% of all US adults have prediabetes and are therefore at high risk for future development of type 2 diabetes (T2D) and cardiovascular disease (CVD, [1]). Regular exercise can help prevent the progression of prediabetes to T2D [2]. Landmark trials, including the Diabetes Prevention Program, have shown that a lifestyle intervention including 150 minutes of moderate-intensity physical activity (primarily walking) per week can reduce the incidence of T2D by ~58% [3]. These findings have contributed to the development of physical activity guidelines in several countries, which typically recommend at least 150 minutes per week of moderate-intensity activity to improve health [4–6]. Unfortunately, the vast majority of individuals fail to achieve this target. Specifically, large population-based studies in the US [7], UK [8], and Canada [9] that objectively measure physical activity by accelerometer suggest that only 15–20% of adults accumulate 150 minutes of moderate-intensity physical activity per week. Alternative forms of physical activity that can increase exercise adherence may therefore be attractive for the prevention of T2D. High-intensity interval training (HIIT) involves brief bursts of vigorous exercise separated by periods of rest or recovery. HIIT has garnered attention in recent years because it promotes cardiometabolic adaptations that are often superior to moderate-intensity continuous training (MICT) across a range of clinical populations [10, 11]. The benefits of HIIT are intriguing because adaptations to HIIT appear to occur with considerably less exercise time commitment than traditional exercise guidelines [12]. For example, time-efficient HIIT has been shown to rapidly improve glucose control in individuals with prediabetes [13, 14] and T2D [12, 15]. Despite the evidence for promising health benefits in individuals with prediabetes (for review see [16, 17]) and suggestions that HIIT may represent a time-efficient health promoting exercise strategy [18], there are no studies that have assessed adherence to HIIT in individuals at high risk for developing T2D. Epidemiological data suggests that more vigorous-intensity physical activity may confer greater benefits to metabolic health [19]. In support of the efficacy of high-intensity exercise, the American College of Sports Medicine and American Heart Association guidelines for maintaining fitness and health recommend 150 minutes of moderate or 75 minutes of vigorous physical activity for optimal health. These recommendations imply that equivalent health benefits are achieved in less time, provided the intensity of exercise is high. Advocating this lower volume of vigorous physical activity is of potential importance for exercise adherence as "lack of time" is the most commonly cited perceived barrier to regular exercise participation [20]. However, suggestions of prescribing vigorous-intensity physical activity for health promotion and T2D prevention have been met with reluctance in the scientific, medical, and lay communities, as it is presumed to be too intense and aversive (and thus less likely to be adhered to) for individuals who are sedentary and/or at increased risk of chronic disease [21, 22]. By virtue of the built-in rest periods and break in monotony, HIIT is different than high-intensity continuous exercise and may represent an alternative and more feasible option for introducing vigorous activity for health promotion in individuals with prediabetes. In fact, we have recently shown that a majority of inactive adults (n = 44) prefer HIIT (62%) to MICT (20%) and high-intensity continuous training (3%) after experiencing a single bout of each type of exercise [23]. The objective of this feasibility study was to determine the utility of HIIT as an exercise strategy for promoting short-term exercise adherence in comparison to traditional MICT. Individuals with prediabetes were randomized to a two-week supervised exercise intervention involving either HIIT or MICT. After the supervised phase, they were prescribed to maintain, on their own, thrice-weekly exercise sessions of their respective modality. The primary outcome was exercise adherence assessed by accelerometer and training logs after one month of independent exercise. As this preliminary pilot study was the first to examine independent exercise adherence to HIIT in comparison to traditionally prescribed MICT, the time point of one month was chosen a priori because it was deemed important to ascertain the feasibility of prescribing HIIT prior to conducting longer-term adherence trials. A secondary objective was to compare fitness and anthropometric changes in response to HIIT or MICT to determine which type of exercise might lead to greater cardiometabolic benefits. We hypothesized that HIIT would lead to greater adherence and improvements in cardiometabolic risk when compared to MICT. BODY.2. MATERIALS AND METHODS.2.1. PARTICIPANTS: Participants with prediabetes between the ages of 30 and 60 years were recruited from posters, online message boards, and word of mouth. After expressing interest, a phone interview was conducted to assess preliminary eligibility. Participants were considered to have prediabetes based on one of the following criteria: (1) physician-diagnosed, (2) HbA1c values between 5.7 and 6.4% (American Diabetes Association [24]) assessed using a clinically validated point-of-care monitor (HbA1c Now, Bayer Inc., Ontario, Canada), (3) fasting blood glucose of 5.6–6.9 mmol/L [24], and/or (4) a CANRISK questionnaire score of moderate/high (>21 [25]). To be eligible, participants also had to be inactive based on completion of less than two 30-minute bouts of moderate-intensity physical activity per week over the past six months. Participants completed the Canadian Society for Exercise Physiology (CSEP) Physical Activity Readiness Questionnaire-Plus (PAR-Q+ [26]) and were cleared for participation in vigorous activity by a CSEP Certified Exercise Physiologist. Exclusion criteria included diagnosed diabetes, glucose lowering medications, uncontrolled hypertension (blood pressure > 160/90), history of heart disease, previous myocardial infarction or stroke, and any contraindications to exercise. Thirty-two participants met the eligibility criteria and were enrolled in the study after providing written informed consent. Flow of participants is depicted in Figure 1. BODY.2. MATERIALS AND METHODS.2.2. PROCEDURES: The study design was approved by the Institutional Clinical Research Ethics Board. Following baseline testing (including accelerometry, cardiorespiratory fitness, blood pressure, and anthropometrics), eligible participants were randomized to HIIT or MICT conditions involving ten sessions of exercise performed over a 12-day period (i.e., Monday-Friday over two weeks with Saturday and Sunday as rest days). Exercise prescriptions for each condition were progressive in nature and were designed to be matched for external work [13]. Specifically, individuals randomized to HIIT began with four intervals lasting 1 minute each at an intensity that elicited ~90% peak heart rate (HRpeak) separated by 1-minute of low intensity recovery and increased to 10 × 1 min intervals by day 10, which was based on previously published studies in individuals with, and at risk for, T2D [12, 13, 15]. A 3-minute warm-up and 2-minute cool-down was incorporated into the HIIT sessions. Individuals randomized to MICT began with 20 minutes of continuous activity at ~65% HRpeak and gradually increased duration to 50 minutes by day 10. Participants self-selected exercise modality (walking outdoors, elliptical machine, treadmill walking, or stationary cycling) for each bout to encourage autonomy. Two trained research assistants (RAs) supervised participants during the training phase. To decrease reliance on staff and encourage the practice of independent exercise, participants completed 3 of the 10 training days (days 4, 7, and 9) at home unsupervised. Participants wore a heart rate monitor during each exercise session (supervised and unsupervised) to monitor exercise intensity and ensure compliance. In addition, participants recorded their exercise bouts in a logbook on both lab and home training days. Participants in both groups received 10 minutes of behavioural counselling each day they trained in the lab (for a total of 70 minutes). The aim of this one-on-one counselling was to further prepare participants for transition from lab-based training to independent exercise. The behavioural counselling was grounded in social cognitive theory [27] and identical in content for both conditions. Topics covered during counselling included overcoming exercise barriers, bolstering self-regulatory self-efficacy, planning, and increasing awareness for the physical and psychological benefits of exercise. Taken together, the short-term intervention served to introduce participants to their assigned exercise modality (HIIT or MICT) while providing evidence-based strategies and skills in attempts to bolster continual exercise adherence. During the supervised training phase, average HRpeak during HIIT (including rest intervals, warm-up, and cool-down) was 82 ± 3% HRpeak, confirming that HIIT sessions were in the vigorous domain. Average HRpeak during MICT was 67 ± 5%, confirming that MICT sessions were in the moderate domain. Following the supervised training phase, participants were instructed to maintain HIIT or MICT three days per week independently. Specifically, individuals randomized to HIIT were prescribed three exercise sessions per week involving 10 × 1-minute intervals at an interval intensity of ~90% HRpeak separated by 1 minute of easy recovery with a 3-minute warm-up and 2-minute cool down (for a total of 25 minutes of vigorous exercise), while participants randomized to MICT were prescribed three sessions per week of 50-minute continuous exercise at an intensity of ~65% HRpeak. Therefore, the HIIT group was prescribed vigorous exercise requiring 75 minutes of time commitment per week whereas the MICT group was prescribed 150 minutes of moderate activity per week. Training logs were provided and participants were instructed to estimate exercise intensity based on physiological cues and ratings of perceived exertion (RPE) taught during supervised training days. Accelerometers were used to objectively measure physical activity over seven days after four weeks of independent exercise. Adherence was assessed based on accelerometers and training logs. BODY.2. MATERIALS AND METHODS.2.3. MEASURES.2.3.1. HEART RATE: During the supervised training intervention, heart rate was recorded using downloadable Polar heart rate monitors (Polar FT7, Finland) to ensure that participants were working at the prescribed exercise intensity. BODY.2. MATERIALS AND METHODS.2.3. MEASURES.2.3.2. TRAINING LOGBOOKS: Participants were provided with a training logbook to complete during the intervention as well as during the subsequent four weeks of independent exercise. Participants were asked to record (a) the activity performed, (b) the minutes spent engaging in the activity, (c) the number of intervals conducted (for the HIIT condition exclusively), and (d) how hard the session was (RPE, CR-10 scale, [28]) for the session. Training logbooks were returned at the four-week follow-up and were analyzed by calculating the percentage adherence (i.e., number of exercise sessions divided by the number of prescribed sessions times 100% [29]) and average RPE. BODY.2. MATERIALS AND METHODS.2.3. MEASURES.2.3.3. ACCELEROMETERS: The Actigraph GT1M accelerometer (Actigraph, LCC, Fort Walton Beach, FL, USA) was used to objectively measure physical activity. This device is a dual-axis motion sensor that records vertical and horizontal accelerations allowing researchers to identify time spent in various physical activity intensities on a daily basis. Participants were instructed to wear the accelerometer for all waking hours of the day and to only remove it for sleeping or water-based activities. The monitor was worn on, or just above, their right hip for seven consecutive days prior to the intervention (baseline) and after 4 weeks of independent exercise. Accelerometer data were also collected during the intervention (days 7, 8, and 9) as a manipulation check. Each time participants wore an accelerometer, they were also asked to monitor their wear time with a self-report log. Epoch lengths were specified at 5 seconds and were summed as counts per minute. Valid wear time was ascertained using Choi and colleagues [30] parameters (i.e., nonwear is classified as 90 minutes of consecutive zeros, allowing for nonzero counts up to 2 minutes, if no counts are detected during the 30-minute counts before and after this interval). A trained researcher verified valid wear time using the raw data and self-report logs. Freedson et al. [31] cut-points were used to identify time spent in moderate (≥1,952 counts/min), vigorous (≥5,725 counts/min), and moderate-to-vigorous physical activity (MVPA, sum of moderate and vigorous) during wear time on valid days (i.e., ≥10 hours of wear time per day, without any excessive counts ≥20,000 counts/min). Purposeful exercise was operationalized as minutes spent in MVPA in bouts of at least 10 minutes (MVPA10+ [31]) based on physical activity guidelines, which specify bouts, should be accumulated in bouts of 10 minutes or more. MVPA10+ is a more appropriate measure of purposeful exercise [32] and therefore allows a more direct objective assessment of exercise adherence. Participants were required to have at least 3 valid days to be included in the analyses at baseline and one month [33]. Time spent in moderate activity and vigorous activity, total MVPA, and MVPA10+ were calculated for each valid day independently. Time spent in the various intensities was averaged across valid wear days and multiplied by seven to provide a weekly estimate of physical activity at baseline and one-month follow-up. BODY.2. MATERIALS AND METHODS.2.3. MEASURES.2.3.4. CARDIORESPIRATORY FITNESS: Participants performed a continuous incremental ramp maximal exercise test on an electronically braked cycle ergometer (Lode Excalibur, Netherlands) before intervention and at one-month follow-up to determine peak oxygen uptake (VO2peak), HRpeak, and peak power output (W peak). Expired gas was collected continuously by a metabolic cart (Parvomedics TrueOne 2400, Salt Lake City, Utah, USA) that was calibrated with gases of known concentration and a 3.0 L syringe prior to every test. The test started at 50 Watts and increased by 15 Watts/min. Verbal encouragement was provided to participants throughout the test, which was terminated upon volitional exhaustion or when revolutions per minute fell below 50. VO2peak was defined as the highest 30 sec average for VO2 (in L/min and mL/kg/min). Criteria for achieving VO2peak were (i) respiratory exchange ratio >1.15; (ii) plateau in VO2; (iii) reaching age-predicted HRpeak (220-age); and/or (iv) volitional exhaustion. BODY.2. MATERIALS AND METHODS.2.3. MEASURES.2.3.5. ANTHROPOMETRICS AND BLOOD PRESSURE: Body mass, height (SECA, 700 SECA, Hamburg, Germany), waist circumference (WC, measured at the level of the umbilicus [34]), and blood pressure (BP [35]) were measured in the morning after an overnight fast at baseline testing and one-month follow-up using standard procedures. BODY.2. MATERIALS AND METHODS.2.3. MEASURES.2.3.6. ANALYTICAL PLAN: Data were analyzed using SPSS Statistics (v22, 2013) on a per-protocol basis. Independent samples t-tests were conducted to examine differences between the two exercise conditions on (a) percentage adherence to the exercise prescription and (b) total number of minutes of purposeful physical activity, reported over the four weeks of independent exercise in the training logbooks. Analyses of covariance (ANCOVA) were conducted to examine differences in accelerometer-derived moderate physical activity, vigorous physical activity, total MVPA, and MVPA10+ between the conditions during the fifth week of independent exercise, after controlling for baseline measures as covariates. In order to examine changes in cardiorespiratory fitness, anthropometry, and blood pressure between baseline and one-month follow-up, a series of two-way (group × time) repeated measures ANOVA were conducted. Independent sample t-tests were conducted prior to running the repeated measures ANOVA to ensure there were no differences in baseline activity levels or fitness data between conditions. Significance was set at P ≤ 0.05. BODY.3. RESULTS: All 32 participants (17 in MICT, 15 in HIIT) completed the short-term supervised intervention with no complications (see Figure 1 for participation flow through the study). Descriptive statistics for these individuals are provided in Table 1. One participant in MICT did not return for one-month testing and was deemed lost to follow-up as she could not be reached after repeated attempts to contact. The remaining 16 MICT participants returned for 1-month follow-up. In the HIIT condition, all 15 participants completed the 2-week intervention. At one-month follow-up, two participants were deemed lost to follow-up and did not provide specific reasons. Three participants were unable to complete one-month follow-up for reasons unrelated to the study (car accident, work-related injury, diagnosis of depression, and change in medication). Therefore, a total of 16 and 10 participants completed one-month follow-up for MICT and HIIT, respectively. Of the individuals that completed one-month follow-up, accelerometer data were available for 10 individuals in the HIIT condition and 13 individuals in the MICT condition (two participants did not accrue the minimum of valid wear days and one accelerometer malfunctioned displaying counts >20,000 during wear time). Mean (±SD) daily wear time was 855 (±175) minutes at baseline and 846 (±63) minutes at follow-up. Eighty-six percent and 98% of participants provided at least 5 valid wear days at baseline and one-month follow-up, respectively. BODY.3. RESULTS.3.1. MANIPULATION CHECK: Accelerometer data collected during the intervention revealed that the accelerometers were able to clearly detect intermittent patterns of HIIT in comparison to the continuous moderate activity characteristic of MICT (see Figures 2(a) and 2(b)). Visual inspection of the accelerometer data (days 7 and 9) and confirmation of exercise heart rate data (days 4, 7, and 9) revealed that all participants were working at the prescribed exercise intensity during the unsupervised training days. BODY.3. RESULTS.3.2. SELF-REPORT PHYSICAL ACTIVITY BEHAVIOUR: To examine differences in percentage of overall adherence to the exercise prescription over one-month follow-up, an independent t-test was conducted. Results revealed a significant difference in adherence rates between HIIT and MICT, t(18.96) = 2.08, P = 0.05, Cohen's d = 0.75. Specifically, individuals in the HIIT condition (89 ± 11%) adhered to their prescribed protocol, to a greater extent than individuals in the MICT condition (71 ± 31%). The same analysis revealed a significant difference in average ratings of perceived exertion between the two conditions, t(23) = 2.80, P = 0.01, Cohen's d = 1.19. As expected, HIIT was perceived to be of greater intensity than MICT (HIIT = 7.4 ± 1.7, MICT = 5.4 ± 1.8). BODY.3. RESULTS.3.3. OBJECTIVE PHYSICAL ACTIVITY BEHAVIOUR: A series of ANCOVA revealed no significant differences between the HIIT and MICT conditions in the average number of minutes spent in (a) purposeful bouts of MVPA per week (i.e., MVPA10+), (b) overall MVPA per week, and (c) moderate activity per week (P's > 0.05). Means and standard deviations for each condition are displayed in Table 2. There was, however, a significant difference between the average number of minutes spent in vigorous physical activity per week between the two conditions, F(1, 20) = 4.41, P = 0.049, Cohen's d = 0.92, with HIIT being greater than MICT. BODY.3. RESULTS.3.4. CHANGES IN FITNESS PARAMETERS: Changes in fitness and anthropometric measures from baseline to one-month follow-up for HIIT and MICT are shown in Table 3. Repeated measures ANOVA revealed a significant main effect of time for absolute VO2peak, F(1, 24) = 20.96, P < 0.001, Cohen's d = 1.92; relative VO2peak, F(1, 24) = 14.82, P < 0.001, Cohen's d = 1.62; and peak power output, F(1, 24) = 14.08, P < 0.001, Cohen's d = 1.57, indicating an increase in cardiorespiratory fitness assessed at one-month follow-up. There were no main effects for group or group by time interactions for these fitness parameter variables, indicating that there were no differences in the degree of change experienced between HIIT and MICT over time. Maximal heart rate achieved at the end of the graded exercise test, body mass, and waist circumference did not change significantly from baseline to one-month follow-up in either HIIT or MICT (P's > 0.05). There was a significant main effect of time for systolic blood pressure, F(1, 24) = 17.36, P < 0.001, Cohen's d = 1.75, such that it decreased at one-month follow-up for both conditions. Diastolic blood pressure tended to be lower at one-month follow-up; however, this main effect for time did not reach statistical significance (P = 0.11, Cohen's d = 0.13). BODY.4. DISCUSSION: Physical activity is key in the prevention of T2D and cardiovascular disease in individuals with prediabetes; yet exercise adherence is low in this population [36]. HIIT has recently been touted as an effective and time-efficient exercise option for improving cardiometabolic health [18, 37]. However, adherence to HIIT outside of a supervised lab-based intervention has not been tested. The present study demonstrates that individuals with prediabetes can adhere to HIIT independently for one month following a very brief supervised laboratory intervention. Interestingly, adherence to HIIT, assessed by self-report in free-living conditions, was greater than standard care exercise involving MICT. In addition, only those individuals randomized to HIIT increased their vigorous physical activity (>6 METS) from baseline to one-month after intervention. Current physical activity guidelines recognize that vigorous physical activity can elicit health benefits in less time when compared to moderate-intensity physical activity. Indeed, guidelines imply that 75 minutes of vigorous activity is equivalent to 150 minutes of moderate activity [38]. However, it is generally assumed that adherence to vigorous exercise will be lower than moderate exercise. This appears based on studies examining the affective response to acute bouts of exercise at different intensities [39]. Our findings provide initial evidence that short-term adherence to vigorous exercise performed as HIIT may be superior to traditional moderate-intensity continuous exercise. In addition to time-efficiency, vigorous exercise may elicit benefits over and above those accrued through moderate-intensity activity. Specifically, NHANES data suggests that individuals accumulating more vigorous-intensity physical activity have reduced odds of metabolic syndrome (closely related to prediabetes), independent of total physical activity levels [19]. Further, when matched for energy expenditure, vigorous physical activity had a greater influence on metabolic syndrome than did moderate intensity physical activity. In other words, it appears that vigorous-intensity physical activity has an important independent role in the prevention of cardiometabolic disease. By design, only those randomized to HIIT in this feasibility study would be expected to increase their minutes of vigorous-intensity physical activity. Indeed, those randomized to HIIT increased their vigorous-intensity physical activity by ~12 minutes per week while individuals randomized to MICT in this study experienced no increase. There were no significant differences between conditions in minutes spent in moderate-intensity physical activity or MVPA10+ assessed at one-month follow-up. This was despite individuals in MICT being prescribed 150 minutes of moderate-intensity physical activity per week as compared to individuals in HIIT being prescribed 75 minutes of intermittent vigorous-intensity physical activity. This suggests that objectively measured moderate and total physical activity increased to a similar extent in both groups despite those in the HIIT group being prescribed only ~50% the amount of time to exercise as those in MICT. Taken together, individuals in HIIT effectively increased time spent in vigorous-intensity physical activity and also saw increases in their moderate-intensity physical activity that were comparable to individuals in MICT. Assessing levels of physical activity objectively by accelerometer following supervised training interventions remains largely unexplored. Our findings indicate that physical activity levels can be increased for up to one month following a brief (10-day) exercise intervention as measured by accelerometry. Specifically, time spent in MVPA10+ was increased following both HIIT and MICT. This was further confirmed with the training logbook data collected in our sample, with >70% adherence measured in both groups. Both HIIT and MICT increased VO2peak assessed at one-month follow-up, indicating an improvement in fitness. This is promising considering all participants were training under their own volition for one month. A recent meta-analyses reported that the increase in VO2peak following 12–16 weeks of supervised HIIT is approximately double the increase seen following MICT [17]. Thus, the increase in adherence and vigorous physical activity in the HIIT group may, if adhered to for longer periods of time, lead to greater reductions in cardiometabolic risk when compared to MICT. The present feasibility study is the first of its kind to assess adherence to HIIT following a supervised laboratory intervention. Such research is needed to determine whether HIIT is a viable health-enhancing exercise strategy in the real world. However, our study is not without limitations. First, we examined independent exercise over a short time period. The results are promising but certainly more research is needed to determine whether individuals with prediabetes can adhere to HIIT over the long term. Second, our sample size was small and larger studies are warranted to confirm and expand our preliminary findings. Loss to follow-up was similar in HIIT (2 participants) versus MICT (1 participant), although we were limited by the fact that a further 3 participants in the HIIT group did not complete one-month follow-up testing due to factors unrelated to the study protocol. There are natural safety concerns when implementing vigorous exercise. All participants in our study completed two weeks of supervised HIIT without any reported complications but our study was not designed to examine safety and musculoskeletal injuries in response to HIIT compared to traditional MICT. The small sample size of the current investigation does not permit an accurate assessment of the safety or injury risk potential of HIIT or MICT. BODY.5. CONCLUSION: Adherence to exercise in individuals at risk for T2D is remarkably low. HIIT has recently gained popularity as a potential health-enhancing exercise strategy that is time-efficient and distinct from traditional MICT [17, 18]. However, the application of HIIT for persons at risk of chronic disease has been questioned due to perceptions that adherence to vigorous-intensity physical activity is unlikely. In this feasibility study, we provide preliminary evidence that individuals with prediabetes can adhere to HIIT over the short term and do so at a level that is greater than MICT. These findings support the potential utility of HIIT as an alternative exercise strategy that could bolster exercise adherence. Future studies are warranted to assess long-term adherence, cardiometabolic benefits, and safety of HIIT in individuals with prediabetes.

TITLE: A Randomised Trial of Subcutaneous Intermittent Interleukin-2 without Antiretroviral Therapy in HIV-Infected Patients: The UK–Vanguard Study ABSTRACT.OBJECTIVE:: The objective of the trial was to evaluate in a pilot setting the safety and efficacy of interleukin-2 (IL-2) therapy when used without concomitant antiretroviral therapy as a treatment for HIV infection. ABSTRACT.DESIGN AND SETTING:: This was a multicentre randomised three-arm trial conducted between September 1998 and March 2001 at three clinical centres in the United Kingdom. ABSTRACT.PARTICIPANTS:: Participants were 36 antiretroviral treatment naïve HIV-1-infected patients with baseline CD4 T lymphocyte counts of at least 350 cells/mm3. ABSTRACT.INTERVENTIONS:: Participants were randomly assigned to receive IL-2 at 15 million international units (MIU) per day (12 participants) or 9 MIU/day (12 participants) or no treatment (12 participants). IL-2 was administered by twice-daily subcutaneous injections for five consecutive days every 8 wk. ABSTRACT.OUTCOME MEASURES:: Primary outcome was the change from baseline CD4 T lymphocyte count at 24 wk. Safety and plasma HIV RNA levels were also monitored every 4 wk through 24 wk. The two IL-2 dose groups were combined for the primary analysis. ABSTRACT.RESULTS:: Area under curve (AUC) for change in the mean CD4 T lymphocyte count through 24 wk was 129 cells/mm3 for those assigned IL-2 (both dose groups combined) and 13 cells/mm3 for control participants (95% CI for difference, 51.3–181.2 cells/mm3; p = 0.0009). Compared to the control group, significant increases in CD4 cell count were observed for both IL-2 dose groups: 104.2/mm3 (p = 0.008) and 128.4 cells/mm3 (p = 0.002) for the 4.5 and 7.5 MIU dose groups, respectively. There were no significant differences between the IL-2 (0.13 log10 copies/ml) and control (0.09 log10 copies/ml) groups for AUC of change in plasma HIV RNA over the 24-wk period of follow-up (95% CI for difference, −0.17 to 0.26; p = 0.70). Grade 4 and dose-limiting side effects were in keeping with those previously reported for IL-2 therapy. ABSTRACT.CONCLUSIONS:: In participants with HIV infection and baseline CD4 T lymphocyte counts of at least 350 cells/mm3, intermittent subcutaneous IL-2 without concomitant antiretroviral therapy was well tolerated and produced significant increases in CD4 T lymphocyte counts and did not adversely affect plasma HIV RNA levels. BODY.INTRODUCTION: The development of combination antiretroviral therapy for the treatment of HIV infection has produced a marked decline in AIDS and death, but enthusiasm for these treatments in patients with early stages of HIV infection has been tempered by long-term toxicity, such as lipodystrophy and lactic acidosis, difficulties with maintaining rigorous compliance, and the evolution of drug resistant HIV [1–5]. The use of these treatments for prolonged periods may not be achievable, and treatment guidelines continue to change [6–8]. For these reasons, the development of alternate therapies or treatment strategies continues. One such strategy is the administration of intermittent interleukin-2 (IL-2) to augment or preserve immune function [9–11]. IL-2 is a cytokine that in vivo is secreted by activated T lymphocytes. IL-2 regulates the proliferation, differentiation, and survival of lymphocytes, including CD4 T cells [12]. Increases in CD4 T lymphocyte count arising from the use of intermittent IL-2 in combination with antiretroviral therapy have been demonstrated consistently in a number of randomised clinical trials [13–21]. The use of recombinant IL-2 has been associated with transient rises of plasma HIV RNA levels in some patients [13–14]. However, no significant persistent increase in HIV RNA has been observed in IL-2 recipients when compared to controls treated with combination antiretroviral therapy [13–21]. In fact, a pooled analysis of long-term follow-up data from the first three randomised controlled trials of intermittent IL-2 suggested that IL-2 in combination with antiretroviral therapy produced larger decreases in viral load than antiretroviral therapy alone [22]. One randomised study similarly found that IL-2 in combination with antiretroviral therapy produced larger decreases in viral load than antiretroviral therapy alone [18], although these findings were not observed in other randomised studies of short duration [13–17,19–21]. The purpose of this randomised controlled pilot trial was to determine whether intermittent IL-2 therapy administered without concomitant antiretroviral therapy safely increased CD4 T lymphocyte counts. Ultimately, if this strategy were to be successful, it might lead to a delay in the time at which chronic antiretroviral therapy would need to be initiated. Further trials would be required from which to draw any definitive conclusions. BODY.METHODS.PARTICIPANTS: Patients 18 y or older who had HIV-1 infection and CD4 T lymphocyte counts of at least 350 cells/mm3 at screening were eligible for enrollment if they had never received IL-2 or antiretroviral therapy. Additional eligibility criteria required that participants had no history of AIDS-defining illness and had received no corticosteroids, cytotoxic chemotherapy, or experimental cytotoxic therapy in the preceding 4 wk. Participants were required to have blood cell profiles and serum chemistry values within acceptable ranges. The study was approved by the National Institute of Allergy and Infectious Diseases' institutional review board and also by each site's research ethics committee. All participants provided written informed consent. An independent data and safety monitoring board reviewed safety and efficacy data on one occasion during the conduct of the trial. Patients were recruited and followed, and one of three participants' sites provided primary healthcare. BODY.METHODS.INTERVENTIONS: Participants were randomly assigned in equal proportions to intermittent subcutaneous injections of IL-2 at two dosage levels (4.5 million international units [MIU] or 7.5 MIU twice daily for five consecutive days every 8 wk) or no treatment. The study was not placebo controlled, as the constitutional side effects of IL-2 make blinding impractical. IL-2 (aldesleukin [Proleukin], Chiron, Emeryville, California, United States) was administered either in a hospital clinic or through an outpatient department. Dose modifications in decrements of 1.5 MIU were allowed for the management of clinical or laboratory toxicities. BODY.METHODS.OBJECTIVES: Our primary hypothesis was that intermittent cycles of IL-2 would result in significant increases in CD4 T lymphocyte counts relative to no therapy. Our secondary hypotheses were that there would be no significant increases in plasma HIV RNA between treatment groups and that IL-2 cycles would be safe and well tolerated, in keeping with the experience of earlier studies. BODY.METHODS.OUTCOMES: Over the 24-wk study period, participants were evaluated monthly, with additional visits of the IL-2-treated participants at day 5 of each cycle. Clinical assessments involved physical examination, complete blood counts with differentials and platelet counts, serum chemistry profiles, T lymphocyte subset enumeration, and plasma HIV RNA quantitation. Absolute CD4 and CD8 T lymphocyte counts were determined from 100 μl of fresh EDTA blood, by direct immunofluorescence using the Ortho-Trio method [23,24]. Particle-associated plasma HIV RNA concentrations were determined using a branch-chain DNA assay (Chiron) with a lower limit of detection at 50 copies/ml plasma [25,26]. Flow cytometry and HIV quantitation were performed at a single laboratory throughout the study. A treatment failure was prospectively defined as any patient who experienced either at least a 1-log increase in plasma HIV RNA on two consecutive occasions more than 29 d apart in the absence of an intercurrent illness, a greater than 25% reduction from the baseline CD4 T lymphocyte count on two occasions more than 29 d apart in the absence of intercurrent illness, and/or initiation of antiretroviral therapy for any reason. BODY.METHODS.SAMPLE SIZE: Sample size was specified as 36 participants in order to provide 80% power to detect a difference between both IL-2 dose groups combined and the control group of 150 CD4 T lymphocytes/mm3 at a two-sided significance level of 5%. With this sample size, power was also 80% to detect a difference of 0.7-log copies of HIV RNA per cubic millimeter between treatment groups. These estimates were considered conservative because they did not consider the averaging of multiple follow-up measurements of CD4 and HIV RNA that were to be used for the primary analysis. BODY.METHODS.RANDOMISATION: Randomisation was performed through using a central randomisation office located at the University of Minnesota. Computer-generated randomisation lists were generated at this office using a blocking factor of 6. Allocation of patients was by facsimile request from participant sites to the University of Minnesota randomisation office. BODY.METHODS.STATISTICAL METHODS: The primary end points of the study were area under the curve (AUC) for CD4 T lymphocyte count change from baseline and AUC for plasma HIV RNA change from baseline over the 24 wk of the study. The AUC estimates were standardised for the timing of the last measurement for each person [27]. Secondary end points included the comparative frequency of protocol-defined treatment failure, changes in percentage of CD4 T lymphocytes, the number and percentage of CD8 T lymphocytes, the CD4/CD8 T lymphocyte ratio, and safety data. Plasma HIV RNA data were log10 transformed prior to analyses. Baseline was the average of three measurements made within 30 d of randomisation. Levels at 24 wk were the average of three measurements within 2 wk of one another. Follow-up levels at other weeks were based on a single reading. Data for patients assigned IL-2 were analysed on an intention-to-treat basis; i.e., all follow-up measurements were included even if the patient was not taking IL-2. Data from one participant in the control group who initiated antiretroviral therapy are only included up to the time antiretroviral therapy commenced. As stated in the protocol, for the primary analyses, the combined data from the two IL-2 dose groups were compared with control participants. Pairwise comparisons between each of the randomly assigned treatment arms and the control group and with one another were also carried out. In addition to AUC analyses, longitudinal regression methods that take into account correlations within and between participants were used to estimate the average difference between treatment groups (IL-2 and control) over follow-up and to estimate the differences in CD4 T lymphocytes and plasma HIV RNA at each follow-up timepoint [28]. These analyses were carried out using the PROC Mixed procedure of the SAS Institute (Cary, North Carolina, United States). Other analyses were also carried out with the use of the SAS Version 8 software. All statistical tests were two-sided, with a p-value of <0.05, indicating statistical significance. BODY.RESULTS.PATIENT DISPOSITION, RECRUITMENT, AND BASELINE CHARACTERISTICS: A total of 45 patients were screened for participation. Disposition of the cohort over the entire period of follow-up is shown in Figure 1. Prior AIDS diagnoses (one participant) and low CD4 cell counts (eight participants) accounted for all ineligible screens. The remaining 36 participants were enrolled between September 1998 and August 1999. Of these, 12 were randomly assigned to each of the IL-2 treatment groups (giving a total of 24 IL-2 recipients) and 12 to the control group. No patients were lost to follow-up for the purposes of clinical assessment. However, five control patients and seven IL-2 recipients did not contribute laboratory data to the week 24 assessments. The baseline characteristics of the three groups were similar (Table 1). Figure 1Flow Diagram of Study Design and Patient Disposition Table 1 Demography and Baseline Characteristics BODY.RESULTS.EXPOSURE TO IL-2: The exposure of participants randomised to receive IL-2 is summarised in Table 2. During the 24-wk study period, most participants in either treatment group completed three treatments with IL-2. However, participants randomised to receive the 7.5 MIU dose reduced more frequently than those randomised to 4.5 MIU, and at week 24 the average unit dose of IL-2 was only 5.8 MIU. This is in contrast with the 4.2 MIU average unit dose for patients randomised to receive IL-2 at 4.5 MIU/dose. Table 2 Exposure of Participants to IL-2, Shown by Number of Participants Commencing Each Cycle and Average Unit Dose in Each Cycle by Treatment Group BODY.RESULTS.IMMUNOLOGIC MEASURES: CD4 T lymphocyte counts over follow-up for each treatment group, based on longitudinal regression, are illustrated in Figure 2. At each follow-up visit except the 8-wk visit, CD4 T lymphocyte increases from baseline were significantly greater for those assigned IL-2 compared to control. At 24 wk, this difference was 132 cells/mm3 (p = 0.02). AUC for change in the mean CD4 T lymphocyte count through 24 wk was 129 cells/mm3 for those assigned IL-2 (both dose groups combined) and 13 cells/mm3 for control participants (95% CI for difference, 51.3–181.2 cells/mm3; p = 0.0009). As shown in Table 3, the AUC for change in CD4 T lymphocyte count from baseline was significantly greater for those assigned IL-2 (+129.4 cells/mm3) compared to control (+13.1 cells/mm3) (difference, 116.2 cells/mm3; 95% CI for difference, 51.3–181.2 cells/mm3). Longitudinal regression analysis yielded a similar difference between treatment groups (132.3 cells/mm3; p = 0.0001). Statistically significant differences were also observed for the pairwise comparisons of each IL-2 dose with control (p = 0.002 for 7.5 MIU versus control; p = 0.008 for 4.5 MIU versus control), but not for the comparison of IL-2 doses 4.5 MIU versus 7.5 MIU (p = 0.57). The numbers of CD8 T lymphocytes in each treatment group remained stable throughout the period of observation in each treatment group (unpublished data). Figure 2CD4+ Cell Count with 95% CI and Number of Patients with CD4 Cell Count Used in the Analysis Table 3 AUC for CD4 T Lymphocyte Count and HIV RNA Change from Baseline BODY.RESULTS.VIROLOGIC MEASURES: The AUC for change in plasma HIV RNA from baseline was similar for those assigned IL-2 (0.13 log10 copies/ml) and control (0.09 log10 copies/ml) (95% CI for difference, −0.17 to 0.26; p = 0.70) (Table 3). Similarly, there were no significant differences between treatment groups for mean changes in log10 HIV RNA at any timepoint in this parameter. Analyses based on longitudinal regression produced results similar to AUC (average difference, 0.11; p = 0.32). Evaluation of plasma HIV RNA at the end of each 5-d cycle of IL-2 administration revealed a transient increase of at least 0.5 log10 HIV RNA in 32% of patients in cycle 1, 40% of patients in cycle 2, and 37% of patients in cycle 3. These increases ranged from 0.5 to 1.6 log10 HIV RNA. There were no statistically significant differences between the 4.5 MIU and 7.5 MIU dose groups (unpublished data). BODY.RESULTS.TOXICITIES AND SAFETY DATA: Following randomisation, one participant in each IL-2 dose group declined IL-2 therapy before receiving their first cycle, but their data have been included in the 24-wk analyses. One additional participant in the 4.5 MIU dose group permanently discontinued IL-2 after completing one cycle, citing personal reasons. Two additional participants in the 7.5 MIU dose group discontinued therapy, citing toxicity (one participant after completing two cycles), and multiple reasons, including toxicity and CD4 T lymphocyte count decrease (one participant after completing three cycles), as the reasons for discontinuation. No new side effects were encountered. The most numerous dose-limiting events were constitutional signs and symptoms, including fever and nausea. Five grade 4 events were reported during the trial as follows: diarrhea (4.5 MIU), pancreatitis (7.5 MIU), abdominal pain with hyperamylasemia (7.5 MIU), elevated alanine aminotransferase (4.5 MIU), and abdominal cramps with diarrhea (control). There were no deaths during follow-up. BODY.RESULTS.PROTOCOL-DEFINED TREATMENT FAILURES AND CLINICAL DISEASE PROGRESSION: On the basis of the protocol definition, three patients experienced treatment failure during the 24-wk follow-up period: two control participants (one commenced antiretroviral therapy at week 16 after being diagnosed with nonvisceral Kaposi's sarcoma and one had a decreased CD4 count); and one participant assigned 7.5 MIU IL-2 (who commenced antiretroviral therapy at week 24 after being diagnosed with visceral Kaposi's sarcoma). BODY.DISCUSSION.OVERALL EVIDENCE: Studies of intermittent administration of IL-2 in combination with antiretroviral therapy have demonstrated significant and sustained increases in CD4+ T cell count resulting from a preferential increase in CD4+ T cell survival and decreased cell turnover in the setting of decreased immune activation [29,30]. In this pilot study, CD4 T lymphocyte counts increased significantly in the IL-2 monotherapy arm, compared with the control group, at week 24. Importantly, these increases were not associated with sustained increases in HIV RNA load. Measurements of plasma HIV RNA in IL-2 recipients at the end of each 5-d cycle of IL-2 indicated that transient bursts of viremia were consistent with those observed in previous trials in which patients were treated with what would currently be regarded as suboptimal regimens of antiretroviral therapy [3–15]. Despite these transient bursts, there were no long-term changes in viral load. The clinical significance of the increase in CD4 T lymphocytes that are produced under the influence of IL-2 therapy is uncertain and has led to the initiation of two large clinical endpoint studies (SILCAAT and ESPRIT [31]) to assess the clinical consequences of IL-2 in combination with antiretroviral therapy. SILCAAT and ESPRIT are sister studies, the former assessing HIV-infected participants with between 50 and 300 cells/mm3 and HIV RNA levels of <10,000 copies/ml, and the latter in participants with ≥300 cells/mm3 and no restriction on viral load. Prior to ESPRIT, four Vanguard studies were conducted to address methodological and operational issues for studies of IL-2 therapy [19–21]. The pilot study reported here, the UK–Vanguard, was initiated to examine IL-2 treatment without antiretroviral medication. A striking feature of the data from this study relative to that from the others is a relatively blunted CD4 T lymphocyte count response. The mean increase in CD4 T lymphocyte count observed at 24 wk compared to control was 132 cells/mm3, considerably less than that observed in the other three Vanguard studies (an average increase of 328, 459, and 347 from baseline above those achieved by the control groups [19–21]. BODY.DISCUSSION.INTERPRETATION: The reasons for the clearly blunted response in the current trial are not known, but one possible explanation is that, in patients with ongoing uncontrolled virus replication, a larger proportion of the newly emerging CD4 T lymphocytes are eliminated. It is less likely that these differences in CD4 T lymphocyte count increases are due to the doses of IL-2 actually administered. In the other Vanguard studies, the average total doses of IL-2 given over the first three cycles to the 7.5 MIU arm (maximum of 225 MIU) were about 198–225 MIU, while in this study the average total dose of IL-2 given over the first three cycles to the 7.5 MIU arm was only minimally lower (192 MIU). In this protocol we did not assess T-cell function. Neither did we choose to examine distinct subsets of CD4+ T lymphocytes that are believed to be of significance in the immunopathogenesis of HIV disease. In many other trials of subcutaneous IL-2, a consistent observation has been that the functionality and/or immunophenotype of T cells present prior to IL-2 administration remains following IL-2 treatment, although in some settings there appears to be a preferential expansion of naïve CD4+ T lymphocytes [29–30,32]. A role for IL-2 in restoring or perhaps inducing anti-HIV-specific immune responses has not been part of the hypothesis behind the therapeutic evaluation of IL-2 in our hands. In the current study we were encouraged by the absence of any within- or between-treatment group differences in HIV RNA plasma load. We infer from this observation that any active anti-HIV-specific immune responses were unaffected by the administration of IL-2 in this study. BODY.DISCUSSION.GENERALISABILITY: As observed in other trials, this study demonstrates that IL-2 is well tolerated at doses that produce significant increases in CD4 T lymphocyte counts. Toxicities occurred only during the intermittent cycles of IL-2, were mostly mild to moderate in severity, and were managed with a comprehensive approach that included dose modification and the use of medications to control signs and symptoms. Grade 4 events occurred once in four IL-2 recipients and once in a control patient. Across the 66 treatment cycles initiated in this trial, this number of grade 4 events indicates a prevalence of approximately 6% for events of this severity. No novel toxicities were observed. While the sample size for this study was only 36 and not all patients provided data at the final timepoint for the primary measure of interest, the lower limit of the 95% CI for CD4 T lymphocyte (51 cells/mm3) and the upper limit for plasma HIV RNA difference from control (0.32 copies/ml) indicate that at least modest CD4 T lymphocyte increases are possible without adversely affecting viral load. Thus, these findings are sufficiently encouraging to plan other studies of intermittent monotherapy with IL-2 to study its potential for increasing or maintaining CD4 T cell counts and prolonging the time to initiation of antiretroviral therapy. In summary, this pilot study demonstrated that intermittent IL-2 therapy alone can be used to safely and significantly improve CD4 T lymphocyte counts in HIV-infected individuals with baseline CD4 T lymphocyte counts >350 cells/mm3 with no detrimental effect on HIV replication as measured by plasma HIV RNA load. Ongoing studies are addressing the clinical consequences of these CD4 T lymphocyte rises and the significance of these findings in terms of current models used to describe the interplay between virus and host in the setting of HIV infection. BODY.SUPPORTING INFORMATION: CONSORT ChecklistClick here for additional data file.(54 KB DOC) Trial ProtocolClick here for additional data file.(337 KB DOC)

TITLE: L-lysine as adjunctive treatment in patients with schizophrenia: a single-blinded, randomized, cross-over pilot study ABSTRACT.BACKGROUND: Accumulating evidence suggests that the brain's nitric oxide (NO) signalling system may be involved in the pathophysiology of schizophrenia and could thus constitute a novel treatment target. The study was designed to investigate the benefit of L-lysine, an amino acid that interferes with NO production, as an add-on treatment for schizophrenia. ABSTRACT.METHODS: L-lysine, 6 g/day, was administered to 10 patients with schizophrenia as an adjunctive to their conventional antipsychotic medication. The study was designed as a single-blinded, cross-over study where patients were randomly assigned to initial treatment with either L-lysine or placebo and screened at baseline, after four weeks when treatment was crossed over, and after eight weeks. ABSTRACT.RESULTS: L-lysine treatment caused a significant increase in blood concentration of L-lysine and was well tolerated. A significant decrease in positive symptom severity, measured by the Positive And Negative Syndrome Scale (PANSS), was detected. A certain decrease in score was also observed during placebo treatment and the effects on PANSS could not unequivocally be assigned to the L-lysine treatment. Furthermore, performance on the Wisconsin Card Sorting Test was significantly improved compared to baseline, an effect probably biased by training. Subjective reports from three of the patients indicated decreased symptom severity and enhanced cognitive functioning. ABSTRACT.CONCLUSIONS: Four-week L-lysine treatment of 6 g/day caused a significant increase in blood concentration of L-lysine that was well tolerated. Patients showed a significant decrease in positive symptoms as assessed by PANSS in addition to self-reported symptom improvement by three patients. The NO-signalling pathway is an interesting, potentially new treatment target for schizophrenia; however, the effects of L-lysine need further evaluation to decide the amino acid's potentially beneficial effects on symptom severity in schizophrenia. ABSTRACT.TRIAL REGISTRATION: NCT00996242 BODY.BACKGROUND: Schizophrenia is a severely debilitating brain disorder that poses a serious healthcare problem worldwide. Available antipsychotics show efficacy in alleviating psychotic symptoms. However, negative symptoms and the cognitive deficits are to a large extent resistant to antipsychotic treatment [1,2]. Thus, there is a need to find new treatment strategies to improve the treatment of these symptoms and deficits. One such candidate target for novel treatments may be the nitric oxide (NO) signalling system of the brain. As such, translational evidence for this contention can be derived from the observations that methylene blue, which blocks NO-dependent soluble guanylate cyclase-mediated intracellular signalling, was shown to exert therapeutic effects as an adjuvant to established antipsychotics in the treatment for schizophrenia in humans [3]. In addition, a more recent study showed that methylene blue attenuated psychotomimetic-, that is, phencyclidine (PCP), induced behavioural alterations in mice [4]. Despite that the principle mechanism of action of PCP is glutamatergic N-methyl D-aspartate (NMDA) receptor antagonism, PCP has secondary effects on several other neurotransmitter systems (see, for example, [5]) as well as on NO-signalling [6]. Furthermore, the inducible NO synthase inhibitor, minocycline, was recently suggested to have beneficial effects as an add-on treatment in patients with schizophrenia [7,8]. Thus, accumulating evidence indicates that alterations in NO function may be involved in the pathophysiology of schizophrenia and these original findings motivate further investigations of the potential utility of NO modulation as a novel pharmacological treatment rationale for schizophrenia (for review, see [9]). BODY.BACKGROUND.PRECLINICAL FINDINGS SUPPORTING A "NO DYSREGULATION HYPOTHESIS FOR SCHIZOPHRENIA": To better understand the underlying pathophysiology of schizophrenia, several methods have been developed to model schizophrenia in humans and experimental animals. To this end, pharmacological challenge with PCP has been shown to produce a psychotic condition in humans that includes all major symptoms of schizophrenia. Thus, the "PCP model of schizophrenia" has proved to be an important tool for increasing our understanding of the disorder and is considered to have significant heuristic value in the development of novel therapeutic treatment strategies [10]. Interestingly, our preclinical studies have shown that pre-treatment with NO synthase (NOS) inhibitors effectively block the disruptive effect of PCP on behaviours involving several cognitive domains such as pre-attentive information processing, non-associative learning, selective attention, cognitive flexibility, working and long-term memory, as well as deficits in social interaction in rodents [11-20]. These observations suggest that inhibition of NOS is able to counteract very complex behavioural effects of PCP. BODY.BACKGROUND.INHIBITION OF NO PRODUCTION BY L-LYSINE: A NEW TREATMENT OPTION FOR PATIENTS WITH SCHIZOPHRENIA?: NO is produced from the amino acid L-arginine and molecular oxygen in a chemical reaction catalyzed by NOS. Interestingly, the essential amino acids L-lysine and L-arginine share a highly specific membrane bound transport system, the cationic amino acid transporter [21]. This transport system seems critical in mediating the influx of L-arginine across the blood-brain barrier [22]. Furthermore, in vitro studies have shown that saturation of the transporter with L-lysine inhibits transport of L-arginine, depletes intra-cellular stores of L-arginine [23], and reduces NO production [24]. As noted above, our previous findings suggest that the schizophrenia-like behavioural effects of PCP in experimental animals can be ameliorated by pre-treatment with a NOS inhibitor. In analogy it may be speculated that inhibition of L-arginine transport by L-lysine treatment would decrease NO levels and, thereby, attenuate PCP-induced behavioural effects. In support of this argument we recently showed that sub-chronic pre-treatment with L-lysine ameliorates PCP-induced disruption of prepulse inhibition, a measure of pre-attentive information processing, in mice in a dose-related manner without affecting basal prepulse inhibition [25]. Moreover, using a NO selective microsensor and in vivo voltammetry in awake freely moving rats, we recently found that acute L-lysine administration decreased NO levels in the rat prefrontal cortex [26]. A competitive antagonism of L-arginine transport across the blood-brain barrier and a depletion of L-arginine supply for NO synthesis may explain these findings. A relative lack of substrate for NO production would prevent a hypothesized PCP-induced increase in NO levels and thus the disruptive effect of PCP on cognitive function. The aim of the present study was to investigate the relevance of these contentions in humans. To this end, the effect of adjunctive L-lysine treatment on symptom severity and cognition was studied in patients with schizophrenia. Ten patients with a diagnosis of schizophrenia were treated with L-lysine, 6 grams daily, or placebo for four weeks in a cross-over design. Outcome measures were assessed at baseline, after four weeks and after eight weeks. BODY.METHODS.SUBJECTS: Eleven well-defined psychiatric outpatients were recruited from the following three clinics: Nå-Ut Teamet, Psykosteamet Järntorget, and Psykosvård i Väster, the Sahlgrenska University Hospital, Gothenburg, Sweden. Inclusion criteria consisted of a DSM-IV diagnosis of schizophrenia (F295.xx) and that the patient had been in a stable phase of illness (that is, no psychotic episode) during the last two months preceding study participation. Furthermore, all subjects were required to be on a stable dose of antipsychotic medication, in addition to any other prescribed medication, for at least three months before the start of the study (for specification of prescribed antipsychotic drugs, see Table 1). No major medical or neurological conditions or other psychiatric diagnosis (besides DSM-IV, F295.xx) were allowed as assessed by a comprehensive medical history and a medical examination. Furthermore, all subjects had to show normal admission laboratory tests and vital signs. Substance abuse, apart from smoking, was grounds for exclusion. The study was approved by the Swedish Medical Products Agency and the Internal Review Board at the University of Gothenburg, Sweden. One participant (subject number 9) terminated participation after four weeks of treatment due to non-compliance with the drug administration procedure, thus resulting in a total of 10 patients completing the study. Table 1 Demographic data from all patients participating in the study. Case Age (years) Sex Subtype (DSM-IV) Duration of illness (years) Antipsychotic treatment, dosage 1 56 M Schizophrenia, undifferentiated 9 Risperdal consta 25 mg/2w 2 23 F Schizophrenia, undifferentiated 3 Leponex 700 mg/day, 3 29 M Schizophrenia, unspecified 8 Risperdal 2 mg/day, Abilify 25 mg/day 4 45 M Schizophrenia, paranoid 6 NA 5 35 M Schizophrenia, unspecified 11 Leponex 200 mg/day, Abilify 15 mg/day 6 54 M Schizophrenia, undifferentiated 29 Zyprexa 15 mg/day 7 51 M Schizophrenia, paranoid 23 Leponex 100 mg/day, Abilify 10 mg/day 8 56 M Schizophrenia, undifferentiated > 15 Zyprexa 10 mg/day, Cisordinol 140 mg inj 1/4w 9 - - - - - 10 39 F Schizophrenia, paranoid 8 Risperdal 4 mg/day 11 44 M Schizophrenia, unspecified 26 Leponex 800 mg/day, Abilify 10 mg/day Case 9 dropped out due to difficulties ingesting the study medicine. BODY.METHODS.DEMOGRAPHIC DATA: The demographic data of the patients enrolled in the present study are shown in Table 1. BODY.METHODS.STUDY DESIGN: The study was designed as a single-blinded, placebo-controlled, eight-week cross-over study. A written consent was signed before patients entered the study and were randomly assigned to initial treatment with either L-lysine dissolved in a soft drink or a soft drink only (the placebo) and screened for the outcome measures at baseline, after 28 days of treatment when treatment was crossed over, and after 56 days when treatment was terminated. BODY.METHODS.STUDY COMPOUND: L-lysine is a basic amino acid (that is, it carries a positive net charge at physiological pH) with a high nutritional value. It is readily absorbed from the intestine and metabolized by the liver. High levels of L-lysine are found in muscle, and increased intake of L-lysine is more readily distributed to this compartment than to, for example, blood. L-lysine supplementation has been tried as treatment for, for example, recurrent herpes simplex infection [27] and osteoporosis [28]. It is an essential amino acid and has very good oral bioavailability [29] and brain penetration [22]. L-lysine monohydrochloride is soluble in water and has a mild salty flavour. In the present study the compound was mixed with juice or a soft drink and ingested once a day. The dose of L-lysine was set to 6 g/day, a dose above dietary levels (dietary intake of L-lysine rich foods may reach 3 g/day) but within known safety limits [30]. L-lysine monohydrochloride was synthesized by Apoteket Produktion & Laboratorier, APL Stockholm. BODY.METHODS.SAFETY ASPECTS: The only reported adverse effects of L-lysine treatment in humans are transient gastrointestinal problems in a few subjects. Based on published studies, a long-term addition of 6 g of L-lysine to the daily diet should be safe [30]. In short-term human studies doses as high as 40 g/day have been tested and again the only reported adverse effects were abdominal cramps and transient diarrhoea that resolved as the dose was decreased [31,32]. Toxicological studies in rats indicate that the lethal dose 50 (LD50) of intravenous L-lysine is 4 mg/kg [33]. No lethal dosage could be reached by oral administration. Long-term studies on L-lysine administration (up to two years) have not revealed any adverse effects [30]. BODY.METHODS.CLINICAL ASSESSMENTS: Patients were screened for outcome measures at baseline, after four weeks, and after eight weeks. Symptom severity and functional outcome was assessed with the PANSS, and the UKU-Scale for monitoring side effects of psychiatric medication. Measures of physical status included blood sample analysis for L-lysine concentration and amino acid composition, blood pressure, pregnancy test and weight, all obtained by a trained research psychiatrist. Tests of cognitive performance included the Ray Auditory Verbal Learning Test (RAVLT), Continuous Performance Test (CPT; vigilance and attention), Trail Making Test A and B (speed of processing), Letter and Number Span test (working memory) and Wisconsin Card Sorting Test (WCST; working memory and executive functioning). All tests were carried out by a trained behavioural scientist. BODY.METHODS.AMINO-ACID MEASURES: Blood samples (4 ml) for assessment of essential amino acid profiling were obtained at baseline after L-lysine treatment, and after placebo treatment. The blood samples were sent to the Laboratory for Clinical Chemistry, the Sahlgrenska University Hospital, Gothenburg, Sweden, for analysis using a ninhydrin reagent for photometric determination of amino acids (for procedure, see [34]). BODY.METHODS.STATISTICS: Statistical analysis was performed by one- or two-way ANOVA when appropriate and multiple testing was subjected to Bonferroni correction with the significance level set to 0.05. Linear regression analysis was also performed to test if blood level of L-lysine concentration could predict functional outcome of any of the outcome measures collected. BODY.RESULTS.L-LYSINE TREATMENT SIGNIFICANTLY INCREASED BLOOD CONCENTRATION OF L-LYSINE WITHOUT INDUCING ADVERSE SIDE EFFECTS: Analysis of L-lysine concentration in blood showed a significant effect of treatment such that concentration increased after L-lysine treatment (6 g/day for four weeks) compared to baseline and placebo levels (F(2,14) = 7.84, P < 0.05, Table 2), in 8 out of the 10 patients. Two patients showed no change in L-lysine concentration and were treated as non-responders and included in an "Intention to Treat" group (n = 10), which was analyzed in parallel with the responders (n = 8) in the statistical analysis. Furthermore, the L-lysine treatment was not found to induce any adverse side effects including extrapyramidal effects. Neither was L-lysine treatment found to significantly alter blood concentration of any of the other amino acids analysed, such as citrulline, arginine, proline, glutamate and alanine. Table 2 Results before, after four weeks of L-lysine treatment, and after four weeks of placebo treatment. Outcome measure Baseline Post L-lysine Post placebo F value P- value* L-lysine blood conc (μmol/l) 196 ± 14 (201 ± 15) 401 ± 74 (363 ± 64) 195 ± 13 (195 ± 10) 7.84 (6.57) 0.024 (0.027) Symptom severity scales PANSS Positive symptoms 14.6 ± 0.7 (16.0 ± 1.1) 11.9 ± 0.6 (13.5 ± 1.3) 11.6 ± 0.8 (13.4 ± 1.4) 13.11 (12.49) 0.001 (0.000) Negative symptoms 18.4 ± 2.4 (19.7 ± 2.2) 17.4 ± 2.2 (19.4 ± 2.2) 17.3 ± 2.4 (18.8 ± 2.2) 1.53 (0.69) 0.25 (0.51) General symptoms 31.3 ± 2.5 (33.5 ± 2.5) 29.5 ± 1.9 (32.3 ± 2.4) 29.1 ± 2.0 (30.9 ± 2.0) 2.14 (2.84) 0.16 (0.09) Total 64.3 ± 4.2 (69.2 ± 4.7) 58.8 ± 4.0 (65.2 ± 5.4) 58.0 ± 4.5 (63.1 ± 5.0) 13.36 (8.59) 0.001 (0.002) GAF 43.8 ± 1.8 (42.0 ± 1.9) 46.9 ± 1.6 (45.8 ± 1.6) 44.1 ± 2.3 (42.8 ± 2.1) 4.29 (7.34) 0.035 (0.005) CGI 3.63 ± 0.18 (3.80 ± 0.20) 3.63 ± 0.18 (3.80 ± 0.20) 3.50 ± 0.27 (3.70 ± 0.26) 1.00 (1.00) 0.39 (0.39) Depression scale MADRS 8.50 ± 2.09 (9.10 ± 1.74) 7.50 ± 1.90 (7.90 ± 1.57) 8.38 ± 1.74 (8.40 ± 1.38) 0.54 (0.89) 0.59 (0.43) Cognitive tests RAVLT 45.9 ± 4.3 (43.5 ± 4.2) 46.0 ± 4.6 (43.3 ± 4.4) 48.8 ± 3.5 (45.2 ± 4.1) 0.79 (0.48) 0.47 (0.62) CPT 0.76 ± 0.16 (0.67 ± 0.13) 0.81 ± 0.13 (0.76 ± 0.12) 0.86 ± 0.13 (0.71 ± 0.14) 0.25 (0.21) 0.77 (0.81) Trail Making Test A 33.8 ± 1.7 (39.2 ± 5.9) 31.3 ± 2.5 (39.9 ± 8.9) 35.3 ± 4.5 (40.8 ± 6.3) 0.44 (0.07) 0.58 (0.93) Trail Making Test B 77.4 ± 7.2 (134 ± 56) 70.0 ± 5.2 (119 ± 48) 71.3 ± 6.4 (124 ± 50) 0.40 (1.61) 0.68 (0.24) Letter and number span 8.13 ± 0.90 (7.00 ± 1.03) 9.00 ± 0.66 (8.10 ± 0.82) 9.13 ± 0.92 (8.50 ± 0.95) 0.74 (1.92) 0.50 (0.18) Wisconsin Card Sorting Number of errors 33.9 ± 10.0 (31.9 ± 9.0) 21.0 ± 7.2 (19.7 ± 6.5) 24.4 ± 10.1 (23.2 ± 9.0) 4.91 (5.45) 0.024 (0.016) Perseverant errors 20.4 ± 7.6 (19.0 ± 6.8) 11.5 ± 5.0 (10.7 ± 4.4) 14.8 ± 8.1 (13.4 ± 7.3) 4.46 (5.00) 0.032 (0.021) Perseverant answers 23.6 ± 9.6 (21.9 ± 8.7) 13.1 ± 6.6 (21.1 ± 5.9) 17.1 ± 10.3 (15.6 ± 9.2) 4.77 (5.20) 0.026 (0.018) Mean values outside parenthesis refers to analysis including individuals who did display an increase in blood levels of L-lysine during the L-lysine treatment period (n = 8). Mean values within parenthesis refer to statistical analysis performed on all individuals, that is, also those not displaying increased blood levels of L-lysine during the L-lysine treatment period (total n = 10). *Corrected by the Huynh-Feldt procedure as required. BODY.RESULTS.POSITIVE SYMPTOM SCORES WERE SIGNIFICANTLY IMPROVED DURING TREATMENT: Symptom severity as measured by the positive symptoms sub-scale of PANSS showed a significant decrease (F(2,14) = 13.11, P < 0.001). This effect was mainly due to a significant change in scores for the sub-scales assessing delusions and suspiciousness/persecution. It should be noted that the patients tended to improve their performance on the positive symptoms PANSS measure regardless of treatment, an effect that may be explained by the increased attention the patients received simply by taking part in the study. Since such an effect would jeopardize the interpretation of the results, measures were taken to control for this effect by applying a two-way ANOVA to the data analyzing the interaction between treatment order (L-lysine treatment during first or second testing period) and test session (Baseline, Test 1 and Test 2). The rationale behind this approach was that given a biologically significant effect of L-lysine, a major part of the improvement in the testing score should be attributed to the L-lysine treatment period, and less to the placebo period and this effect should be detected in the interaction analysis. Albeit close to a significant interaction (F(2,12) = 3.08, P = 0.08), this analysis did not reach statistical significance and consequently it could not be excluded that a significant part of the effects obtained on the PANSS score was caused by factors not related to L-lysine treatment. BODY.RESULTS.PROBLEM SOLVING CAPACITY WAS SIGNIFICANTLY IMPROVED DURING TREATMENT: Problem solving capacity and cognitive flexibility, as assessed by the WCST, was significantly improved following treatment. This effect was statistically significant measured as decrease in the number of errors on the WCST (F(2,14) = 4.91, P < 0.05, Table 2), measured as perseverant errors (F(2,14) = 4.46, P < 0.05), and measured as perseverant answers (F(2,14) = 4.77, P < 0.05). A similar tendency of the patients to improve their WCST performance regardless of treatment was observed. Statistical analysis did not show a significant interaction between treatment order and test session and consequently the effects obtained were caused by factors not related to L-lysine treatment. The RAVLT, CPT-IP, TRAIL MAKING A and B, and Letter and Number Span test outcome scores were not significantly affected by treatment. Besides the outcome measures, patients self-reported on their condition, most commonly once a week when receiving the study drug. As such, no patient reported any adverse side-effects or worsening of symptoms. Interestingly though, three patients self-reported improvements of their symptoms. Two patients reported a decrease in positive symptoms, such as attenuated auditory hallucinations (i.e. hearing voices), an improvement that disappeared after trial termination. One of these patients also experienced improved attention following L-lysine treatment and one other patient reported increased mental stability and memory capacity following L-lysine treatment, an effect that remained several weeks after trial termination. BODY.DISCUSSION: Evidence was obtained suggesting that 6 g/day of adjunctive L-lysine treatment is a sufficient dose for increasing blood L-lysine levels above the nutritional, naturally occurring levels, without inducing adverse side-effects. In addition, there was a significant decrease in the positive PANSS scores and WCST over the whole study period. These two measures represent different functional abilities in the individual; PANSS is used to measure psychosis severity and WCST cognitive functionality. Thus, evidence was obtained for symptom improvement in both of these domains; however, accounts for placebo effects, such as training on WCST, cannot be excluded. Given the limited number of patients enrolled in the study, difficulties arise as to the possibilities of making reliable conclusions about the efficacy of L-lysine treatment. However, it should be noted that despite the low number of participants statistically significant effects of treatment on outcome measures were in fact obtained. Furthermore, albeit the relatively small changes in PANSS and WCST scores observed, statistical analysis showed fairly high effect sizes and observed powers for these changes (positive PANSS (n = 10): effect size (η) = 0.76, observed power (1-β) = 0.99; WCST number of errors (n = 10): effect size (η) = 0.64, observed power (1-β) = 0.77) indicating that they were of significant magnitude and detectable also in small sample sizes. Needless to say, it is also possible that a higher dose of L-lysine or a longer treatment period may have provided more clear-cut results. The reason that two participants (one male and one female) did not get an elevation in their blood concentration of L-lysine is unknown. In order to assess adherence to the L-lysine treatment the L-lysine containers were carefully labelled with dates and empty containers were collected weekly. However, there was no control for actual intake of the study medication beyond the assessed blood L-lysine concentrations. Metabolic individual differences, due to i.a. intense physical activity or gender, or simply non-adherence to L-lysine treatment, cannot be ruled out as one of the contributing factors to these findings. Cautions should be taken when interpreting the data. Both the PANSS and the WCST scores seemed to improve over repeated testing, and consequently this placebo and/or training effect could explain the effects obtained. The study design presently used did not provide a satisfactory control for such effects, but the statistical measures, that is, analysis of interaction between treatment order and test session on outcome measure, that were taken to address this issue confirmed the possibility of placebo and training effects. Indeed, the WCST has been shown to be sensitive to training effects (see, for example, [35]) and should be used with care. Ideally, a test less sensitive to training and with higher test-retest reliability may be advisable for future studies, as well as a pre-testing cognitive training session (before the baseline assessment) in order to control for training artefacts. The fact that the patients enrolled in the study were in a stable phase of their illness and were all treated with atypical antipsychotics may also have made it more difficult to detect beneficial effects of L-lysine treatment on cognitive functioning and symptom severity. In this respect, special attention may be paid to the self-reported improvements of positive symptoms, attention and memory capacity by three of the patients, since these potential effects of the treatment would be important but unfortunately could not be addressed by statistical analysis. It should also be noted that the patient who experienced improved memory capacity continuously trained his memory using a computerized training program. There is an ongoing debate in the literature as to how to disentangle training effects of repeated cognitive testing, from "real" drug effects, as well as how to establish consensus regarding a reliable cognitive test battery [36-38]. The cognitive tests chosen for the purpose of the present study are some of the most frequently applied in research and several of them have been shown to be resistant to training effects [39,40]. The initiatives taken to pharmacologically enhance cognition are plentiful; however, the results of such studies are diverse and few studies have been able to demonstrate replicable cognitive enhancement in patients with schizophrenia [38]. One contributing factor to these shortcomings, may be that schizophrenia encompasses abnormal progressive loss in brain volume [41], possibly counteracting any cognitive enhancing potential. However, cognitive enhancing therapy was recently demonstrated to counteract gray matter loss in patients with schizophrenia [42]. Cognition is a highly specific and complex phenomenon that develops from early life into adulthood, depending on both biological pre-disposition as well as environmental input [36]. Perhaps the lack of development of cognitive enhancing agents is due to an overly optimistic belief in cognitive enhancement being viable without concurrent cognitive training. The plasticity of the brain is a complex, time- and stimulus-dependent process by which the strengthening of synapses has to be modulated in order for learning to occur [43]. Thus, it may in fact not be possible to find improvements in cognition by solely adding cognitive enhancing compounds without simultaneous cognitive training. Therefore, it is interesting that L-lysine treatment in combination with training was perceived to improve memory capacity in one patient. Moreover, adjunctive L-lysine treatment needs to be investigated in a larger placebo-controlled, double-blinded study further limiting possible training effects. Taken together, preclinical and clinical studies have provided evidence for the involvement of a brain NO imbalance in schizophrenia. Furthermore, preliminary in vivo studies in rats have indicated that NO production is decreased by L-lysine [26]. Consequently, L-lysine may have a beneficial potential in the treatment of the disorder. In addition, targeting the NO pathway may represent a new therapeutic approach that is pharmacologically fundamentally different from that of the traditional antipsychotics and, hence, may be beneficial in targeting symptoms that are currently treatment resistant. BODY.CONCLUSIONS: To our knowledge, the present study is the first to investigate L-lysine treatment as a potential new add-on treatment for patients with schizophrenia, and as a novel means of targeting the NO pathway in order to treat a psychiatric disorder. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS' CONTRIBUTIONS: This study is to the best of our knowledge the first investigation to ever evaluate the effects of L-lysine treatment in patients with schizophrenia. CW wrote the study protocol, coordinated the study, carried out cognitive tests and wrote the first version of the manuscript. DK planned and initiated the study, analyzed and interpreted data as well as co-authored the manuscript. EK was the physician carrying out the clinical assessments including PANSS, MADRAS and CGI together with responsible senior specialist in psychiatry BR; they have both co-authored the paper. KF and EP did the ethical application, analyzed and interpreted the data as well as wrote the report. In addition, EP planned and made necessary pharmacological and pharmacokinetic research to determine the dosing of L-lysine in the present investigation. LS wrote and managed the application to the Swedish Medical Products Agency and carried out all the statistical analyses, and co-authored the manuscript. I-BB carried out cognitive testing, supervised CW and helped interpret the results. JAE provided financial means as the majority of grants were sought by him, as a professor in pharmacology and an M.D. JAE provided medical expertise in the planning and managing of the study as well as during analysis and interpretation of the data. JAE also co-authored the report. 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/1741-7015/9/40/prepub

TITLE: Effect of Improving Dietary Quality on Arterial Stiffness in Subjects with Type 1 and Type 2 Diabetes: A 12 Months Randomised Controlled Trial ABSTRACT: People with diabetes have accelerated arterial stiffening. The aim of this study was to determine the effect of increasing fruit, vegetable and dairy intake for 12 months on carotid femoral pulse wave velocity (cfPWV), augmentation index (AIx), and central blood pressure (cBP), compared to a usual diet control, in people with type 1 and type 2 diabetes. In a 12 months randomised controlled trial, cfPWV, AIx and cBP were measured every 3 months. The intervention group received dietary counselling to increase consumption of fruit (+1 serving/day; 150 g/day), vegetables (+2 servings/day; 150 g/day) and dairy (+1 serving/day; 200–250 g/day) at baseline, 1, 3, 6 and 9 months. The control group continued on their usual diet. One hundred and nine participants were randomised and 92 (intervention n = 45; control n = 47) completed. At 3 months, fruit (184 g/day; p = 0.001) and dairy (83 g/day; p = 0.037) intake increased in the intervention group compared with the control group but this increase was not maintained at 12 months. After adjustment for baseline measurements there was no time by treatment effect for central systolic or diastolic BP, AIx or cfPWV. A time effect existed for AIx which modestly increased over time. Peripheral diastolic BP and central pulse pressure were improved in the intervention group compared with the control group at 12 months. In the cohort with type 1 and type 2 diabetes, improving dietary quality by increasing consumption of fruit, vegetables and dairy did not improve cBP, AIx or cfPWV, compared with a control group continuing on their usual diet, after 12 months. BODY.1. INTRODUCTION: Individuals with type 1 and type 2 diabetes are two to three times more likely to develop cardiovascular disease (CVD) compared to the general population [1,2]. In Australia, in 2010 approximately 30% of all deaths in people with type 1 and type 2 diabetes were due to CVD [3]. Improving dietary quality may be a strategy to reduce the burden of CVD in people with diabetes. Epidemiological studies show that better dietary quality is associated with lower rates of CVD in the general population [4] and in people with type 1 and type 2 diabetes [5,6]. We have previously shown that people with type 1 and type 2 diabetes have similar dietary quality, as defined by a priori indices, to age, sex and body mass index (BMI) matched non-diabetic subjects [7]. Furthermore, when dietary quality was improved for 12 months it was shown that common carotid artery intima media thickness (CCA IMT) regressed to a greater extent than was observed in the control group [8]. Carotid femoral pulse wave velocity (cfPWV) is considered the gold standard for determining arterial stiffness [9] and is an independent predictor of cardiovascular and all-cause mortality [10]. Furthermore, the addition of cfPWV to conventional Framingham risk factors improves 10 years CVD risk prediction in those at intermediate risk of CVD [10]. Augmentation index (AIx) is another indirect measure of arterial stiffness and has been associated with cardiovascular events and total mortality [11]. People with type 1 and type 2 diabetes have accelerated arterial stiffening when compared to their non-diabetic counterparts [12,13]. We have shown that greater consumption of reduced fat dairy and vegetables was correlated with lower cfPWV in a population with type 1 and type 2 diabetes [14]. Previous epidemiological studies in non-diabetic populations suggest that fruit, vegetables and dairy intake may improve PWV and/or AIx [15,16] but randomised controlled trials have failed to support this observational evidence. The aim of this study was to determine the effect of increasing fruit (+1 serving/day; 150 g/day), vegetable (+2 servings/day; 150 g/day) and dairy (+1 serving/day; 200–250 g/day) intake on cfPWV, AIx and central blood pressure (cBP) over a 12 months period, compared to a control group continuing on their usual diet, in people with type 1 and type 2 diabetes. These data are secondary endpoints from a trial that has previously been reported [8]. BODY.2. MATERIAL AND METHODS.2.1. STUDY DESIGN: A 12 months randomised controlled trial was conducted as previously described [8]. This analysis comprises a sub-group (n = 109) whereby cBP, AIx and cfPWV were measured. Ethics approval was obtained from the University of South Australian Human Research Ethics Committee and participants provided written informed consent. The trial was registered with the Australian New Zealand Clinical Trials Registry (ACTRN12613000251729). Subjects were above 18 years of age with diagnosed type 1 or type 2 diabetes for any duration managed with diet, oral hypoglycaemic agents (OHA) and/or insulin. Recruitment was conducted from August 2012 until December 2013 from a database of volunteers, public advertisements and a recruitment company (Intuito Market Research, Adelaide, SA, Australia). Exclusion criteria were: unstable CVD requiring active intervention, heart failure, significant renal impairment (eGFR < 30 mL/min), liver disease, cancer or allergic/intolerant/dislike of fruit, vegetables or dairy. In a parallel design, participants were randomised to either the intervention group (improved diet quality) or the control group (continue usual intake) using an online generated balanced random number allocation sequence [17] stratified by diabetes type and sex, by a person independent of the study. The details of the dietary intervention have previously been reported [8]. Briefly, nutritional counselling to increase consumption of fruit (+1 serving/day; 150 g/day), vegetables (+2 servings/day; 150 g/day) and dairy (+1 serving/day; 200–250 g/day), regardless of usual intake, was provided to the intervention group. The control group were not given any dietary advice. At 1, 3, 6, 9 and 12 months, participants met with a qualified dietician and completed a checklist to determine on how many days of the week they complied with the intervention. A random spot urine sample was provided by the participants at the 3 monthly appointments for measurement of potassium and creatinine. A food frequency questionnaire (FFQ) was administered at 3 and 12 months to determine if food and nutrient intake had changed from baseline. BODY.2. MATERIAL AND METHODS.2.2. MEASUREMENTS: At 3 monthly intervals cfPWV, AIx and cBP were measured. Anthropometric measures, peripheral blood pressure (BP), blood lipids, fasting glucose, C reactive protein (CRP) and spot urinary sodium and potassium excretion were also measured every 3 months. All of the measurements were completed after an overnight fast and the operator completing the vascular measurements was blinded to the participant's randomisation. BODY.2. MATERIAL AND METHODS.2.3. CENTRAL BLOOD PRESSURE AND AUGMENTATION INDEX: A SphygmoCor® XCEL (AtCor Medical, West Ryde, Sydney, Australia) was used to measure cBP and AIx. A cuff was placed over the brachial artery on the right arm and measurements were completed after the participants had been quietly resting for 5 min. Three consecutive measurements were taken and the average calculated. All of the measurements were taken by one operator with a CV of 4.2% (n = 28). BODY.2. MATERIAL AND METHODS.2.4. PULSE WAVE VELOCITY: A SphygmoCor® XCEL (AtCor Medical, West Ryde, Sydney, Australia) was used to measure cfPWV. The tonometer was placed on the right carotid artery and the cuff on the right femoral artery. A 10 s recording of the carotid-femoral waveform was taken. Three measurements were performed at each time-point and an average was taken. The measurements were taken by two operators; the intra-observer CVs were 4.2% (n = 28) and 7.3% (n = 11), respectively and the inter-observer CV was 5.0% (n = 18). BODY.2. MATERIAL AND METHODS.2.5. ANTHROPOMETRIC MEASUREMENTS: Height was measured using a stadiometer (SECA, Hamburg, Germany) to the nearest 0.1 cm while barefoot/flat footwear. Weight was measured to the nearest 0.05 kg using calibrated electronic scales (SECA, Hamburg, Germany) while the participants were barefoot/wore light footwear and wore light clothing. BODY.2. MATERIAL AND METHODS.2.6. PERIPHERAL BLOOD PRESSURE: Peripheral BP was measured using an automated sphygmomanometer (SureSigns VS3; Philips, North Ryde, Sydney, Australia) once the participant had been seated for 5 min. A normal sleeve (16 × 52 cm) was used for an arm circumference of 24–32 cm and a large sleeve (16 × 70 cm) for an arm circumference of 32–42 cm. A minimum of four consecutive readings were taken at 1 min intervals. The first reading was discarded and the following three consistent measurements, i.e., systolic BP within a range of 10 mmHg, were used. BODY.2. MATERIAL AND METHODS.2.7. BIOLOGICAL MEASUREMENTS.2.7.1. SPOT URINE SAMPLE: A spot urine sample was provided by participants when they attended the University of South Australia. Analysis of sodium, potassium, creatinine and albumin was done by SA Pathology (Adelaide, SA, Australia). The albumin to creatinine ratio was calculated from one spot urine sample at baseline to determine the presence of micro-albuminuria. BODY.2. MATERIAL AND METHODS.2.7. BIOLOGICAL MEASUREMENTS.2.7.2. FASTING BLOOD SAMPLE: A fasting venous blood sample was taken at each time-point by a trained phlebotomist. Total cholesterol, HDL cholesterol, triglycerides, CRP and glucose were measured using a Konelab 20XTi automatic analyzer (Thermo Electron Corporation, Louisville, CO, USA) with reagents from ThermoFisher Scientific (Melbourne, Australia). LDL cholesterol was calculated using the Friedewald formula ((total cholesterol − HDL cholesterol) − (triglycerides × 0.45)) [18]. Serum MMP-7 was measured in duplicate by ELISA (Quantikine Human Total MMP-7, R & D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The inter-assay CV was 5.6%. BODY.2. MATERIAL AND METHODS.2.7. BIOLOGICAL MEASUREMENTS.2.7.3. HBA1C: The participants were asked to provide the pathology report from their most recent haemoglobin A1c (HbA1c) measurement or the result was sourced from their general practitioner or the pathology company. BODY.2. MATERIAL AND METHODS.2.7. BIOLOGICAL MEASUREMENTS.2.7.4. DIETARY INTAKE: Dietary intake was measured using the electronic version of the Dietary Questionnaire for Epidemiological Studies version 2 FFQ, as previously described [19]. BODY.2. MATERIAL AND METHODS.2.8. STATISTICAL ANALYSIS: Data are presented as mean ± standard deviation (SD) or median (interquartile range) depending on the distribution. Data were checked for normality using Shapiro-Wilk and Kolmogorov-Smirnov values. Independent samples t tests were used to determine between group differences at baseline for continuous variables and chi squared tests were used for categorical variables. Mixed effect modelling was used to determine between group changes over time and post hoc analyses were adjusted for multiple comparisons using the Bonferroni method. For the vascular measurements the results of the mixed effect models are presented as both unadjusted and adjusted for baseline measurements. The twelve months change in anti-hypertensive medication, lipid lowering medication, OHA and insulin were calculated using the following formula: mean (percentage dose change from baseline for each medication)/number of different medications [20]. Independent samples t tests were used to determine if there was a difference in medication change between the groups at 12 months. In addition, participants were categorised into the following groups for anti-hypertensive medication, lipid lowering medication, OHA and insulin: medication increased, decreased or no change and chi squared tests were conducted to determine if there was any difference between the groups. A per protocol analysis was conducted to determine if there was any difference in vascular measurements between participants that achieved any increased in fruit, vegetable and dairy consumption at 3 or 12 months and those that decreased consumption. This study was powered on CCA IMT which was the primary outcome but a post-hoc power analysis showed that with 78 participants we had 80% power (p < 0.05) to detect a 1.1 m/s difference in cfPWV between the groups. With 92 participants we had 80% power (p < 0.05) to detect a 9/6 mmHg difference in central systolic and diastolic blood pressure. All of the analyses were performed using SPSS (version 19, 2010, SPSS Inc., Chicago, IL, USA). Statistical significance was set at p < 0.05. BODY.3. RESULTS.3.1. SUBJECTS: One hundred and nine participants were randomised (intervention n = 55; control n = 54). The characteristics of the participants are presented in Table 1. There were no statistically significant differences in age, anthropometric measures, sex, diabetes type and time since diagnosis, BP, lipids, CRP, glycaemic control or medication prescription between the groups at baseline. Ninety two participants (intervention n = 45; control n = 47) completed the 12 months study. BODY.3. RESULTS.3.2. DIETARY INTAKE AND COMPLIANCE: Food and nutrient data measured using the FFQ are available in the online supplemental material (Table 1 and Table 2). Compliance with the intervention measured using the FFQ showed that there was a significant time by treatment effect for fruit (p = 0.001) and total dairy consumption (p = 0.001) and a non-significant time by treatment effect for vegetable intake (p = 0.056). Post-hoc testing showed that fruit intake was 184 g/day (95% CI 113, 254 g/day) greater in the intervention group compared with the control group at 3 months (p = 0.001); there was no significant difference at 12 months. The intervention group consumed 83 g/day (95% CI 5, 60 g/day) more dairy at 3 months compared with the control group (p = 0.037) and this was accounted for by higher yoghurt intake (49 g/day; 95% CI 19, 80 g/day; p = 0.002). At 12 months the control group reported a higher consumption of dairy products (90 g/day; 95% CI 14, 166 g/day; p = 0.02) due to greater milk consumption (97 g/day; 95% CI 28, 166 g/day; p = 0.006). A significant time by treatment effect existed for fibre, calcium, potassium and magnesium. A time effect was present for vegetable and legume (p = 0.038), fresh fruit (p = 0.03) and yoghurt consumption (p = 0.017). At 12 months vegetable and legume intake was 24 g/day higher than at baseline in the whole group (95% CI −1, 49 g/day; p = 0.065) and consumption of fresh fruit had increased by 47 g/day (95% CI −3, 67 g/day; p = 0.07). A time effect was observed for the sodium to potassium ratio which increased, see online supplemental material (Table 3). No time by treatment effects existed. Participants reported that the extra serving of fruit and dairy were consumed on a median of 5.5 days per week throughout the study and at 9 months, participants reported that the extra serve of fruit was consumed on 7 days per week. The two extra servings of vegetables were consumed 5.5 days per week until 9 months and 7 days per week at 12 months. BODY.3. RESULTS.3.3. BLOOD PRESSURE AND VASCULAR MEASUREMENTS: AIx, cfPWV, cBP and peripheral BP measures are presented in Table 2. Measurements of cfPWV were completed on 78 participants (intervention n = 36; control n = 42) due to technical difficulties because of obesity. A borderline non-significant time effect was observed for peripheral systolic BP (p = 0.053) and diastolic BP was reduced over time (p = 0.027) with a non-significant time by treatment effect (p = 0.053). Post-hoc testing showed that diastolic BP was reduced by 1.8 mmHg (95% CI −3.6, −0.06 mmHg; p = 0.037) at 6 months compared with baseline; pairwise testing showed no other significant differences between any of the time-points. Central systolic BP and diastolic BP were reduced over time (p = 0.001), however there was no time by treatment effect. In the whole group the reduction in central systolic and diastolic BP at 12 months was 5.3 mmHg (95% CI −1.4, −9.2 mmHg; p = 0.002) and 3.0 mmHg (95% CI −0.5, −5.4 mmHg; p = 0.009), respectively. In addition, central pulse pressure was lowered over time (p = 0.003) with no time by treatment effect (p = 0.71). After adjustment for the baseline value there were no significant time effects for peripheral or central BP but a time by treatment effect existed for peripheral diastolic BP and central pulse pressure. Post-hoc testing showed that at 12 months peripheral diastolic BP was 3.6 mmHg (95% CI −6.4, −0.7) lower in the intervention group compared with the control group (p = 0.015). There was no significant difference in central pulse pressure between the groups at any of the time points. AIx (p = 0.002) and central augmented pressure (p = 0.02) however increased with time and there was no time by treatment effect. Post-hoc testing showed that compared with baseline AIx was increased at 3 (2.7%; 95% CI −0.01, 5.5%; p = 0.051), 6 (5.1%; 95% CI 1.5, 8.7%; p = 0.001), 9 (2.9%; 95% CI 0.2, 5.5%; p = 0.03) and 12 months (2.9%, 95% CI 0.2, 5.5%; p = 0.02) compared with baseline in the whole group. Similarly, there was a time effect (p = 0.048) for cfPWV with a small increase observed but no time by treatment effect. Post-hoc testing showed no statistically significant difference in cfPWV between any of the time-points. After adjustment for baseline values a time effect was present for central augmented pressure and AIx but the time effect present for cfPWV was attenuated to non-significance after adjustment for baseline cfPWV. Post-hoc testing showed no significant difference in central augmented pressure or augmentation index between the groups at any of the time-points. At three months 15 participants increased fruit, vegetables and dairy consumption and 66 participants decreased consumption. At 12 months 20 participants increased consumption of fruit, vegetables and dairy and the remaining 69 decreased consumption. Per protocol analyses showed no between group differences for any of the outcome measurements. BODY.3. RESULTS.3.4. WEIGHT AND BIOCHEMISTRY: Weight, total cholesterol, LDL and HDL cholesterol, triglycerides and glucose remained unchanged over time and there was no time by treatment effect, see Table 3. A time effect existed for CRP (p = 0.016) which decreased to a small degree, see Table 3. MMP-7 was measured at baseline and 12 months in a subgroup of the cohort (n = 54) and there was no change over time or by treatment, see Table 3. BODY.3. RESULTS.3.5. CHANGE IN MEDICATION: There was no statistically significant difference between the groups for the change in medication dose or the number of participants that increased, decreased or did not change their anti-hypertensive medication (82% of participants' dose was unchanged), lipid lowering medication (89% of participants dose was unchanged), OHA (78% of participants dose was unchanged) and insulin (83% of participants dose was unchanged). BODY.4. DISCUSSION: In this cohort of individuals with type 1 and type 2 diabetes cBP and arterial stiffness measures were not improved in the intervention group, compared with the control group at 12 months. Fruit (184 g/day) and dairy intake (83 g/day) were increased at 3 months in the intervention group, but this increase was not maintained at 12 months. There was a border-line non-significant increase in vegetable intake in the intervention group (p = 0.056). Central systolic and diastolic BP and pulse pressure were improved over time in the whole cohort, although after adjustment for baseline values the time effect was attenuated to non-significance for cBP. After adjustment for baseline measurements there was a significant reduction in peripheral diastolic BP in the intervention group compared with the control group and a time by treatment effect existed for central pulse pressure. There was no time by treatment effect for AIx or cfPWV but there was a time effect for AIx such that it modestly increased (or deteriorated) over time. In this study there was a time by treatment effect present for central pulse pressure such that there was a non-significant reduction in the intervention group at 12 months. It has been shown that a 10 mmHg increase in central pulse pressure increases the risk of a cardiovascular event by ~14% [11]. After adjustment for baseline values there was no significant change in cBP over the 12 months study but AIx increased (or worsened) in the absence of any change in cfPWV. This suggests that there was no change in arterial stiffness and the change in AIx was driven by a change in the wave reflection. Fruit and vegetables have previously been shown to have BP lowering properties which may be explained by their nutritional composition [21]. However, there are a lack of studies investigating the effects of fruit and vegetables or their compounds on cBP. Jennings et al. [22] showed in a cross-sectional study that greater anthocyanin intake is associated with lower central systolic blood pressure. In this study central pulse pressure was improved in the intervention group compared with the control group in the absence of a change in central systolic or diastolic blood pressure. The findings of this study suggest that increasing consumption of fruit and vegetable by small amounts (approximately one third of a serve) which is achievable for free-living people may improve central pulse pressure. The unchanged cfPWV and MMP-7 levels suggest that vascular remodelling did not occur. MMP-7 acts on collagen and elastin to cause degradation of the extracellular matrix resulting in structural changes and increased arterial stiffness [23]. A recent prospective study with a 4.2 years follow-up, conducted in a cohort with type 2 diabetes, showed that HbA1c during follow-up predicted cfPWV. A greater reduction in HbA1c during the first year of follow-up was associated with less of an increase in cfPWV during follow-up [24]. The authors of this study speculated this finding may be explained by lower advanced glycation end product (AGE) formation with better glycaemic control. AGEs have been shown to be positively associated with cfPWV in subjects with type 1 diabetes [25]. The results of the trial by Ferreira et al. [24] suggest that we may not have observed an effect on cfPWV because glycaemic control was not altered. A previously conducted randomised controlled trial of people at high risk of CVD with habitually low intake of fruit and vegetables, also showed that greater intake of fruit and vegetables (+2, +4 and +6 portions/day above habitual intake of high or low flavonoid fruit and vegetables) did not affect cfPWV or AIx after 18 weeks, once the data was adjusted for heart rate [26]. A recent randomised controlled trial showed that supplementation with 350 mg of magnesium for 6 months reduced cfPWV by 1 m/s. In the current study the extra serves of fruit, vegetables and dairy provided approximately 80–90 mg of magnesium [27]. Intervention trials examining the effect of dairy on arterial stiffness are lacking. Dietary intake reported in the FFQ showed that intake of fruit and dairy increased in the intervention group, compared with the control group at 3 months; however, this was not maintained at 12 months. In the compliance checklists the participants reported consuming the extra serves of fruit, vegetables and dairy on greater than 5 days per week throughout the 12 months intervention period. However, the change in fruit, vegetable and dairy intake derived from the FFQ data did not correspond with these checklists, especially for dairy. There was no difference in the urinary sodium to potassium ratio or potassium to creatinine ratio by treatment. Surprisingly the sodium to potassium ratio increased over the 12 months in both groups. This suggests the increase in sodium from dairy products outweighed the increase in potassium from fruit and vegetables despite the FFQ computed intakes. This study is limited by the poor compliance of the participants with the intervention as shown by the FFQ although biomarkers of fruit, vegetables and dairy intake were not measured to provide an objective measure of compliance. In addition, this work comprises analyses of secondary endpoints and Post-hoc power analyses show we had reduced power to detect effects. In this study dietary intake changed in the whole cohort over the study period which may have reduced our capability to detect between group changes. Finally, random spot urine samples taken on one occasion were used to estimate sodium and potassium intake. BODY.5. CONCLUSIONS: In a cohort with type 1 and type 2 diabetes improving dietary quality by increasing consumption of fruit, vegetables and dairy did not improve cBP, AIx or cfPWV, compared with a control group continuing on their usual diet, after 12 months. Peripheral diastolic BP and central pulse pressure were improved in the intervention group compared with the control group at 12 months.

TITLE: The impact of urinary bladder catheterisation after ureterorenoscopic stone removal on the postoperative course ABSTRACT.INTRODUCTION: The most frequent reason for ureterorenoscopy is the necessity to remove calculi from the ureter and/or kidney. After completing this procedure the Foley catheter is inserted in the bladder. The aim of the study is to show whether catheterisation of the bladder after ureterorenoscopic stone removal in patients with a low-risk of complications is necessary and indicated. ABSTRACT.MATERIAL AND METHODS: This is a comparative, prospective and randomized study. 100 patients meeting the assumed criteria, subjected to the ureterorenoscopy due to ureter and/or kidney stones participated in the study. The patients were divided into the experimental (Group I) and control (Group II) groups. Group I did not have a catheter, Group II was catheterised. There were two subgroups: female and male in each group. Mean values of the following parameters were calculated: intensity of postoperative pain measured by Visual Analog Pain Scale, the number of additional doses of painkillers administered after the procedure, hospital stay, occurrence of fever, significant bacteriuria, acute urinary retention and post- void retention greater than 30 ml. ABSTRACT.RESULTS: Intensity of pain measured by the Visual Analog Scale was higher in Group II. Catheterisation does not influence: the number of additional doses of ketoprofen and pethidine administered during the 1st day after the operation, the duration of hospitalization, the occurrence of fever, significant bacteriuria, the postoperative acute urinary retention and the post-void residual urine volume. ABSTRACT.CONCLUSIONS: In patients with a low risk of postoperative complications who did not have any intraoperative complications, catheterisation of the urinary bladder increases discomfort without bringing any benefits. BODY.INTRODUCTION: Removal of calculi from the urinary tract by means of ureterorenoscopy (URS) is one of the most frequent surgical procedures in urology. A catheter is usually left in the urinary bladder after the procedure. A question thus arises, whether catheterisation is an integral part of the procedure or results from acquired routine. The goal of the study was to provide evidence whether, urinary bladder catheterisation, applied after ureterorenoscopic removal of stones from the ureter and/or the kidney in patients with a low risk of complications from the lower urinary tract, has any impact on selected aspects in the post-operative follow-up. BODY.MATERIAL AND METHODS: The study was designed as a prospective, randomized trial. The written informed consent was obtained from all patients. The approval of the Bioethics Committee was obtained. The patients were divided in two groups: experimental and control. After the surgery, patients from the experimental group (Group I) were not catheterised, while patients from the control group (Group II) had catheters left in their bladders. Both groups were gender subdivided into female and male (FI and MI vs. FII and MII, respectively). Each of the qualified patients had to meet the predefined inclusion criteria, while not meeting any of the exclusion criteria. Inclusion criteria were as follows: age >18 years, successful URS under subarachnoid spinal block, application of the JJ stent, unified analgesic therapy, unified anti- bacterial prophylaxis and in the control group presence Foley catheter. The exclusion criteria were: urinary tract infection, urinalysis positive for infection, post-void residual (PVR) >30 ml, alcoholism, neurological conditions with disturbed uresis, post-operative or post-radiotherapy condition of the pelvis minor and/or the retroperitoneal space, immunodeficiency, diabetes mellitus, significant clinical complications of URS, surgical procedures in the urinary system other than URS and no patient willingness to collaborate. In the course of hospitalisation of each included patient, the following data were acquired concerning his/her therapy: the highest severity of post-operative pain, determined by means of the Visual Analogue Scale (VAS) of Pain on the first day after operation, the number of doses of ketoprofen and pethidine administered in the first day after surgery, post-operative hospitalisation period in days, post-operative fever, post-operative bacteriuria, post-operative acute urinary retention (AUR) and post-operative PVR >30 ml. Statistical analysis was performed with the chi-square, Fisher's exact and U Mann-Whitney exact tests. It was assumed that the difference was significant under P <0.05, and highly significant when P <0.01. BODY.RESULTS: The mean severity of post-operative pain on the first day after surgery is presented in Table 1 and Figure 1. It was confirmed that the catheterised patients had sensed more intensive pain than the non-catheterised subjects. Both in the group of women and in the group of men, a significant correlation of the highest post-operative pain severity, determined by means of the Visual Analogue Scale (VAS) of Pain, with catheterisation was found. Table 1 Distribution of the most severe post-operative pain on the first day after surgery in VAS, with regards to catheterisation Pain severity in VAS Total Women Men Catheterisation Line Total Catheterisation Line Total Catheterisation Line Total no yes no yes no yes 0 27 9 36 14 4 18 13 5 18 54% 18% 56% 16% 52% 20% 1 3 3 6 0 1 1 3 2 5 6% 6% 0% 4% 12% 8% 2 9 12 21 4 7 11 5 5 10 18% 24% 16% 28% 20% 20% 3 7 11 18 5 6 11 2 5 7 14% 22% 20% 24% 8% 20% 4 3 3 6 2 1 3 1 2 3 6% 6% 8% 4% 4% 8% 5 1 4 5 0 3 3 1 1 2 2% 8% 0% 12% 4% 4% 6 0 4 4 0 1 1 0 3 3 0% 8% 0% 4% 0% 12% 8 0 4 4 0 2 2 0 2 2 0% 8% 0% 8% 0% 8% Total 50 50 100 25 25 50 25 25 50 Figure 1Distribution of the most severe post-operative pain on the first day after surgery in VAS, depending on catheterisation. No significant differences were found in the amount of pethidine or ketoprofen additionally administered on the 1st day after catheterisation, both in total as well as separately in men and women. There was also no significant difference in the post-operative hospital stay between the groups, both in total and separately in women and in men. No fever occurred after surgery in any of the patients – there were no differences among the groups. Presence of the post-operative positive urine culture was not significantly dependent on catheterisation. No significant correlation was found in the post operative AUR and PVR >30 ml on the day of discharge depending on catheterisation, both in total and separately in women and in men. BODY.DISCUSSION: Post-surgical catheterisation of the urinary bladder is carried out for a certain number of causes. The main reason for bladder catheterisation is the risk of acute urinary retention (AUR) as a complication of anaesthesia. The percent of post-operative urinary retention varies from 2.1 to 2.52% for all types of surgery [1, 2], while for surgical procedures under spinal anaesthesia it is 0–79% [3, 4]. The occurrence of AUR under subarachnoid anaesthesia depends, among others, on the type and dose of an intrathecally administered drug [3, 5–10]. Intralumbar anaesthesia with amide derivatives results in atony of the urinary bladder, mainly as a result of blockade of the S2–S4 parasympathetic sacral segments. Post-operative urinary retention (POUR) may also be a consequence of urinary bladder atony, caused by excessive bladder wall extension by urine accumulating in the bladder during surgery. The risk increases with operation time, the volume of administered fluids and urine volume in the bladder when the patient was transferred to the post-operative unit [8, 9, 11, 12]. Oedema and/or lesions of the urethra, the prostate gland, the urinary bladder neck or of the genital organ in women may cause a secondary subvesical obstruction [2, 9, 12]. The risk increases if the patients presented with lower urinary tract symptoms (LUTS) or had been treated for bladder outlet obstruction before surgery [7, 8, 12, 13, 14]. Following certain reports, diabetes mellitus, renal insufficiency, neuropathies and depression increase the risk of POUR [1, 12–15], while other reports do not confirm such correlations [16]. Some publications mention the male gender and age as the risk factors [1, 2, 13, 14, 16], while other do not confirm such correlations [8, 12, 15, 16]. In the study all patients were submitted to subarachnoid anaesthesia, using heavy lidocaine, which assured homogeneity in the aspect of POUR as an anaesthetic complication. It is assumed that a lower percent of POUR is observed after the administration of lidocaine and procaine than after bupivacaine or tetracaine [9]. Bladder emptying during and after URS is an integral part of the procedure. Significant clinical, intraoperative lesions of the urinary tract were among the exclusion criteria, similarly to diabetes mellitus, diseases of the nervous system with miction disorders, identified and confirmed, considerable, preoperative urine retention after miction as an important indicator of bladder outlet obstruction, as well as previous surgical operations and/or therapeutic irradiation, which could have impaired the innervation of the lower urinary tract. Their role as a risk factor for POUR had no effect on the results of the study. A urinary bladder catheter allows for monitoring of haematuria and reduces the risk of urine drainage block by clots [17]. Scarce haematuria occurred in 40% of URS procedures, carried out for urolithiasis of the upper urinary tract [18]. In turn, Tanriverdi and Geavlete reported 0.1% to 3.2% of medium degree haematuria cases [19, 20]. No severe cases of haematuria were noted. In line with literature reports, no cases of severe haematuria were observed in the study, thus it also could not be an indication to apply a urinary catheter or have any potential effect on the actual POUR. Urinary bladder catheterisation ensures drainage of urine which may contain microorganisms and elements of a substrate for biofilm [21, 22, 23] and thus prevents infection complications. Preoperative urinary tract infections (UTI), identified by clinical symptoms or confirmed in urine culture, were among the exclusion criteria. Thus, since there were no cases of severe UTI, it could not be regarded as an indication for catheterisation of the patients included into the study. A bladder catheter facilitates monitoring of diuresis after surgery [5, 15]. It is applied mainly in patients with a high risk of post-operative occurrence of renal or prerenal kidney failure [24, 25]. In the other cases, the anaesthetic indications for urinary bladder catheterisations are rather ambiguous. None of the patients from the study groups required urine output measurements as an element of post-operative monitoring. The following complications of short-term urinary bladder catheterisation are mentioned: 1) A syndrome of complaints of various character and intensity, in response to the presence of the catheter in the bladder is called catheter-related bladder discomfort (CRBD). Catheterised patients complain of pains and burning sensation at the region of the urethral orifice or dull pain in the lower abdominal part. Men sometimes complain of pain in the entire hanging penile urethra. However, the main discomfort results from painful, uncontrolled urinary bladder contractions, induced by the presence of the catheter tip in the bladder lumen. These contractions very much resemble the pain which occur in the overactive bladder (OAB) and depend, among others, on stimulation of the muscarinic receptors [26, 27, 28]. In as much as the former components of CRBD can be alleviated with commonly applied analgesics, the OAB-like components poorly respond to medications from the NSAID group or to opioids [29, 30]. Even a short-term catheterisation of the urinary bladder, applied as a procedure, closing all types of operations, may considerably increase the post-operative discomfort of the patient [28, 29, 31].2) UTI as a result of urinary bladder catheter installation. Taking into account the high number of catheterised patients, the term CAUTI (Catheter Associated Urinary Tract Infection) has been defined [32, 33, 34, 35]. It is assumed that CAUTIs are the most frequent hospital infections in the world [36]. The number of CAUTIs of all hospitalised patients varies from 1.8% up to 4% [33, 37]. The incidence of CAUTIs among patients, submitted to all kinds of surgical interventions, is higher by 5% with every subsequent day of catheterisation [33, 38].3) Urethral lesions. According to Kashefi and Leuck, 0.32% and 0.5% of hospitalised and catheterised patients, respectively, present with iatrogenic lesions in this context [39, 40]. Thomas, in his analysis of urethral lesions from iatrogenic causes, writes that 6% of urological consultations at a teaching (university) hospital are carried out with complications of urinary bladder catheterisation in the background. As much as 67% of these complications are associated with urethral lesions [41]. The most severe consequence of iatrogenic urethral lesions is urethrostenosis, most often caused by direct injuries during catheterisation [42, 43].4) Other complications, observed in catheterisation of the urinary bladder, such as lesions preventing fluid removal from the catheter balloon or other defects of the Foley catheter, are much less frequent [44, 45]. There is some evidence for the assumption that the male gender is an independent risk factor with a powerful impact on the incidence of CRBD or discomfort induced by catheter presence after surgical procedures [46]. This assumption resulted in the subdivision into male and female subgroups in Group I and II. An analysis of acquired data demonstrated a statistically significant difference in post-operative pain intensity. The patients from Group II had higher pain sensations than the patients from Group I, which was a clear correlation to catheterisation. The harmonised scheme of post-operative pain treatment was based on the intravenous administration of ketoprofen and metamizole in equally divided doses, applied in regular time intervals: metamizole every 4–6 hours and ketoprofen every 8–12 hours. Daily doses of these medications were calculated by an anaesthesiologist with consideration of factors affecting pharmacokinetics. No statistically significant differences were found in the observed demands for additional analgesic doses of the patients from Group I and II, including the subdivision into male and female subgroups. The mean post-operative hospitalisation stay after surgery lasted 1.19 days and was the same in both groups. It may then be concluded that the lack of Foley catheter in patients from the experimental group was not associated with hospitalisation stay extension by complications of direct invasiveness of the catheter in the urinary bladder. None of the patients included into the study, presented with post-operative fever. Therefore, one may assume that neither the presence nor the absence of urinary catheter in the patients included into the study could in any way be associated with fever-inducing factors. All the patients were submitted to urine sampling on the day of discharge. UTI after URS was possible in the following two circumstances: by exo- or endogenous contamination during surgery (Group I and II) and as a result of urinary bladder catheterisation (Group II). There were no statistically significant differences between Group I and Group II, regarding the mean number and percent of patients with significant bacteriuria during post-operative hospitalisation, as confirmed by urine culture tests. It is assumed that catheterisation in a single patient increases the risk of UTI by 1 to 5%, while every subsequent day of the catheter's presence in the urinary bladder increases the risk by 5% [37, 40]. The studies of post-operative CAUTIs most often analyse the correlation between the number of CAUTIs and the time period of catheterisation, without reference to any control group without catheter [38, 47]. Some of these studies demonstrated a much lower incidence of CAUTI cases after shorter catheterisation vs. its longer periods [47]. One case of POUR was noted in MI group. Thus there was no statistically significant difference in the number of AUR cases after surgery, depending on catheterisation. Wu and Kang are the authors of reports based on the highest number of patients. The percent of POUR in their studies oscillates between and 2.52% [1, 2]. It would mean then that the number of POUR cases in our study was not much different from its average value. It should simultaneously be noted that the criteria of inclusion into the study, the modes of anaesthesia and the types of procedures significantly reduced the number of described in literature risk factors for POUR. Besides, lidocaine was used for the subarachnoid anaesthesia, which very rarely causes POUR. The time period of surgery (with spinal anaesthesia with lidocaine) did not, as a rule, exceed 2 hours. Therefore, no urinary bladder overfilling took place and the patients did not exhibit any intraoperative or intraoperatively diagnosed cases of pyonephrosis. The results of the reported study suggest then that the lack of Foley catheter after ureterorenoscopic removal of deposits from the ureter and/or from the kidney does not increase the risk in patients from the group of low risk for POUR. The volume of urine, retained after miction was determined by transabdominal ultrasound on the day of discharge from the ward. In the literature, one may come across a theme of lack of clear criteria for PVR [48, 49]. Kolman published a report dealing with the quantitative distribution of PVR in the population of healthy men. Its upper limit is 28 ml of urine retained in the bladder [50]. For this reason, in this reported study, the criterion for PVR diagnosis was 30 ml of urine retained in the bladder after miction. None of the discharged patients presented with urine volumes, retained after miction, above 30 ml. Thus it may be assumed that urinary bladder catheterisation after URS in patients with low risk for POUR has no effect on the risk for a clinically significant condition of PVR. BODY.CONCLUSIONS: Pain sensations, measured by VAS and by the quantities of additional analgesic medications, administered on the first day after the surgery of stone removal (URS) are higher in patients with a Foley catheter placed in their urinary bladder. Catheterisation of the urinary bladder after URS in patients with low risk, defined as no obstructions in urine drainage, and with a low risk for urinary tract infections and for metabolic and neurological conditions, has no effect on the post-operative hospitalisation time period nor on the number of post- operative complications, such as fever, urinary tract infections, urine retention after micturition or the risk for acute urine retention on the last day of hospitalisation. In patients with a low risk for post-operative complications and in those without any serious intraoperative complications, the urinary bladder catheterisation enhances patients' discomfort, while bringing no expected benefits. In such patients, the routine application of urinary bladder catheterisation after successful, uncomplicated URS, should become a subject of thorough and careful consideration. BODY.CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

TITLE: Effect of adding insulin degludec to treatment in patients with type 2 diabetes inadequately controlled with metformin and liraglutide: a double‐blind randomized controlled trial ( ABSTRACT.AIM: To evaluate the efficacy and safety of adding insulin degludec (IDeg) to treatment in patients with type 2 diabetes receiving liraglutide and metformin and qualifying for treatment intensification because of inadequate glycaemic control. ABSTRACT.METHODS: In this 26‐week, double‐blind trial, patients who still had inadequate glycaemic control after a 15‐week run‐in period with initiation and dose escalation of liraglutide to 1.8 mg in combination with metformin (≥1500 mg) were randomized to addition of once‐daily IDeg ('IDeg add‐on to liraglutide' arm; n = 174) or placebo ('placebo add‐on to liraglutide' arm; n = 172), with dosing of both IDeg and placebo based on titration guidelines. ABSTRACT.RESULTS: At 26 weeks, the mean change in glycated haemoglobin level was greater in the IDeg add‐on to liraglutide arm (−1.04%) than in the placebo add‐on to liraglutide arm (−0.16%; p < 0.0001). Similarly, the mean fasting plasma glucose reduction was greater, and self‐measured plasma glucose values were lower at all eight time points, with IDeg add‐on versus placebo add‐on (both p < 0.0001). At 26 weeks, the IDeg dose was 51 U (0.54 U/kg). During the run‐in period with liraglutide, body weight decreased by ∼3 kg in both groups. After 26 weeks, the mean change was +2.0 kg (IDeg add‐on to liraglutide) and −1.3 kg (placebo add‐on to liraglutide). Confirmed hypoglycaemia rates were low in both groups, although higher with IDeg than with placebo (0.57 vs. 0.12 episodes/patient‐years of exposure; p = 0.0002). Nocturnal confirmed hypoglycaemia was infrequent in both groups, with no episodes of severe hypoglycaemia, and no marked differences in adverse events with either treatment approach. ABSTRACT.CONCLUSION: The addition of liraglutide and IDeg to patients sub‐optimally treated with metformin and liraglutide and requiring treatment intensification was found to be effective and well‐tolerated. BODY.INTRODUCTION: Current guidelines on the management of type 2 diabetes (T2D) recommend metformin as the initial pharmacological therapy, followed by combination therapy with other oral antihyperglycaemic agents, glucagon‐like peptide‐1 (GLP‐1) receptor agonists (RAs) or insulin 1, 2. In patients who do not achieve control with metformin, one possible strategy is to add a GLP‐1 RA, followed by a basal insulin analogue if required 3, 4. GLP‐1 RAs and basal insulin address different but complementary physiological approaches to treating hyperglycaemia. GLP‐1 RAs mimic the effect of native GLP‐1 by stimulating insulin secretion in response to the absorption of orally ingested glucose (the so‐called incretin effect) 5. Treatment with GLP‐1 RAs benefits both postprandial and fasting plasma glucose (FPG) levels. Basal insulin addresses insufficient pancreatic β‐cell function in T2D by providing a constant concentration of insulin over an extended period, resulting in improved FPG control. In addition, the weight‐lowering effect of GLP‐1 RAs may limit the weight gain associated with insulin 3. Liraglutide is a long‐acting, once‐daily GLP‐1 RA that offers efficacious glycaemic control with minimal risk of hypoglycaemia accompanied by weight loss. Its efficacy and safety across the continuum of T2D was demonstrated in the LEAD programme of phase III clinical trials 6, 7, 8, 9, 10, 11, 12, 13. Insulin degludec (IDeg) is a once‐daily basal insulin analogue with a long duration of action 14. In randomized controlled trials, IDeg was as effective in achieving glycaemic control in T2D as insulin glargine, with fewer episodes of nocturnal hypoglycaemia 15, 16, 17, 18, 19, 20, 21, 22. The aim of the present study (BEGIN: ADD TO GLP‐1) was to determine the efficacy and safety of the addition of IDeg, compared with placebo, in patients who had not reached the glycated haemoglobin (HbA1c) target of <7.0% (<53 mmol/mol) with metformin and maximum‐dose liraglutide (1.8 mg). BODY.METHODS: A detailed description of the methods is given in the Supporting Information (Appendix S1). BODY.METHODS.TRIAL DESIGN AND PARTICIPANTS: This was a randomized (1 : 1), parallel‐group, double‐blind, multinational, controlled trial with a 15‐week run‐in phase, followed by a 26‐week core phase. Patients were eligible if they were aged ≥18 years and had T2D not previously treated with insulin and a body mass index (BMI) ≤45 kg/m2. Patients had to be receiving ongoing therapy with metformin ± a sulphonylurea, glinide, a dipeptidyl peptidase‐4 inhibitor or exenatide (twice daily only; details of regimens are provided in Table S1, Supporting Information), and had to have an HbA1c level of 7.5–10.0% (58–86 mmol/mol) inclusive (patients on metformin monotherapy) or 7.0–9.0% (53–75 mmol/mol) inclusive (patients on metformin combination therapy). At entry into the run‐in period, all previous antihyperglycaemic therapies except for metformin were discontinued. Key exclusion criteria were: a calcitonin level ≥50 ng/l; a history of chronic pancreatitis or idiopathic acute pancreatitis; and a current or past malignant neoplasm (except basal cell and squamous cell carcinoma). BODY.METHODS.TREATMENT: During the 15‐week run‐in period, liraglutide was initiated at 0.6 mg daily and increased to 1.8 mg daily over 2 weeks as recommended in the prescribing information 23. Metformin was continued at ≥1500 mg daily or the maximum tolerated dose (Figure 1). During run‐in and treatment, the daily doses of metformin and liraglutide (after the dose increase to 1.8 mg) were to remain unchanged. Patients were discontinued during run‐in if they could not tolerate liraglutide 1.8 mg daily. Figure 1Trial design. FPG, fasting plasma glucose; HbA1c, glycated haemoglobin; IDeg, insulin degludec; Lira, liraglutide; Met, metformin; SMPG, self‐measured plasma glucose; V, visit.DOM-12661-FIG-0001-c After completion of run‐in, patients whose HbA1c was still 7.0–9.0% (53–75 mmol/mol) were randomized to receive IDeg 10 U or placebo once daily subcutaneously in addition to liraglutide and metformin. IDeg was adjusted according to a titration guideline (Figure 1), aiming at self‐measured plasma glucose (SMPG) values of 4.0–5.0 mmol/l. Patients receiving placebo followed the same titration guideline, with placebo dispensed from an identical pen and measured in 'dummy units' as if it were insulin. Withdrawal criteria were prescribed in case of hyperglycaemia (Appendix S1, Supporting Information). BODY.METHODS.ENDPOINTS AND ASSESSMENTS: The primary endpoint was change from baseline in HbA1c after 26 weeks of randomized treatment. Secondary efficacy endpoints at 26 weeks included: change in FPG; percentage of responders achieving HbA1c <7.0% (<53 mmol/mol); and change from baseline in the following: mean pre‐breakfast SMPG measurements used for titration of IDeg/placebo dose, 8‐point SMPG profile, and mean of the 8‐point profile. Changes from baseline in body weight and health‐related quality of life (HRQoL), and dose of IDeg/placebo, were also assessed. Safety assessments included adverse events (AEs), hypoglycaemic episodes and changes from baseline in clinical evaluations and central laboratory assessments. Confirmed hypoglycaemic episodes included asymptomatic and symptomatic episodes confirmed by a measured plasma glucose value <3.1 mmol/l or severe episodes requiring assistance. Hypoglycaemic episodes occurring from 00:01 to 05:59 hours (inclusive) were classified as nocturnal. Prespecified medical events of special interest included thyroid disease, any confirmed episode of calcitonin levels ≥20 ng/l, all neoplasms (excluding thyroid neoplasms), pancreatitis or clinical suspicion of pancreatitis, lipase and/or amylase >3 × upper limit of normal range, and AEs leading to withdrawal (for a full list see Table S2, Supporting Information). Selected AEs as follows underwent adjudication by an independent External Adjudication Committee: neoplasms; thyroid disease; all types of stroke; acute coronary syndrome; hospitalization for unstable angina pectoris; all types of myocardial infarction; and fatal events. BODY.METHODS.STATISTICAL ANALYSIS: The sample size required to meet the primary objective with ≥90% power, using an assumed mean treatment difference of 0.4% and an estimate for the standard deviation (s.d.) of 1.1% for HbA1c (based on previous phase III trials in patients with T2D) was calculated as 320 participants. Changes from baseline in HbA1c, FPG, mean of the 8‐point SMPG profile, mean pre‐breakfast SMPG and HRQoL scores were analysed using analysis of covariance, with treatment, sex and region as fixed factors, and age and baseline value of the relevant variable as covariates. For change in HbA1c, superiority was to be considered confirmed if the upper bound of the two‐sided 95% confidence interval (CI) was below 0%. The percentages of responders achieving HbA1c <7.0% [<53 mmol/mol] were analysed based on a logistic regression model using the same fixed factors and covariates. The number of treatment‐emergent hypoglycaemic episodes was analysed using a negative binomial regression model with a log‐link function and the logarithm of the time period for which a hypoglycaemic episode is considered treatment‐emergent as offset. The model included treatment, sex and region as fixed factors, and age as a covariate. Other safety data were compared using descriptive statistics. Analyses of all efficacy endpoints and hypoglycaemia were based on the full analysis set (all randomized patients). Safety endpoints were summarized using the safety analysis set (all patients receiving at least one dose of study drug). Missing values were imputed using the last observation carried forward method, as recommended in US Food and Drug Administration (FDA) guidance 24. BODY.RESULTS.PATIENT DISPOSITION AND CHARACTERISTICS: Of 1504 patients screened, 970 met the selection criteria and entered the run‐in period (Figure 2). During the run‐in period, 389 patients (40% of those who entered run‐in) reached an HbA1c level of <7.0% (<53 mmol/mol); among these, 181 patients had previously been treated with metformin only, and 208 had been treated with combination therapy. After the run‐in, the 346 patients meeting HbA1c eligibility criteria were randomized to receive IDeg (n = 174) or placebo (n = 172), both added on to liraglutide 1.8 mg and metformin. Of these, 173 and 170 patients, respectively, received at least one dose of study drug, and 160 and 131 patients, respectively, completed the study. The withdrawal rate was greater with placebo (23.8%) than with IDeg (8.0%; Figure 2). Figure 2Patient disposition. AE, adverse event; FAS, full analysis set; HbA1c, glycated haemoglobin; IDeg, insulin degludec; Lira, liraglutide; SAS, safety analysis set. FAS: all randomized patients. SAS, all patients receiving at least one dose of study drug. aDuring the run‐in period patients were ineligible for randomization for the following reasons: AE, n = 76; non‐compliance with protocol, n = 29; randomization criteria (including HbA1c <7.0% [<53 mmol/mol]), n = 426; withdrawal criteria, n = 2; other, n = 91. bDuring the treatment phase: withdrawals due to 'other' reasons were caused by erroneous randomization, inefficient therapy (only in placebo + liraglutide group) and personal reasons such as patient not able to attend visits or unspecified withdrawn consent.DOM-12661-FIG-0002-c The characteristics of the two study groups were well matched at week 0 of the study (i.e. after the run‐in period; Table 1). The two study groups were also generally well matched with regard to antihyperglycaemic treatment at screening (Table S1, Supporting Information). At screening, ∼50% of patients were receiving a combination of metformin and sulphonylurea, and 30% metformin only. Table 1 Baseline characteristics of participants randomized at week 0. Characteristic IDeg add‐on to liraglutide Placebo add‐on to liraglutide (n = 174) (n = 172) Female/male, % 43.7/56.3 39.5/60.5 Race: white/black/Asian/other, % 80.5/12.6/5.2/1.7 87.8/6.4/2.3/3.5 Ethnicity: Hispanic or Latin American, % 9.8 11.0 Age, years 57.0 (±10.0) 57.3 (±9.4) Weight, kg 90.7 (±18.2) 94.0 (±19.1) BMI, kg/m 2 32.0 (±5.7) 32.4 (±5.4) Duration of diabetes, years 9.7 (±5.8) 9.3 (±5.4) HbA1c, % 7.6 (±0.6) 7.6 (±0.6) HbA1c * , mmol/mol 59.6 59.6 FPG mmol/l 8.7 (±2.1) 9.1 (±2.2) mg/dl 156.2 (±37.8) 164.0 (±39.5) BMI, body mass index; FPG , fasting plasma glucose; HbA1c , glycated haemoglobin; ID eg, insulin degludec. Data are mean ± standard deviation unless otherwise indicated. * Calculated values. The 389 patients who successfully achieved an HbA1c level <7.0% (<53 mmol/mol) during run‐in had the following characteristics at initial screening (week –16): mean (s.d.) HbA1c 8.1 (0.7)% [65 (8) mmol/mol], weight 95.9 (17.7) kg, BMI 33.6 (5.5) kg/m2 and duration of diabetes 7.9 (5.3) years. Values for the patients who did not achieve HbA1c levels <7.0% and were randomized were similar: HbA1c 8.3 (0.7)% [67 (8) mmol/mol], weight 95.4 (18.6) kg, and BMI 33.2 (5.5) kg/m2. Only duration of diabetes differed: 9.5 (5.6) years. BODY.RESULTS.GLYCAEMIC CONTROL: The observed mean (s.d.) change in HbA1c values from baseline to week 26 was −1.04 (0.89) percentage points with IDeg and −0.16 (0.86) percentage points with placebo, both on a continued background of liraglutide and metformin (HbA1c values over time are shown in Figure 3a). This resulted in an estimated treatment difference (ETD) of −0.92% (95% CI −1.10; −0.75; p < 0.0001). Thus, treatment with IDeg was confirmed to be superior to placebo for reduction in HbA1c. Figure 3(a) Glycated haemoglobin (HbA1c), (b) fasting plasma glucose (FPG), (c) body weight and (d) hypoglycaemia over time. (FPG values were not available for week −16.) CI, confidence interval; ETD, estimated treatment difference; FPG, fasting plasma glucose; IDeg, insulin degludec; Lira, liraglutide; n, number of patients with events; Rate, number of events per patient‐year of exposure; % patients, proportion of patients with events. HbA1c and FPG are mean values ± standard error (s.e.). Full analysis set: last observation carried forward. Comparisons: estimates adjusted for multiple covariates. Body weight (mean values ± s.e.) and hypoglycaemia are safety analysis set. The statistical comparisons for hypoglycaemia are based on the full analysis set.DOM-12661-FIG-0003-c At 26 weeks, 77.6% of patients receiving IDeg and 35.5% of those receiving placebo had achieved an HbA1c level <7.0% (<53 mmol/mol); the likelihood of achieving HbA1c <7.0% was significantly higher for participants in the IDeg add‐on group [odds ratio 7.79 (95% CI 4.57; 13.27); p < 0.0001]. The mean reduction from baseline in FPG at week 26 was greater with IDeg than with placebo [ETD –2.55 mmol/l (95% CI −3.07; −2.02); p < 0.0001 (Figure 3b)]. After 26 weeks, pre‐breakfast SMPG values were significantly lower with IDeg than with placebo [ETD –2.34 mmol/l (95% CI −2.67; −2.01); p < 0.0001, for the mean SMPG levels used for dose adjustment (Figure S1, Supporting Information)]. At 26 weeks, plasma glucose levels at all eight time points were significantly lower with IDeg versus placebo (p < 0.0001; Figure S2, Supporting Information). The mean of the 8‐point SMPG profile was significantly lower at week 26 with IDeg versus placebo, with an ETD of –1.95 mmol/l (95% CI −2.29; −1.60; p < 0.0001). BODY.RESULTS.OTHER ASSESSMENTS: Physical and mental HRQoL patient‐reported outcome scores changed marginally in both treatment groups, with no statistically significant differences between groups from baseline to week 26. At screening, body weight in patients subsequently randomized to IDeg [mean (s.d.) 93.7 (18.1) kg] was lower than in those subsequently randomized to placebo [97.2 (19.0) kg]; mean body weight decreased by ∼3 kg during the liraglutide run‐in in both groups (Figure 3c). During randomized treatment, mean body weight increased by 2.0 kg in the IDeg group and decreased by 1.3 kg in the placebo group. The mean dose of IDeg and placebo was 10 U (0.11 U/kg) at baseline in both groups. Mean doses increased steadily during the initial weeks in both groups, but to a greater extent with placebo (Figure S3, Supporting Information). After 10–12 weeks of treatment, the increase in mean daily dose of IDeg levelled off, reaching 51 U (0.54 U/kg) at week 26. The dose of placebo continued to increase and reached 105 U (1.18 U/kg) at week 26. BODY.RESULTS.SAFETY ENDPOINTS. : The number of patients experiencing confirmed hypoglycaemia during randomized treatment was higher with IDeg (17.3%) than with placebo (4.7%; Table S3, Supporting Information). The rate of confirmed hypoglycaemia was significantly higher with IDeg [0.57 episodes/patient‐years of exposure (PYE)] than with placebo [0.12 episodes/PYE; p = 0.0002 (Figure 3d)]. No severe hypoglycaemia was reported during the randomized treatment period. The proportion of patients with, and the rates of, nocturnal confirmed hypoglycaemia episodes during randomized treatment were low, at 1.7% (0.05 episodes/PYE) with IDeg and 1.2% (0.03 episodes/PYE) with placebo (not significant). During the run‐in period, plasma‐glucose‐confirmed non‐severe hypoglycaemia was reported by six patients (1.7%). Three episodes of severe hypoglycaemia were reported in two individuals: one episode with a blood glucose level of 2.2 mmol/l in a female patient later randomized to IDeg, who subsequently withdrew because of non‐compliance that was not further described; and two episodes that were not supported by blood glucose measurements, occurring in one female patient who was considered ineligible for randomization because she was not testing blood glucose during possible hypoglycaemic events. Adverse events reported by ≥5% of patients during the run‐in period were primarily gastrointestinal, with no unexpected clustering of events. Rates of diarrhoea and vomiting were low in both phases. The incidence of nausea during run‐in was 27.2 and 22.9% for participants subsequently randomized to IDeg or placebo, respectively, but decreased to 4.6 and 3.5%, respectively, during the randomized period. During run‐in, 76 patients withdrew because of AEs, of whom 57 withdrew because of gastrointestinal AEs. During the randomized period, the proportion of patients reporting treatment‐emergent AEs and the rates of treatment‐emergent AEs were similar in the two groups (Table S4, Supporting Information). Proportions reporting treatment‐emergent AEs were 55% (IDeg add‐on to liraglutide) and 52% (placebo add‐on to liraglutide), and rates were 344 and 335 events per 100 PYE, respectively. The most frequently reported AEs were nasopharyngitis, diarrhoea and elevated lipase. Serious AEs were reported by 3.5% of IDeg patients and 5.3% of placebo patients; no deaths were reported. The proportion of patients who withdrew from the trial because of AEs was 2.9% (IDeg) and 1.7% (placebo). Medical events of special interest were few, with no difference between treatment groups. No pancreatitis or pancreatic neoplasms were reported over the entire trial period, including run‐in, and all pancreas‐related AEs were non‐serious. Mean amylase and lipase values increased in both groups, with wide fluctuations between individual patients. Two patients withdrew from the trial because of elevated amylase and lipase values (both in the IDeg group). Two patients were withdrawn because of increased calcitonin values (Appendix S1, Supporting Information). Calcitonin levels normalized after 3 weeks in one individual; the other had a normal thyroid on examination, and continued to have elevated calcitonin levels. Further information on pancreas‐ and thyroid‐related AEs is available in Appendix S1, Supporting Information. Treatment‐emergent neoplasms were identified in four participants by the External Adjudication Committee (two in each group); none were considered related to study drugs (Table S5, Supporting Information). No event was confirmed by the External Adjudication Committee as a major adverse cardiovascular event. No clinically relevant differences in mean blood pressure between the two groups were seen at screening or end of treatment. From week 0 to week 26, mean blood pressure decreased slightly in both treatment groups; mean pulse increased slightly with IDeg (2.5 beats/min) but remained almost unchanged with placebo (0.6 beats/min). BODY.DISCUSSION: In the present study we evaluated the sequential addition of a GLP‐1 RA, liraglutide, to metformin, followed by either a basal insulin, IDeg, or placebo in patients requiring treatment intensification after treatment with liraglutide. Adding IDeg resulted in the majority of patients reaching the glycaemic goal within 6 months. HbA1c and FPG levels fell significantly more in patients who received add‐on IDeg than in those who received add‐on placebo. After 26 weeks, 78% of the patients receiving IDeg had achieved HbA1c <7.0% (<53 mmol/mol) versus 36% of patients with placebo. In comparison, a 43% response rate was reported in a study evaluating the addition of insulin detemir to liraglutide plus metformin in patients with T2D and HbA1c levels ≥7.0% (≥53 mmol/mol) 25. Rates of plasma‐glucose‐confirmed hypoglycaemia were higher with IDeg versus placebo (events/PYE: 0.57 vs. 0.12, respectively); rates of nocturnal confirmed hypoglycaemia were low in both treatment groups (higher with IDeg but not statistically significant). The reduction in HbA1c and FPG observed with IDeg was not accompanied by severe hypoglycaemia. The rates observed are relatively low for insulin‐treated patients when compared with other IDeg trials in the phase IIIa programme (BEGIN). In two trials, confirmed hypoglycaemia rates (using the definition used in the present study) in previously insulin‐naïve patients were 1.2 and 1.5 events/PYE with IDeg and 1.4 and 1.9 events/PYE with insulin glargine, respectively, used at similar insulin dose levels to those in the present study (0.53 and 0.59 U/kg for IDeg, and 0.60 U/kg for insulin glargine in both trials) 15, 18. The present study also confirmed the efficacy of liraglutide as add‐on to metformin 6: among the 624 patients ineligible for randomization after the run‐in period, 389 [62%; 40% of all 970 run‐in patients (Figure 2)] had reached HbA1c <7.0% (<53 mmol/mol) with the addition of liraglutide. This is a notable result given that, among these 389 patients, 208 (53%) had discontinued an additional oral antihyperglycaemic drug or twice‐daily exenatide from their metformin combination therapy before entering the run‐in phase. In the present trial, patients also lost ∼3 kg during the run‐in period and there was no unexpected clustering of AEs. The low incidence of hypoglycaemia during run‐in is consistent with rates observed in the LEAD development programme for liraglutide, and specifically in LEAD‐2 6. Patients who successfully reached HbA1c levels <7.0% (<53 mmol/mol) at the completion of the run‐in period had a mean HbA1c of 8.1% (65 mmol/mol) compared with 8.3% (67 mmol/mol) in those who did not reach target, and a shorter disease duration of 7.9 years (vs. 9.5 years), while mean weight and BMI did not differ. A run‐in period of 15 weeks was chosen to allow the initiation and dose escalation of liraglutide to the maximum dose of 1.8 mg daily. This was long enough to identify a population qualifying for treatment intensification in accordance with current guidelines 1, while also avoiding a confounding effect of any additional increase in dose of liraglutide during the trial. In order to justify the addition of IDeg or placebo, only patients who failed to reach the glycaemic target on the highest dose of liraglutide (1.8 mg), and therefore needed treatment intensification, were included. Weight increased slowly over 26 weeks in patients receiving IDeg, by a mean of 2.0 kg; however, final mean weight did not reach the pre‐run‐in value, suggesting that the weight gain associated with insulin may have been mitigated by the concomitant use of liraglutide. In this regard, it is of interest to note the results of a recent study, BEGIN VICTOZA ADD‐ON, which employed IDeg and liraglutide in the opposite sequence to that used in the present study 26. Patients with HbA1c >7.0% (>53 mmol/mol) after 104 weeks of treatment with metformin + IDeg were randomized to add either liraglutide once daily (dose‐escalated to 1.2 or 1.8 mg) or insulin aspart once daily (starting at 4 U and titrated as needed). The mean weight change at 26 weeks was −2.8 kg (liraglutide) and +0.9 kg (insulin aspart; p < 0.0001). While the patients in this study had more advanced diabetes, and the comparator group included a prandial insulin, both studies show a weight‐mitigating effect of liraglutide administered together with IDeg. In the placebo group, HbA1c remained more or less stable [mean value decreased from 7.6 to 7.5% (60–58 mmol/mol)] and weight decreased from 94.2 to 92.7 kg. These favourable results can be attributed to ongoing treatment with liraglutide. In the present study, there were no confirmed cases of pancreatitis or pancreatic cancer. Other AEs reported in this study were as expected considering the component drugs of the regimen, and patterns of AEs were similar in the two treatment groups. The AEs reported during the run‐in period were primarily gastrointestinal‐related, as was seen in the liraglutide development programme 5, but the relatively high incidence of these gastrointestinal AEs decreased over time, as has previously been reported with GLP‐1 RAs 25. The present carefully designed study had the advantage of being double‐blinded, unlike many studies using insulin. Limitations include the fact that placebo rather than an active drug was the comparator; this was decided so that the absolute effect of adding IDeg to liraglutide could be assessed, in line with FDA guidance that phase III studies of investigational agents as add‐on therapy are typically designed as placebo‐controlled superiority or active‐controlled non‐inferiority trials 24. Other limitations were the fact that only patients able to tolerate the maximum dose of liraglutide were randomized, and the withdrawal rate in the placebo add‐on to liraglutide arm was notably greater than that in the IDeg add‐on to liraglutide arm – driven in part by withdrawals as a result of inefficient therapy in the placebo arm. In conclusion, physicians can now choose from a number of strategies that involve the complementary actions of a GLP‐1 RA and basal insulin, according to the individual patient's needs: GLP‐1 RA followed by basal insulin; or basal insulin followed by GLP‐1 RA; or use of the two in combination. In this multicentre, randomized, placebo‐controlled trial, the sequential addition of a GLP‐1 RA, liraglutide, followed by a basal insulin, IDeg, to patients with T2D requiring treatment intensification was found to be an effective and well‐tolerated treatment regimen. BODY.CONFLICT OF INTEREST: V. R. A. has received research grants from Amylin, AstraZeneca, Boehringer Ingelheim, Hanmi, Eisai, GI Dynamics, Novo Nordisk, Sanofi‐Aventis and Takeda (all to employer institution) and served as a consultant for Janssen, Novo Nordisk, Sanofi (to employer institution). T. B. has received research grants from Abbott, ACON, Alere, Animas, Bayer, BD, Cebix, Bristol‐Myers Squibb, Dexcom, GlaxoSmithKline, Halozyme, Insulet, Lifescan, Eli Lilly & Co, Mannkind, Medtronic, Merck, Novo Nordisk, Orexigen, Sanofi and Tandem, has acted as a consultant for Bayer, BD, Medtronic, Novo Nordisk and Sanofi, and attended speakers' bureaux for Novo Nordisk. B. C. has received research grants from Sanofi‐Regeneron and acted as a consultant for Amgen, AstraZeneca, Debiopharm, Janssen, Eli Lilly & Co., Genfit, Novo Nordisk and Sanofi‐Regeneron. S. K. has received research grants from, and attended advisory boards for Novo Nordisk. L. A. L. has received research grants from AstraZeneca Pharmaceuticals LP, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly & Co., GlaxoSmithKline, Janssen Pharmaceuticals, Merck, Novo Nordisk, Pfizer, Sanofi, Servier; attended advisory boards for AstraZeneca Pharmaceuticals LP, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly & Co., Janssen Pharmaceuticals, Merck, Novo Nordisk, Sanofi, Servier, and attended speakers' bureaux for AstraZeneca Pharmaceuticals LP, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly & Co., Janssen Pharmaceuticals, Merck, Novo Nordisk and Sanofi. P. R. has received research grants from Novo Nordisk, Eli Lilly & Co., Sanofi, Pfizer, AstraZeneca Pharmaceuticals LP, Boehringer Ingelheim Pharmaceuticals, Inc., Gilead Sciences, Inc., Intarcia; acted as a consultant for Janssen Pharmaceuticals, Inc., GlaxoSmithKline Pharmaceuticals, and attended speakers' bureaux for Janssen. J. Z. is an employee of Novo Nordisk and holds shares in the company. T. H. A. was an employee of Novo Nordisk at the time of this study and holds shares in the company. A. P.‐T. has received research grants from Merck, Novo Nordisk, Sanofi, Lilly, Amylin, AstraZeneca, Pfizer, Janssen and Genentech, and acted as an advisory panel member for Lilly, Novo Nordisk and Sanofi on behalf of her employer institution. All authors (V. R. A, T. B., B. C, S. K., L. A. L, P. R., J. Z., T. H. A and A. P. ‐T.) were involved in critical analysis and interpretation of the data, drafting/critically revising the article and shared in the final responsibility for the content of the manuscript and the decision to submit it for publication. BODY.SUPPORTING INFORMATION: Appendix S1. Supplementary methods. Table S1. Antihyperglycaemic treatment at screening. Table S2. Adverse events classified as medical events of special interest. Table S3. Hypoglycaemic episodes. Table S4. Treatment‐emergent adverse events. Table S5. Neoplasms by system organ class and preferred term: safety analysis set. Figure S1. Mean pre‐breakfast self‐measured plasma glucose for dose adjustment by treatment week: mean plot, full analysis set. Figure S2. Eight‐point self‐measured plasma glucose profile at week 26: mean plot, full analysis set. Figure S3. Insulin doses.Click here for additional data file.

TITLE: The effect of school meals with fatty fish on adolescents’ self-reported symptoms for mental health: FINS-TEENS - a randomized controlled intervention trial ABSTRACT.ABSTRACT: There is a growing body of evidence linking fish consumption and n-3 LCPUFAs to mental health. Still, the results from randomized trials with n-3 LCPUFAs show conflicting results, and it is possible that the combined effect of several nutrients in fish may explain the observed associations. To aim of the present study was to investigate if school meals with fatty fish three times per week for 12 weeks could alter mental health in a sample of typically developing adolescents. In the Fish Intervention Studies-TEENS (FINS-TEENS), adolescents from eight secondary schools (n=425) in Norway, were randomized to receive school meals with fatty fish, meat or n-3 LCPUFA supplements. Mental health was assessed with the Strengths and Difficulties Questionnaire (SDQ) and the differences between the groups were assessed with linear mixed effect models, unadjusted and adjusted for baseline and dietary compliance. The results showed no effects of school meals with fatty fish compared to similar meals with meat or n-3 LCPUFAs on the adolescents' self-reported symptom scores for mental health. Among adolescents scoring above the SDQ cut-offs (high-scorers), the fish- improved less than the meat group in the self-reported symptom scores for total difficulties- and emotional problems. However, the findings should be regarded as preliminary, as the analyses for the high-scorer group were underpowered. In conclusion, serving school meals with fatty fish did not alter mental health in a typically developing sample of adolescents. It is possible that serving healthy school meals with meat is more beneficial than similar meals with fatty fish in adolescents scoring high on mental health problems. However, the results should be seen as preliminary, as the dietary compliance in the fish group was low and the analyses in the high score group underpowered. Thus, further studies should investigate the associations between fish consumption and adolescents' mental health. BODY.INTRODUCTION: Mental health disorders affect a significant proportion of children and adolescents. A recent meta-analysis found a worldwide prevalence of 13% of children and adolescents, with an anxiety disorder being the most prevalent (6.5%) [1]. In Norway, the prevalence is estimated to be around 7% in pre-[2] and primary [3] school children, and a large population-based survey found an increase in self-reported mental health problems during adolescence [4]. In addition, a substantial number report subthreshold symptoms that may lead to functional impairment [5]. An unhealthy dietary pattern has been associated with poor mental health in both adults [6,7], children and adolescents [8], even though the precise mechanisms are unclear [9]. A general concern is given to the effects of the typically modern Western dietary pattern, which is characterized by high levels of meat and saturated fat and low levels of fish and vegetables [10]. Fish is rich in n-3 long chain polyunsaturated fatty acids (n-3 LCPUFAs) and other important nutrients, such as vitamin D, selenium, iodine and high-quality protein [11,12]. Especially the n-3 LCPUFAs have received considerable interest, and there is growing evidence that suboptimal intakes of n-3 LCPUFAs may be associated with mental health over the lifespan [13,14]. The most prominent findings have been revealed for depression [15], but there is also some support for an effect of n-3 LCPUFAs in subgroups of children with attention-deficit hyperactivity disorder (ADHD) [16]. Still, findings from randomized controlled trials (RCT) with clinical populations remain inconclusive [14] and no benefits on mental health were seen in a sample of typically developing children after supplementation with n-3 LCPUFAs for 16 weeks [17]. However, long-term effects were found for both externalizing and internalizing problems, after 6 months supplementation with n-3 LCPUFAs in a community-residing sample of children (8–16 y), suggesting a delayed effect of n-3 LCPUFAs [18]. Recently, evidence has also accumulated for the impact of vitamin D and the overall results from cross-sectional and longitudinal studies suggest that vitamin D plays a role in the pathogenesis of mental health in both children and adolescents [19]. Most studies have focused on the effect of supplementing with single micro- or macro-nutrients and only a few studies have explored the relationship between consumption of fish as food and mental health. One study found an inverse relationship between fish consumption and major depression across countries [20] and two surveys from Finland found an association between infrequent fish intake and depression in women [21,22]. Longitudinal studies have also found a negative association between maternal intake of seafood during pregnancy and suboptimal child outcomes, such as IQ, social development and communication skills [23], as well as a negative association between the n-3 LCPUFAs, docosahexaenoic (DHA) status early in life and child internalizing problems (anxious/depressed) at 7 years of age [24]. However, to our knowledge no study has investigated the possible impact of an increased intake of fatty fish, rich on n-3 LCPUFAs and other important nutrients, on mental health status in a sample of typically developing adolescents. The aim of the present study was to investigate whether lunch meals with fatty fish three times per week for 12 weeks altered mental health status compared to identical control meals with meat or supplements with n-3 LCPUFAs in a typically developing sample of adolescents. BODY.SUBJECTS AND METHODS.SOURCE POPULATION AND PARTICIPANTS: The source population in the Fish Intervention Studies-TEENS (FINS-TEENS) were adolescents attending 9th grade (14–15 years old) at eight secondary schools in Bergen, Norway. All the 26 secondary schools in the municipality were contacted. Three schools never replied, nine refused to participate and six were excluded because they had less than three school classes in 9th grade. Thus, eight secondary schools with 785 adolescents attending 9th grade were invited to take part in the study and written consent was obtained from 481 (61%) adolescents and one of their parents/caregivers. Exclusion criteria were allergy or intolerance to the food or supplements included in the intervention. The trial was conducted between February and May 2015 in accordance with the declaration of Helsinki. Ethical approval was obtained from the Norwegian Data Protection Official for Research (project number: 41030) and the trial is registered in ClinicalTrials.gov (NCT02350322). BODY.SUBJECTS AND METHODS.TRIAL: A three-armed randomized controlled study design was used. The participants received a school meal with either fatty fish (Fish group), a comparable meal with meat/cheese (Meat group) or fish oil supplements containing n-3 LCPUFAs (Supplement group). The meals and supplements were served to participants three times a week for 12 weeks in the classrooms during lunch break. The meals were prepared by a catering agency (Søtt+Salt A/S). The meals in the fish group consisted of salmon, mackerel and herring, whereas the meals in the meat group consisted of chicken, turkey and beefburger (sometimes cheese was served together with the meat). Halal meat was provided on request and pork meat was not used. All meals consisted of vegetables and/or salad in combination with mostly wholegrain pasta, focaccia, baguette or tortilla. The amount of fish and meat was requested to be 80–100 grams per meal, and thus 90 gram fatty fish per servings were used to calculate the weekly intake of n-3 LCPUFAs in the supplement group. Each capsule contained 500 milligrams (mg) of concentrated fish oil (Nycoplus® Omega-3, 500 mg produced by Takeda Nycomed, Asker, Norway) and eight capsules per serving corresponded to 90 gram of oily fish. Trained research assistants were responsible for handing out the meals/supplements in each classroom and monitored compliance by registering the amount of leftovers from every adolescent, on a scale ranging from 0 (none of the served food was consumed) to 4 (all of the served food was consumed). Likewise, the intake of supplements were counted and scored according to the number of capsules (0–7) consumed. Records of dietary compliance revealed that there were significant differences in intake between the three intervention groups, and that the proportion of participants who consumed at least half of the meals/capsules during the trial was 38%, 66% and 87% in the fish, meat and supplement group, respectively [25]. A more detailed description of the design, study meals and dietary compliance are given in Skotheim et al. [25]. BODY.SUBJECTS AND METHODS.PROCEDURE: The SDQ was administered to the participants at school as part of a larger computer-based questionnaire, including a food frequency questionnaire (FFQ) and questions about background characteristics. The participants had access to individual computers and the questionnaire was filled out before lunch break at both pre- and post-intervention. In addition, the participants took part in a concentration and reading- and spelling test, and biological samples were collected (not used in this present article). The same group of researchers was responsible for the data collection at pre- and post-intervention, and was present in the classroom to answer questions from the participants. BODY.SUBJECTS AND METHODS.MENTAL HEALTH: The SDQ is a brief screening questionnaire, measuring mental health during the last 6 months in youths between the age of 3–16 [26]. The instrument consists of 25 items divided on five subscales: Emotional symptoms, conduct problems, hyperactivity/inattention symptoms, peer relationship problems and prosocial behaviour. Each item is rated on a scale from 0–2, and it is possible to get a score on each subscales (0–10), as well as a total difficulties score based on the first four difficulties subscales (0–40). SDQ also includes an impact supplement, which assess the adolescent's level of distress and interference of symptoms and problems on daily life functioning in youths reporting mental health problems. Moreover, a follow-up version of the SDQ has been developed in order to target any changes due to an intervention. The follow-up version is identical to the original SDQ, but asks about the last month as opposed to last 6 months, as in the original SDQ. The SDQ may be completed by several informants (self, parent and teacher) and the present study used the self-completed (SDQ-S) version (11–16 y). The psychometric properties of the Norwegian version of SDQ-S has been investigated in a systematic review [27], that included 39 571 children. The analyses supported its construct validity and the internal consistency for the total difficulties scale and the subscale emotional problems were satisfactory, while the internal consistency for the reaming subscales were somewhat lower, especially for the subscale conduct problems. Norwegian cut-off points for the SDQ-S were used to define the participants that scored high on the SDQ total difficulties or the five subscales (high scorer). The cut-offs were based on a survey, that included 4167 participants (11–16 years old) from Norway, where the 80th percentile were used to determine the participants that scores in the borderline or clinical range on the SDQ-S total difficulties or the five subscales [28]. Thus, high scorers were defined as those who scored ≥ 5 on emotional symptoms, ≥ 4 on conduct problems, ≥ 6 on hyperactivity/inattention symptoms, ≥ 4 on peer problems, or ≥ 15 on total difficulties. As the prosocial scale is inverted compared to the others subscales, 'high scorers' were defines as those scoring ≤ 5 on the scale. The impact supplement was administered but is not reported here. BODY.SUBJECTS AND METHODS.DIETARY HABITS AND BACKGROUND CHARACTERISTICS: A revised and extended version of a validated food frequency questionnaire (FFQ) [29,30] was completed by the adolescents both before and after the intervention to assess their habitual diet during the last 3 months. The purpose of the FFQ was to monitor the participants' habitual diet (i.e. what participants ate besides the intervention meals and supplements). It was a semi-quantitative FFQ, comprising 34 questions, measuring the frequencies of consuming different groups (i.e. milk and dairy products, fruits and vegetables, etc.). In addition, the FFQ included questions related to physical activity and characteristics, such as age, gender, weight, height and ethnicity. A more detailed description of the FFQ used in the present study is given in Handeland et al. [31]. However, as previously shown, there were no changes in the participants' habitual diet during the intervention period [32]. Body mass index (BMI) was calculated by dividing each adolescent's weight (kilogram) by the square of the height (metres). In addition, Cole's age and sex-specific BMI cut-off points for underweight [33] and overweight [34] according to adolescents (14.5 years) were used to define the proportions that where underweight, normal and overweight/obese. At post-intervention the adolescents were instructed to not include the meals/supplements served in the study [31]. In addition, one of the parents/caregivers received an email with a link to an online questionnaire at the same time points, measuring demographic factors, such as education, household income and marital status and the adolescents' mental health status (SDQ-P). BODY.SUBJECTS AND METHODS.RANDOMIZATION: Participants were individually randomized to one of the three groups, stratified by gender. Pieces of papers marked with one of the three intervention groups were put in two boxes: one marked 'girls' and one marked 'boys'. Two researchers assigned every enrolled girl and boy, to either the fish or the meat or the supplement group by drawing lots. The researcher who drew lots was only informed about the participants' gender (blinded), while the other researcher who had access to the list with the participants' names and class affiliation, registered the assigned intervention for each individual in a spreadsheet. BODY.SUBJECTS AND METHODS.SAMPLE SIZE: This three-armed intervention study, had two repeated measurements (pre- and post-intervention) with an assumed correlation of 0.5. Sample size was calculated based on the primary outcome of the trial (d2 test of attention), where a small to moderate effect size (cohens d = 0.35) was applied. Given a power of 80% and a significance level of α = 0.05, it was estimated that a sample size of 119 participants in each group was needed. With the risk of 20% dropout, totally 446 participants ought to be enrolled. BODY.SUBJECTS AND METHODS.STATISTICAL ANALYSES: Continuous variables were expressed in means and standard deviation (SD). Categorical variables were expressed in numbers and percentages. Differences between the groups at baseline were assessed with a one-way ANOVA (continuous variables) or Chi-square test (categorical variables). Differences between completers (valid data pre- and post-intervention) and non-completers (withdrawn or missing SDQ data pre or post), were assessed with independent samples t-test or Chi-square test. Paired-samples t-test was used to analyse differences between pre- and post-scores on the SDQ (total difficulties and the five subscales) within each intervention group. To investigate differences between groups in change SDQ scores (Δ SDQ scores), linear mixed effect models were applied. The participants' school class was included as a random intercept to account for dependency in the data at the level of class affiliation. Two models were presented. In the first model, the currently examined SDQ outcome at post-intervention, was adjusted for the equivalent outcome at baseline. In the second model, dietary compliance was added to the model. The fish group was used as reference. Model assumptions were investigated by visual inspections of residual and normal probability plots. Possible interaction effects between group and compliance were investigated for all SDQ outcomes, but not shown because no significant effects were found. Two-tailed p-values < 0.05 were considered statistically significant. Statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS® Statistics version 24, IBM Corporation, US) and Stata Statistical Software (STATA/IC 14.2). BODY.RESULTS.SUBJECTS: Out of the 481 adolescents who agreed to participate in the trial, three withdrew the day of baseline testing and before randomization. Thus, 478 adolescents were randomized to one of the three treatment groups. During the intervention, 34 pupils withdrew (actively) from the study and 16 were lost to follow-up during the administration of SDQ, either pre or post. In addition, one participant withdrew his consent after study completion and two unusual/extreme change scores on the outcome variable of interest (ΔSDQ Total > 30, scale 0–40, mean change score = -0.12, SD = 4.1) were identified through inspection of boxplot and excluded from the analyses (interpreted as non-valid responses). Thus, the present study included 425 participants (Figure 1).Figure 1.Flow chart over participants. There were no differences between the completers and non-completers (withdrawn and lost to follow-up) in any of the baseline characteristics. Regarding the SDQ, the non-completers scored higher on emotional problems, hyperactivity/inattention and total difficulties than the completers at baseline. However, there were no differences on any of the SDQ scales between the three intervention groups neither for the non-completer or the completer sample at baseline (data not shown). BODY.RESULTS.CHARACTERISTICS OF THE STUDY POPULATION: No differences in baseline characteristics were found between the three intervention groups (Table 1). The participants had a mean age of 14.6 years and 53% were girls. About 79% of the participants had a BMI within the normal range, while 14% was defined as underweight and 7% as overweight/obese. Approximately 11% had a non-Norwegian background. About 71% of the mothers and 59% of the fathers had higher education, and 28% reported a high household income. On average, the participants consumed seafood for dinner once per week and about 17% reported taken n-3 LCPUFAs supplements on a daily basis during the last 3 months.Table 1.Baseline characteristics of the study population and the different intervention groups. nAllFish(n = 137)Meat(n = 145)Supplementa(n = 143)Gender, girls, n (%)425224 (53)77 (56)72 (50)75 (52)Age, mean (SD)42514.6 (.34)14.6 (.34)14.6 (.33)14.6 (.34)BMI category b396 Underweight 55 (14)21 (17)19 (14)15 (11) Normal weight 312 (79)98 (77)105 (78)109 (81) Overweight/obese 29 (7)8 (6)10 (8)11 (8)Ethnicity, non-Norwegianc, n (%)42546 (11)16 (12)16 (11)14 (10)Maternal education level, n (%)346 Elementary/high or vocational school 101 (29)26 (24)40 (35)35 (29)College/University 246 (71)84 (76)75 (65)87 (60)Paternal education level, n (%)345 Elementary/high or vocational school 142 (41)51 (46)47 (41)44 (37) College/University 204 (59)59 (54)68 (59)77 (64)Household income, n (%)345 < 200.000–749.999 73 (21)21 (19)20 (18)32 (26)750.000–1. 249 999 175 (51)57 (52)62 (54)56 (46)1 250 000- >2.000 000 97 (28)32 (29)32 (28)33 (27)Seafood for dinnerd, mean (SD)4254.1 (.95)4.1 (.99)4.0 (.94)4.1 (.90)Omega-3 supplement, n (99%)424 Never 229 (54)67 (49)81 (56)81 (57) 1–3 times/month 53 (13)18 (13)18 (12)17 (12) 1–3 times/week 47 (11)21 (15)14 (10)12 (8) 4–6 times/week 22 (5)7 (5)6 (4)9 (6) Daily 73 (17)24 (18)26 (18)23 (16) BODY.RESULTS.EFFECTS OF THE INTERVENTION ON THE ADOLESCENTS’ SELF-REPORTED SDQ SCORES: There were no differences in any of the SDQ change scores from pre to post between the fish and meat or fish and supplement group, neither before nor after adjusting for the participants' baseline scores or baseline and compliance scores (Table 2).Table 2.Predicted changes in SDQ scores after fish (n = 137), meat (n = 145) or supplement (n = 143) intervention, different models. Crude SDQ scores SDQ scores adjusted for:Baseline scoreBaseline score & complianceSDQ scalesBaselineMean (SD)12 weeksMean (SD)p-withinaChangeMean (95% CI)p-valuebChangeMean (95% CI)p-valuecEmotional problems Fish2.9 ± 2.12.9 ± 2.40.960.07 (-0.18,0.31)-0.12 (-0.15,0.39)-Meat2.6 ± 2.12.6 ± 2.00.770.03 (-0.21,0.27)0.830.02 (-0.22,0.26)0.61Supplement2.5 ± 2.22.6 ± 2.10.470.05 (-0.19,0.29)0.920.01 (-0.26,0.26)0.56Conduct problems Fish1.6 ± 1.41.5 ± 1.40.74−0.07 (-0.27,0.14)-−0.08 (-0.30,0.14)-Meat1.7 ± 1.71.4 ± 1.40.01−0.27 (-.047,-0.07)0.13−0.27 (-0.47,-0.07)0.19Supplement1.6 ± 1.51.7 ± 1.50.260.10 (-0.10,0.30)0.230.11 (-0.10,0.33)0.23Hyperactivity/in attentionFish3.9 ± 2.33.8 ± 2.20.41−0.10 (-0.34,0.16)-−0.17 (-0.44,0.11)-Meat3.9 ± 2.24.0 ± 2.20.580.10 (-0.15, 0.35)0.280.11 (-0.14,0.35)0.14Supplement3.6 ± 2.13.6 ± 2.10.88−0.08 (-0.32,0.17)0.92−0.01 (-0.27,0.26)0.44Peer problemsFish1.5 ± 1.41.5 ± 1.40.88−0.02 (-.22,0.17)-−0.03 (-0.24,0.18)-Meat1.7 ± 1.61.5 ± 1.50.09−0.16 (-0.35, 0.03)0.31−0.16 (-0.35,0.03)0.37Supplement1.4 ± 1.51.5 ± 1.40.80−0.02 (-0.21,0.17)0.99−0.01 (-0.22,0.19)0.93Prosocial behaviourFish7.6 ± 1.87.5 ± 1.70.91−0.02 (-0.25,0.22)-0.10 (-0.16, 0.36)-Meat7.6 ± 1.77.5 ± 1.80.79−0.03 (-0.26,0.19)0.93−0.04 (-0.27,0.19)0.42Supplement7.6 ± 1.77.6 ± 1.80.790.04 (-0.19,0.26)0.75−0.07 (-0.31,0.18)0.39Total difficultiesFish9.9 ± 4.99.7 ± 5.10.52−0.11 (-0.65,0.44)-−0.12 (-0.72,0.48)-Meat9.9 ± 5.39.6 ± 4.60.23−0.33 (-0.90,0.20)0.57−0.32 (-0.85,0.21)0.62Supplement9.2 ± 4.99.4 ± 5.10.470.08 (-0.45,0.62)0.630.10 (-0.48,0.67)0.63 BODY.RESULTS.EFFECTS OF THE INTERVENTION ON HIGH SDQ-SCORERS AT BASELINE: In the high-scorer sample, the fish group improved less in the symptom scores for emotional problems (p = 0.04) and total difficulties (p = 0.02) than the meat group. The difference remained significant for emotional problems (p = 0.03), and was borderline significant for total difficulties (p = 0.06) after adjusting for compliance (Table 3).Table 3.Predicted changes in SDQ scores for participants with high SDQ scores at baseline after fish, meat or supplement intervention, different models. Crude SDQ scores SDQ scores adjusted for:Baseline scoreBaseline score & complianceSDQ scalesnBaselineMean (SD)12 weeksMean (SD)p-withinaChangeMean (95% CI)p-valuebChangeMean (95% CI)p-valuecEmotional problems (≥ 5)Fish266.4 ± 1.36.0 ± 1.90.25−0.31 (-0.92,0.30)-−0.21 (-0.89,0.47)-Meat315.8 ± 1.14.7 ± 1.5<0.01−1.20 (-1.75,-0.64)0.04−1.20 (-1.76,-0.63)0.03Supplement296.1 ± 1.45.2 ± 1.9<0.01−0.82 (-1.39,-0.25)0.23−0.91 (-0.89,0.47)0.17Conduct problems (≥ 4)Fish134.5 ± 1.12.9 ± 1.9<0.01−1.64 (-2.39,-0.89)-−1,38 (-2,23,-0,48)-Meat195.1 ± 1.43.5 ± 1.5<0.01−1.53 (-2.16,-0.91)0.83−1,56 (-2,19,-0.93)0.75Supplement174.5 ± 0.63.5 ± 1.5<0.01−0.98 (-1.63,-0.32)0.18−1,14 (-1,87,-0.41)0.71Hyperactivity/inattention (≥ 6)Fish337.2 ± 1.36.2 ± 1.5<0.01−0.90 (-1.44,-0.37)-−0.88 (-1.50,-0.26)-Meat317.1 ± 1.16.6 ± 1.40.07−0.44 (-0.99,0.11)0.23−0.44 (-1.00,0.12)0.28Supplement296.8 ± 1.05.6 ± 2.1<0.01−1.32 (-1.89,-0.75)0.29−1.35 (-2,0,-0.66)0.37Peer problems(≥ 4)Fish154.3 ± 0.52.9 ± 1.1<0.01−1.47 (-2.28,-0.65)-−1.26 (-2.15,-0.38)-Meat184.8 ± 1.23.1 ± 1.9<0.01−1.95 (-2.70,-1.20)0.78−1.95 (-2.69,-1.22)0.72Supplement115.0 ± 1.03.6 ± 1.20.03−1.31 (-2.23,-0.39)0.32−1.26 (-2.15,-0.38)0.20Prosocial behaviour (≤ 5)Fish204.6 ± 1.05.6 ± 1.4<0.010.84 (0.15,1.52)-0.88 (0.15,1.61)-Meat194.4 ± 1.25.5 ± 1.90.021.05 (0.35,1.75)0.631.06 (0.35,1.77)0.69Supplement214.7 ± 0.65.5 ± 1.30.020.76 (1.0, 1.42)0.860.7 (-0.02,1.45)0.75Total difficulties (≥15)Fish2517.8 ± 2.916.4 ± 3.90.02−1.54 (-3.01,0–08)-−1.88 (-3.60,-0.15)-Meat2618.6 ± 3.514.3 ± 3.6<0.01−4.11 (-5.55,-2.67)0.02−4.10 (-5.54,-2.65)0.06Supplement2018.0 ± 2.615.3 ± 4.7<0.01−2.78 (-4.42,-1.14)0.27−2.38 (-4.34,-0.42)0.73 BODY.DISCUSSION: The overall results from the present study revealed that being served fatty fish three times a week for 12 weeks did not alter the adolescents' self-reported symptom scores for mental health, compared to being served comparable meals with meat or n-3 LCPUFA supplements. In the high SDQ scorer sample the fish improved less than the meat group on the symptom scores for total difficulties and emotional problems, and the findings remained significant for emotional problems and borderline significant for total difficulties after adjusting for compliance. To our knowledge, no RCTs have previously assessed the effect of a dietary intervention with fatty fish on mental health in a sample of typically developing adolescents. However, the lack of findings for both the fatty fish and the meat group, are in concordance with the results from a large cluster randomized intervention trial, showing no reduction in the risk of being in the borderline/abnormal range on any of the SDQ dimensions after a 3 months intervention with school breakfast as rated by the teacher (primary school) or self-report (secondary school) [35]. The findings are also consistent with the results from a randomized intervention trial supplementing with n-3 LCPUFAs or placebo for 16 weeks [17]. In this study, no beneficial effects of n-3 LCPUFAs on mental health scores as measured by the SDQ (parent and teacher reports) were found in 8–10 year old children from a mainstream school population. On the contrary, long-term reductions were found for both externalizing and internalizing behaviour problems in an RCT supplementing with n-3 LCPUFAs for 6 months in a community-residing sample of children and adolescents (8–16 y) [18]. Interestingly, the strongest effects were found 6 months after the end of treatment, suggesting that the accumulation of fatty acids in the brain may take some time before changes can be seen on mental health. Taken together the results indicate that there are no immediate effects on mental health after short-term intervention with either healthy school meals or n-3 LCPUFA supplementation in typically developing children and adolescents. Cross-sectional and prospective studies show that fish consumption is associated with reduced levels of mental health problems in both children and adults [20–24], thus a longer exposure time than 3 months is possibly required in order to influence mental health. Given the low dietary compliance in the fatty fish group compared to the two other intervention groups, it is not certain whether an extension of time in the present study would have yielded a sufficient intake of fatty fish. As we were not able to serve warm lunch meals, future intervention trials with fatty fish should possibly give priority to creating warm meals that highly match adolescents' preferences in order to ensure a higher dietary compliance than achieved in the present study. It is also possible that other research designs are better for investigating the relationship between fatty fish and mental health in a normal sample of adolescents, such as prospective studies over a longer period of time that include validated dietary assessments and that adjust for important confounders [8,36]. The finding that the adolescents with higher levels of emotional problems and total difficulties (emotional problems included here) benefitted more from receiving healthy lunch meals with meat compared to comparable meals with fatty fish, could indicate that micronutrients typically found in meat, such as iron, contributed to a positive change. However, mostly white meat (chicken and turkey) were served in the present study and it is unlikely that iron from the intervention contributed to the present finding. As the dietary compliance in the fish group was low compared to the meat group [25], a possible explanation is therefore that a higher consumption of lunch meals with better nutritional composition than their habitual packed lunch was beneficial for the adolescents with emotional problems. As already reported, the participants' habitual diet was below the recommendations for fish, fruits and vegetables [31], which support this interpretation. In addition, a recent systematic review found evidence for a consistent trend between good quality diet and lower levels of internalizing problems (low mood and anxiety) in children and adolescents, and some evidence for the reverse [8]. However, as this finding was based on a smaller sub-sample that participated in the intervention, the findings should be replicated on a larger sample of adolescents with high levels of emotional problems. One of the most important limitations with the present trial was the low dietary compliance in the fish group compared to the two other intervention groups. Thus, the intake of fatty fish might not have been sufficient to influence mental health. A potential limitation is also the duration of 12 weeks, which might have been too short to alter mental health status. It was also a limitation that that we did not have a second follow-up. The turn-over of fatty acids in the brain is likely to be slower in older children than during the last trimester of pregnancy and the first month after birth, and it is possible that the impact of an increased intake of fatty fish and n-3 LCPUFAs first reveals itself after some time than right after the exposure [18]. The generalizability of the results is also weakened by the fact that 40% of the adolescents from the participating schools refused to participate in the study. Even though we managed to recruit schools from various socio-economic districts of Bergen [25], it is a problem that those who refuse to participate in research often are different from those who participate on important variables, such as socio-economic status, mental health problems and nutritional habits [38,39]. The proportion with a university/college education among the parents/caregivers in the present sample was higher than the general population: between 30-59 years in Norway [40]. In addition, there was a systematic difference between the completers and non-completers with respect to the SDQ symptoms scores, indicating that those with higher mental health problems and who probably would have profited most from the intervention were under-represented in the present study. Moreover, the findings shown for the high SDQ scorer sample should be regarded as preliminary, as the analyses conducted for this sub-sample most likely were underpowered. Strengths in the present study were that the adolescents were randomized independently of class affiliation, reducing the potential bias caused by clusters of data and not the intervention. An important strength is also that we kept detailed registrations of actual intake (dietary compliance) throughout the trial. As dietary compliance might be more demanding when intervening with food instead of supplements, the registrations of dietary compliance made it possible to include this as a covariate in the analyses. BODY.CONCLUSION: In summary, no beneficial effect of an increased intake of fatty fish on mental health as measured with the SDQ were found, in a sample of typically developing adolescents or a high SDQ scorer sample from Norway. Thus, whether there is any causality between fatty fish consumption and mental health in adolescents still remains unestablished. Therefore, further research should investigate the associations between a healthy diet and fish consumption on adolescents' mental health status. This can be carried out either with a prospective study emphasizing validated dietary intake and a longer follow-up period or with an RCT design, prioritizing the serving of warm, tasty meals to ensure acceptable compliance, preferably also in a group of adolescents with suboptimal nutritional status at baseline.

TITLE: Essential Amino Acid Enriched High-Protein Enteral Nutrition Modulates Insulin-Like Growth Factor-1 System Function in a Rat Model of Trauma-Hemorrhagic Shock ABSTRACT.BACKGROUND: Nutrition support for critically ill patients supplemented with additional modular protein may promote skeletal muscle protein anabolism in addition to counteracting acute nitrogen loss. The present study was designed to investigate whether the essential amino acid (EAA) enriched high-protein enteral nutrition (EN) modulates the insulin-like growth factor-1 (IGF-1) system and activates the mammalian target of rapamycin (mTOR) anabolic signaling pathway in a trauma-hemorrhagic shock (T-HS) rat model. ABSTRACT.METHODOLOGY/PRINCIPAL FINDINGS: Male Sprague-Dawley rats (n = 90, 278.18±0.94 g) were randomly assigned to 5 groups: (1) normal control, (2) pair-fed, (3) T-HS, (4) T-HS and standard EN, and (5) T-HS and EAA enriched high-protein EN. Six animals from each group were harvested on days 2, 4, and 6 for serum, gastrocnemius, soleus, and extensor digitorum longus sample collection. T-HS significantly reduced muscle mass. Nutrition support maintained muscle mass, especially the EAA enriched high-protein EN. Meanwhile, a pronounced derangement in IGF-1-IGFBPs axis as well as impaired mTOR transduction was observed in the T-HS group. Compared with animals receiving standard EN, those receiving EAA enriched high-protein EN presented 18% higher serum free IGF-1 levels following 3 days of nutrition support and 22% higher after 5 days. These changes were consistent with the concomitant elevation in serum insulin and reduction in corticosterone levels. In addition, phosphorylations of downstream anabolic signaling effectors - including protein kinase B, mTOR, and ribosomal protein S6 kinase1 - increased significantly in rats receiving EAA enriched high-protein EN. ABSTRACT.CONCLUSION/SIGNIFICANCE: Our findings firstly demonstrate the beneficial effect of EAA enriched high-protein EN on the metabolic modulation of skeletal muscle protein anabolism by regulating the IGF-1 system and downstream anabolic signaling transduction. BODY.INTRODUCTION: Increased nitrogen loss and skeletal muscle wasting are metabolic features in patients under catabolic stress. Nearly 20% of total body protein can be lost in critically ill patients [1], and this severely negative nitrogen balance can adversely affect morbidity and mortality. The erosion of lean body mass is undoubtedly multifactorial [2], [3]; but it is in part attributed to the reduction in circulating insulin-like growth factor-1 (IGF-1) levels or IGF-1 signaling [4], [5]. In normal physiological state, IGF-1 bonds to the type-1 cell-surface receptor and then exerts an anabolic effect by activating the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB/Akt)-mammalian target of rapamycin (mTOR) pathway [6], [7]. Stimulation of mTOR complex1 (mTORC1) contributes to the phosphorylations of two key downstream effectors, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and p70 ribosomal protein S6 kinase1 (p70S6K1), which then induce protein translation initiation [8], [9]. The sustained decrease in circulating IGF-1 levels under catabolic states is thus believed to be responsible, at least in part, for the impaired IGF-1 bioactivity and damaged muscle protein anabolism. IGF-1 circulating in the peripheral vasculature bonds to specific binding proteins. Approximate 95% of circulating IGF-1 forms a ternary complex consisting with insulin-like growth factor binding protein 3 (IGFBP-3) and an acid-labile subunit (ALS) [10]. This ternary complex regulates the biologic effect of IGF-1, functioning as a storage reservoir of IGF-1 in plasma and extending its half-life. Endogenous IGF-1 synthesis is quite sensitive to nutrient availability. In previous work, a 60% reduction in plasma IGF-1 concentrations as well as 8% decrease in gastrocnemius mass was observed in rats receiving a protein-free diet, while recovering to normal after standard casein diet refeeding [11]. Dietary protein intake is also associated with IGF-1 status and whole-body nitrogen economy [12]. In patients with recent hip fracture, a significant elevation of serum IGF-1 levels was observed as early as 7 days after 20 g/(kg·d) protein supplementation [13]. Meanwhile, clinical study revealed that amino acid infusion during anesthesia could attenuate the decrease in IGF-1 levels and maintain glucose homeostasis after surgery [14]. These results collectively indicated that elevated serum IGF-1 levels appeared to be associated with the response to protein or amino acid supplementation during fasting or stress period. In addition, Takenaka et al. [15] reported that dietary restriction of signal essential amino acid (EAA) decreased IGF-1 production, suggesting that adequate EAA in diet might be required for the IGF-1 synthesis. A recent study regarding acute regulation of the IGF-1 system by macronutrients further demonstrated that EAA/carbohydrate mixture ingestion following high-intensity exercise promoted a significant increase in circulating free IGF-1 levels [16]. The essential role of nutrition support for critically ill patients is to promote protein anabolism and protect the lean body mass and function. Historically, providing equivalent amounts of protein or amino acids in nutrition support attempted to counteract the dramatic nitrogen loss [17]. However, it remains to be elucidated whether adequate protein intake modulates the stress response and muscle protein anabolism during critical illness through the IGF-1 system regulation. Therefore, the purpose of the present study was to investigate the role of long-term enteral nutrition (EN), in particular EAA enriched high-protein EN, upon the regulation of IGF-1 system function and downstream mTOR-related signaling transduction in a Sprague-Dawley (SD) rat model of trauma-hemorrhagic shock (T-HS). Serum levels of insulin and corticosterone were also measured, since both factors were implicated in the regulation of IGF-1 by their respective roles in IGFBP regulation [18]. BODY.MATERIALS AND METHODS.ANIMALS: Male SD rats weighted 278.18±0.94 g (Medical Experiment Animal Center of Jinling Hospital, Nanjing, China) were housed in a temperature (25∼28°C) and light (12∶12-hour light-dark cycle) controlled environment for 1 week prior to the experiment. Water and standard chow diet were provided ad libitum. All procedures were carried out in accordance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 86-23 revised 1985) and following protocols approved by the Animal Care and Use Committee of Jinling Hospital. BODY.MATERIALS AND METHODS.SURGICAL PROCEDURE AND EXPERIMENTAL DESIGN (: Following the acclimatization period, rats were fasted overnight (Day-1). All surgeries were performed using sterile technique. At day 0, rats were anesthetized by intraperitoneal injection of ketamine (100 mg/kg body weight (BW)) and placed on a temperature-controlled heating pad. A midline laparotomy (5 cm) was made to insert a catheter (ID 0.8 mm, OD 1.2 mm) into the anterior wall of the stomach followed by suturing to the stomach wall and exteriorizing through the anterolateral abdominal wall. The catheter was then subcutaneously tunneled to the shoulder region, and connected through a customized rotating swivel to a 50 mL syringe pump (Research Center for Analytical Instrument, Zhejiang University, China). An additional groin incision was made. The left femoral artery was catheterized for bleeding, monitoring mean arterial pressure (MAP), and fluid resuscitation through a three-way microvalve. Rats were bled to a target MAP of 30∼35 mmHg within 15 minutes and maintained for 45 minutes. Blood pressure was recorded in 5-minute intervals. At the end of hemorrhagic shock period, rats were resuscitated by infusing their shed blood and lactated Ringer's solution in a 1∶2 ratio. All rats were allowed food and water ad libitum overnight. 10.1371/journal.pone.0077823.g001Figure 1Experimental design.This study includes five groups. After undergoing the trauma-hemorrhagic shock (T-HS) operation, rats received either standard enteral nutrition (EN) (T-HS/SE), essential amino acid (EAA) enriched high-protein EN (T-HS/EAA), or a continue infusion of isotonic saline but on normal chow diet (T-HS/Ctr). Food intake in a pair-fed group (PF) was restricted to the T-HS/Ctr group. A normal control group (NC) without T-HS and fed ad libitum chow was also included. Six individuals in each group were harvested on days 2, 4, and 6, respectively. Animals were randomized to 5 groups. At T-HS 3 groups, rats received either standard EN (T-HS/SE, n = 18), EAA enriched high-protein EN (T-HS/EAA, n = 18), or a continue infusion of isotonic saline but on normal chow diet (T-HS/Ctr, n = 18). One group that was pair-fed (PF, n = 18) with the T-HS/Ctr group was set to control for the influence of reduced food intake post-operatively. Rats in the normal control group (NC, n = 18) were under no treatment and fed standard chow diet ad libitum throughout the study. BODY.MATERIALS AND METHODS.NUTRITION PROGRAM: Following overnight recovery (Day 1), chow diet was removed from rats in the T-HS/SE and T-HS/EAA groups. A continuous EN infusion was started and provided for the subsequent 5 days, providing a non-protein calorie of 250 kcal/(kg BW·d). The T-HS/SE rats received ENSURE® (Lot# 10127NR, Abbott Laboratories B.v., Zwolle, Netherlands) (64.38 g/(kg BW·d), providing 1.65 g N/(kg BW·d)) as the standard enteral formula, in which the ratio of non-protein calorie to nitrogen is 152∶1 ( Table 1 ). Additional nitrogen supplementation in the T-HS/EAA group was provided by leucine enriched EAA solution containing 0.849 g N/(kg BW·d) based on the ENSURE® diet. Therefore, the ratio of non-protein calorie to nitrogen decreases to 100∶1. The composition of the EAA mixture is provided in Table 2 , with minor modifications to previous studies [19], [20]. Half of the estimated requirement based upon individual BW was administrated on the first day and the full requirement was provided subsequently. 10.1371/journal.pone.0077823.t001 Table 1 Nutrition composition of standard diet: ENSURE®. Nutrition Composition /100 g formula Energy, kcal 450 Protein, g 15.9 Fat, g 15.9 Linoleic acid, g 8.7 Carbohydrate, g 60.7 Moisture, g 5 10.1371/journal.pone.0077823.t002 Table 2 The composition of the essential amino acid mixture. Essential amino acids Percentage (%) Lysine 13% Tryptophan 8% Phenylalanine 8% Leucine 38% Isoleucine 9% Valine 11% Threonine 10% Methionine 3% In the T-HS/Ctr group, rats received saline at a constant rate of 0.3 mL/(100 g BW·h) through the enteral routine and had free access to chow diet. Food intake in PF rats was restricted to the T-HS/Ctr group. All rats were allowed free access to water. BW and the amount of EN solution infused were recorded daily. Frequency of diarrhea was also observed to evaluate the tolerance of EN in the T-HS/SE and T-HS/EAA rats. BODY.MATERIALS AND METHODS.SAMPLE COLLECTION: Rats (n = 6) were euthanized from each group on days 2, 4, and 6, respectively. EN was stopped 1 hour before sacrifice in the T-HS/SE and T-HS/EAA groups. After anesthesia, blood was collected by cardiac puncture. Serum was isolated by centrifuge at 4000 rpm at 4°C for 10 minutes and then kept at −80°C until further measurements. The gastrocnemius, soleus, and extensor digitorum longus (EDL) of the right leg were dissected out, weighted, wrapped, and then snap frozen in liquid nitrogen within 3 minutes. All samples were then stored at −80°C until analysis. BODY.MATERIALS AND METHODS.DETERMINATION OF SERUM FREE IGF-1, IGFBP-1, IGFBP-3, INSULIN, AND CORTICOSTERONE LEVELS BY ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): In contrast to measuring total serum IGF-1 levels, we utilized a commercial IGF-1 ELISA kit from R&D Inc. (Minneapolis, MN, USA) to determine the free serum IGF-1 concentrations, which represent the "bioavailable" portion of the circulating IGF-1 pool [16]. Serum concentrations of IGFBP-1, IGFBP-3, insulin, and corticosterone were also determined by ELISA kits (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer's instructions. BODY.MATERIALS AND METHODS.WESTERN BLOT: Muscle preparation and western blotting analysis were performed as previously detailed [21]. Briefly, ∼ 80 mg of frozen muscle sample was homogenized in RIPA lysis buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing 200 mM NaF, 1 mM Na3VO4, 25 mM β-glycerophosphate, 1 mM PMSF, and 1% protein inhibitor Cocktail (Sigma-Aldrich Inc., St. Louis, MO, USA ) for 30 minutes on ice. The lysate was then centrifuged at 12,000 g at 4°C for 8 minutes, and the supernatant was collected. Total protein concentration was determined using the BCA assay (Sangon Biotech Co., Shanghai, China). The supernatant was diluted (4∶1) in a 5× loading buffer (Beyotime Institute of Biotechnology Co., Nantong, China) and then boiled at 100°C for 5 minutes. Equal amounts of total protein (75 ug) were separated by 8% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) determined by the size of target protein and then transferred to polyvinylidene difluoride membrane (Millipore Co., Billerica, MA, USA). Blots were blocked in 5% BSA for 1 hour at room temperature and incubated in specific primary antibodies overnight at 4°C. Blots were then washed in TBST (Tris-buffered saline and 0.05% Tween-20) three times followed by incubation with secondary antibody for 2 hours at room temperature. The protein bands were visualized by a chemiluminescence detection system (Pierce Biotechnology Inc., Rockford, IL, USA) with exposure to Kodak XAR film (Eastman Kodak). Band density was analyzed using Quantity One 4.6.2 software (BioRad). Specific total protein was re-probed after stripping the phospho-primary and secondary antibody. Phosphorylation data are described relative to the total protein expression after normalization by internal loading control. BODY.MATERIALS AND METHODS.ANTIBODIES: Antibodies that detected α-tublin (1∶1000), phospho-mTOR (Ser2448; 1∶500), S6K1 (1∶1000), phospho-S6K1 (Thr389; 1∶500), and phospho-Akt (Ser473; 1∶1000) were from Cell Signaling Technology (Beverly, MA, USA). Akt (1∶1000) and mTOR (1∶1000) antibodies were from Abcam (Cambridge, UK). Anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1∶3000) was from Kirkegaard & Perry Laboratories (KPL) Biotechnology Inc. (Gaithersburg, ML, USA). BODY.MATERIALS AND METHODS.DETERMINATION OF SERUM AMINO ACID CONCENTRATIONS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC): Serum amino acid concentrations were assayed by a Waters Aliance 2695 HPLC system (Waters, MA, USA) after pre-column derivatization with phenylisothicyanate (PITC) (Thermo Scientific (Pierce), Rockford, IL, USA) by our previous method [22]. Data were calculated on the basis of external amino acid standard (Sigma-Aldrich Inc., St. Louis, MO, USA). BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Results are expressed as mean ± SE. Repeated-measures ANOVA was used to characterize dietary intake and BW gain during the 6-day recovery period. For other purposes, statistical evaluation among groups was performed by one-way ANOVA followed by LSD post-hoc analysis, using SPSS software package (SPPS Inc., Chicago, IL, USA). In the case of heterogeneous variances, we used Dunnet's T3 test. A p value <0.05 was considered significant. BODY.RESULTS.1. GROWTH ASSESSMENT DURING STUDY: The average incidence of diarrhea was 0.44/day on the first day following T-HS, and gradually disappeared at day 3 ( Figure 2A ). Early EN failed to increase the incidence of diarrhea, indicating that rats in the T-HS/SE and T-HS/EAA groups tolerated the nutrition support. After T-HS, rats consumed less chow (p<0.05) for 4 days but achieved normal by day 5, when the food intake did not differ between the T-HS/Ctr and NC groups ( Figure 2B ). The daily caloric intake of the T-HS/SE and T-HS/EAA groups is additionally provided in Table 3 . 10.1371/journal.pone.0077823.g002Figure 2Observations of diarrhea and food intake in normal or T-HS injured animals.Diarrhea times (A) and food intake (B) in specific groups were recorded daily during study period. Values are presented as mean ± SE. Marker indicates a significant difference from NC group. *, p<0.05; #, p<0.01. T-HS, trauma-hemorrhagic shock. 10.1371/journal.pone.0077823.t003 Table 3 Daily caloric intake in enterally fed rats (kcal/day). Groups Time point Day 1 Day 2 Day 3 Day 4 Day 5 T-HS/SE 33.03±0.18 65.21±0.98 67.79±0.66 69.25±0.98 69.69±0.87 T-HS/EAA 32.67±0.17 65.75±0.25 68.42±0.17 69.25±0.25 70.67±0.60 Animals weighed an average of 278.18±0.94 g at the beginning of the study. BW was low in all T-HS injured rats throughout the study, but did increase with time ( p<0.01, Figure 3 ). Rats in the T-HS/Ctr group presented a relatively slow gain of BW during the recovery period. Nutrition support was thought to be beneficial for achieving weight gain, especially the EAA enriched high-protein EN, however no significant difference between groups was observed at any time point. 10.1371/journal.pone.0077823.g003Figure 3Changes in body weight of the animals.Values are presented as mean ± SE. Marker indicates a significant difference from other groups across study period. #, p<0.01. T-HS induced significant reductions in gastrocnemius and EDL masses ( Figure 4A, B ). Gastrocnemius masses in the T-HS/SE and T-HS/EAA groups were significantly higher than the T-HS/Ctr group by 2 days post-T-HS (p<0.01), and this trend continued throughout the remainder of the study. In addition, the T-HS/EAA group exhibited a significant increase in gastrocnemius mass compared with the T-HS/SE group on days 4 and 6, respectively (p<0.01). However, both EN treatments had limited improvement in EDL mass maintenance. For soleus mass, a slight but without significant decrease was observed after T-HS ( Figure 4C ). EAA enriched high-protein EN caused a significant increase in soleus wet muscle mass by day 6, which was comparable with the NC group (p<0.05). 10.1371/journal.pone.0077823.g004Figure 4Comparison of skeletal muscle mass.Wet masses of gastrocnemius (A), extensor digitorum longus (B), and soleus (C) in each group were measured following 2, 4, and 6 days of recovery from T-HS. Values are presented as mean ± SE. Groups with different letters at each time point indicate a significant difference (p<0.05). T-HS, trauma-hemorrhagic shock. BODY.RESULTS.2. SERUM FREE IGF-1, IGFBP-3, AND IGFBP-1 CONCENTRATIONS: Serum free IGF-1 concentrations in the PF, T-HS/Ctr, T-HS/SE, and T-HS/EAA groups were significantly decreased by day 2 ( p<0.01, Figure 5A ). Following this time point, free IGF-1 concentrations increased gradually, but most slowly in the T-HS/Ctr group, which remained statistically lower than the other EN-treated groups (vs T-HS/SE at day 4, p<0.05; vs T-HS/EAA at day 4, p<0.01; vs T-HS/SE or T-HS/EAA at day 6, p<0.01). In addition, lower free IGF-1 levels were measured in the T-HS/Ctr group than PF group at day 4 and it reached statistical difference by day 6 (p<0.01), indicating that the depressed IGF-1 expression was sustained 6 days following T-HS after eliminating the interference of reduced food intake. Furthermore, free IGF-1 levels were 18% higher in the T-HS/EAA group than T-HS/SE group following 3 days of nutrition support (p<0.05). And a greater magnitude was observed by day 6 (122%, vs T-HS/SE; p<0.01). 10.1371/journal.pone.0077823.g005Figure 5ELISA analysis of IGF-1-IGFBPs axis expression.Serum concentrations of free IGF-1 (A), IGFBP-3 (B), and IGFBP-1 (C) in each group were measured following 2, 4, and 6 days of recovery from T-HS. Values are presented as mean ± SE. Groups with different letters at each time point indicate a significant difference (p<0.05). T-HS, trauma-hemorrhagic shock. The pattern of serum IGFBP-3 concentrations between groups was similar to that observed in free IGF-1 ( Figure 5B ). In the T-HS/Ctr group, IGFBP-3 levels remained lower than the PF group on days 4 and 6, respectively (p<0.01). Following enteral feeding, there was a significant elevation in IGFBP-3 levels compared with the T-HS/Ctr group (p<0.01). Moreover, EAA enriched high-protein EN-fed rats exhibited a 170% and 140% increase in IGFBP-3 levels compared with animals receiving standard EN on days 4 and 6, respectively (p<0.01). Serum IGFBP-1 levels in the NC group were markedly lower than the other groups at each time point, with the exception of day 6 when IGFBP-1 levels in the T-HS/EAA group decreased to levels comparable with the NC group ( Figure 5C ). T-HS increased IGFBP-1 concentrations in the T-HS/Ctr group by 46% compared with the PF group at day 2 (p<0.01), and this trend was likely to continue throughout the study period. Although both groups of enterally fed rats exhibited significant decreases in IGFBP-1 levels compared with rats in the T-HS/Ctr group at each time point (p<0.01), the reduction was greater in the T-HS/EAA group (p<0.01). BODY.RESULTS.3. SERUM HORMONES (INSULIN AND CORTICOSTERONE) CONCENTRATIONS: Insulin levels in the T-HS/Ctr group were significantly decreased compared with the PF group at all the time points (p<0.05, at day 2; p<0.01, at day 4 or 6; Figure 6A ). Both enterally fed groups showed increase in insulin concentrations, but it failed to reach statistical difference compared with the T-HS/Ctr group at day 2. By day 4, standard and EAA enriched high-protein EN significantly increased insulin levels 1- and 1.3-fold, respectively, compared with the values detected in the T-HS/Ctr rats (p<0.01). Moreover, a greater magnitude of feeding-induced hyperinsulinemia was observed in the T-HS/EAA group than T-HS/SE group on days 4 and 6, respectively (p<0.01, p<0.05). Regression analysis of insulin and IGF-1 further indicated a significantly positive liner relationship (y = 0.038x−1.08; R2 = 0.483; p<0.01). 10.1371/journal.pone.0077823.g006Figure 6ELISA analysis of serum hormones concentrations.Serum concentrations of insulin (A) and corticosterone (B) in each group were measured following 2, 4, and 6 days of recovery from T-HS. Values are presented as mean ± SE. Groups with different letters at each time point indicate a significant difference (p<0.05). T-HS, trauma-hemorrhagic shock. Serum corticosterone concentrations in rats subjected to T-HS significantly increased by day 2 (p<0.01, Figure 6B ). Concentrations in the T-HS/SE and T-HS/EAA groups gradually returned to normal as early as at day 4, whereas corticosterone levels in the T-HS/Ctr group remained statistically elevated (p<0.01). Meanwhile, corticosterone levels in the T-HS/EAA group appeared lower than T-HS/SE group on days 4 and 6, but without statistical difference. BODY.RESULTS.4. CELL SIGNALING: To further assess the signaling transduction associated with muscle protein synthesis, phosphorylations of several key proteins involved in mTOR signaling pathway were determined in gastrocnemius at day 6. After T-HS, phosphorylation of Akt at Ser473 in the T-HS/Ctr group was greatly suppressed (vs NC, p<0.01; vs PF, p<0.01; Figure 7A ). Phospho-Akt levels in the T-HS/EAA group, rather than T-HS/SE group, were significant higher than the T-HS/Ctr group (p<0.01), coinciding with the fact that free IGF-1 levels peaked at day 6. Meanwhile, the phosphorylation of mTOR at Ser2448 in the T-HS/Ctr group decreased to 43.3% and 45.7% compared with the NC and PF groups, respectively (p<0.01, Figure 7B ). A minor increase but without significance in phospho-mTOR levels was observed in rats receiving standard EN formula. However, in the T-HS/EAA group, mTOR phosphorylation at Ser2448 was significantly elevated (176%, vs T-HS/Ctr, p<0.05; 166%, vs T-HS/SE, p<0.05). As the important downstream effector of mTOR signaling, Thr389-phosphorylation of S6K1 was also measured. S6K1 phosphorylations in the PF and T-HS/Ctr groups were similar, but markedly lower than the NC group (p<0.05, p<0.01; Figure 7C ). After EN treatment, S6K1 phosphorylation increased in both groups, but only with statistical significance observed in the T-HS/EAA group compared with the T-HS/Ctr group (p<0.01). Furthermore, phospho-S6K1 levels in the T-HS/EAA group were also higher than the T-HS/SE group (p<0.05). 10.1371/journal.pone.0077823.g007Figure 7Phosphorylations of mTOR related signaling effectors in gastrocnemius at day 6.(A) Akt phosphorylation at Ser473 and total Akt. (B) mTOR phosphorylation at Ser2448 and total mTOR. (C) p70S6K1 phosphorylation at Thr389 and total p70S6K1. The density of each western blot is quantified by Quantity One software and the data are presented as mean ± SE in arbitrary unit (AU) from three independent experiments. Groups with different letters indicate a significant difference (p<0.05). BODY.RESULTS.5. BLOOD EAA CONCENTRATIONS: There was no significant decrease in branched chain amino acid (BCAA) concentrations in the T-HS/Ctr group compared with the NC or PF group at day 6, with the exception of valine (vs NC, p<0.05; Figure 8 ). Treatment with standard EN slightly, without significance, increased the blood leucine and isoleucine levels. In contrast, a pronounced increase in BCAA levels was detected in the T-HS/EAA group (vs T-HS/Ctr, p<0.01), and the leucine and valine concentrations were even higher than that observed in the T-HS/SE group (p<0.01). Similarly, blood levels of other EAA were increased after EN treatment, but with greater in the T-HS/EAA group (vs T-HS/SE, p<0.05). 10.1371/journal.pone.0077823.g008Figure 8Blood essential amino acid concentrations at day 6.Values are presented as mean ± SE. Groups with different letters at each time point indicate a significant difference (p<0.05). BODY.DISCUSSION: Sustained suppression of circulating IGF-1 levels is one important metabolic derangement during critical illness, which is implicated in the pathogenesis of impaired muscle protein anabolism. Our study illustrates that EAA enriched high-protein EN favors early recovery of the IGF-1 system in T-HS injured rats, accompanying with the obvious muscle mass maintenance and downstream mTOR pathway activation, which indicates effective stimulation of muscle protein translation initiation. These results provide valuable insight into the metabolic regulation stimulated by high-protein nutrition support during the stress response to critical illness. The composition of EAA supplementation used in this study contained a large proportion of leucine. Recently, leucine is shown to be capable of stimulating insulin secretion [23]–[25]. This is consistent with our data, which the insulin levels were highest in the T-HS/EAA group by day 4 compared with the T-HS/SE and T-HS/Ctr groups. As reported, insulin is a known regulator to inhibit hepatic IGFBP-1 production [26], and elevated IGFBP-1 levels under catabolic stress can act to inhibit the anabolic effect of IGF-1 [27]. In the present study, serum IGFBP-1 concentrations appeared to correlate well with these data, displaying dramatically decreasing levels that were consistent with the increase in circulating insulin levels observed in enterally fed rats at the time point coinciding with the emergence of elevation in serum free IGF-1 levels. And this response appeared more pronounced in the T-HS/EAA group than T-HS/SE group. Regression analysis of insulin and IGF-1 further indicated a strong positive correlation (y = 0.038x−1.08; R2 = 0.483; p<0.01), which was in line with the previous work [18]. However, Ma et al. [28] found that hepatic IGFBP-1 gene expression was dramatically increased immediately following T-HS along with hyperinsulinemia at 90 and 210 minutes. This does not entirely fit with our results, which appears to depend upon the different time points selected. During the "ebb" phase of injury, insulin levels are commonly decreased or remain unchanged [29], [30]. Insulin requires the PI3K pathway for inhibition of hepatic IGFBP-1 production [31]. Hepatic insulin resistance, characterized by a defect in insulin-induced PI3K-Akt signal transduction, rapidly develops within 90 minutes following T-HS [28], but may have been alleviated within 48 hours post-T-HS in the present study. Endogenous glucocorticoids also mediate IGFBP-1 production. Previous work demonstrated that pretreating animals with glucocorticoid receptor antagonist RU486 partially prevented burn-induced increase in IGFBP-1 and further contributed to the increase in plasma IGF-1 levels [32]. The enterally fed rats in the present study appeared to have an early reduction in serum corticosterone levels following T-HS, which is possibly related to the modulation of host defense by enteral nutrients [33]. This change was correlated with decreased IGFBP-1 as well as increased IGF-1 levels following 3 days of nutrition support. Previous studies using a similar EAA formula or leucine alone to investigate the stimulatory effect upon skeletal muscle protein synthesis did not demonstrate changes in serum IGF-1 [20], [34], [35]. Despite various experimental conditions, those studies measured total IGF-1, instead of free levels. Meanwhile, acute dosing of EAA or leucine solution on the background of inadequate nutrient availability is not sufficient for stimulation of IGF-1 production. In our study, we provided a high-protein diet to the T-HS/EAA animals, with additional EAA supplementation based on the standard EN formula, to investigate the changes in free IGF-1 levels, which represent the "bioavailable" portion of the IGF-1 pool in the circulation [16]. Our study indicated that feeding with EAA enriched high-protein EN greatly increased serum free IGF-1 levels compared with the standard EN-fed rats as early as at day 4. This is a desirable outcome, since it has been concluded that IGF-1 plays an important role in skeletal muscle mass maintenance [36]. In our study, significantly increased muscle mass, especially in the fast-twitch gastrocnemius muscle, was identified 2 days following T-HS in the T-HS/EAA group. We also detected that EAA enriched high-protein EN significantly enhanced the activation of mTOR signaling pathway; phosphorylations of mTOR and its downstream effector S6K1 in rats receiving EAA enriched high-protein formula increased nearly two-fold compared with those receiving standard EN. Recently, a large body of evidence has established that amino acid, especially leucine, can be considered as a signaling molecule to stimulate protein translation initiation through an Akt-independent pathway. Acute leucine ingestion can effectively increase protein synthesis and promote the mTOR-related signaling transduction in both normal and stress conditions [37]–[39]. The stimulation of muscle protein synthesis, as previously reported by Bohé et al. [40], was positively associated with blood EAA concentrations in a dose-response manner. In our study, blood amino acid analysis revealed that leucine and other EAA were significantly increased after EAA enriched high-protein EN treatment. Thus, we speculate that the response to muscle protein translation initiation induced by EAA enriched high-protein EN may be partly attributed to the nutrient-mediated mTOR signaling regulation. The PI3K-Akt pathway, upstream of mTOR, can be phosphorylated by ligands including IGF-1 and insulin and that in turn stimulates the mTOR phosphorylation to induce the muscle protein translation initiation. Previous studies demonstrated the rapid development of insulin resistance in skeletal muscle following T-HS, with the earliest insulin signaling defect occurring at 60 minutes [41], [42]. Zhai et al. [43] further indicated that the T-HS-induced acute muscle insulin resistance was specific to 6- and 10- week old rats, but not the post-weaning. However, the insulin signaling defect is not permanent. Thompson et al. [44] reported that insulin-induced phospho-Akt signaling in skeletal muscle would be gradually restored by 5 and 24 hours following fluid resuscitation. Therefore, it can reasonably be inferred that muscle insulin resistance is a metabolic feature in the acute phase of T-HS, and the diminished signaling transduction associated with the PI3K-Akt pathway may be partially responsible for the increased muscle protein wasting following T-HS. In our study, three later time points following T-HS were selected. Activated Akt was observed at day 6 in both enterally fed groups, but with greater in rats receiving EAA enriched high-protein EN, coinciding with the emergence of significant elevation in both insulin and IGF-1 levels. Previous studies demonstrated the protein retention effect of insulin was likely to be exerted by reducing the protein catabolism after trauma or surgery, because no correlation between changes in muscle protein synthesis and insulin sensitivity was found [45], [46]. However, administration of IGF-1 effectively attenuates the inhibition of protein synthesis in sepsis or trauma and further ameliorates the loss of muscle mass [47], [48]. Therefore, in addition to the nutrient-dependent mechanism described above, the increased mTOR anabolic signaling in the T-HS/EAA group may be partially due to IGF-1 signaling pathway activation. A limitation to the current study is that it is not yet clear whether the increased serum free IGF-1 levels following EAA-enriched high-protein EN are due to the overall increase in IGF-1 production or reduced IGF-1 degradation. As mentioned previously, IGFBP-3 has been shown to be regulated by proteolysis. Increasing proteolytic cleavage of IGFBP-3 causes a decreased affinity for IGF-1, which is responsible for the decrease in circulating IGF-1 levels during stress [49]. In the present study, high serum free IGF-1 levels were observed in the T-HS/EAA group, which were quite consistent with increased IGFBP-3 concentrations. Therefore, further study is required to investigate the IGFBP-3 proteolysis activity and total IGF-1 levels following nutrition support, using a T-HS injured rat model, to make a better interpretation of our data [16]. In summary, our findings firstly demonstrate the effect of EAA enriched high-protein EN upon the metabolic regulation of skeletal muscle protein anabolism by regulating the IGF-1 system and downstream anabolic signaling transduction. We demonstrated that early nutrition support favored the recovery of serum free IGF-1 levels following T-HS, and the EAA enriched high-protein EN treatment appeared more effective. It was beneficial for the muscle mass maintenance in T-HS injured rats. Moreover, long-term treatment with EAA enriched high-protein EN effectively activated the mTOR related translation initiation factors, indicating the stimulation of muscle protein synthesis. And this response might be based on cooperation between IGF-1- and nutrient- mediated signal transduction pathways.

TITLE: The Effect of Anchoring Sutures on Medicinal Leech Mortality ABSTRACT: Objective: The implementation of leech therapy for surgical flaps is not always logistically easy or comfortable for patients or healthcare providers. We examine different methods of placing sutures in the medicinal leech, Hirudo medicinalis, to make the implementation of leech therapy easier. Methods: Sixteen leeches were randomly divided into 3 groups: a control group, a deep anchoring suture group, and a superficial anchoring suture group. The leeches were observed to determine if either of these methods had an adverse effect on survival compared with the control group. Results: No difference in survival time was observed across the different groups. Conclusion: The placement of anchoring sutures in leeches can ease the implementation of leech therapy by allowing for greater control of the leeches and thus increased patient comfort. BODY: The medicinal leech, Hirudo medicinalis, is used by plastic and reconstructive surgeons in the postoperative period to relieve surgical site venous congestion, and this has been shown to improve surgical outcomes.1 However, 2 practical issues limit their use: (1) leech migration often causes patient distress and (2) medical personnel often have difficulties with leech placement and retrieval. Reports of leech migration from the surgical site after feeding has prompted the examination of leech-anchoring methods. A recent study examined a method of anchoring medicinal leeches with a simple suture to limit their migration away from the surgical site and to aid in retrieval when feeding has ended.2 The purpose of the present study was to examine leech mortality when the use of different types of anchoring sutures (superficial vs. deep sutures) were placed through the leech. BODY.METHODS: Sixteen leeches were available for analysis; these were randomly divided into 3 groups from a common container: control (n = 4), deep suture placement (n = 6), and superficial suture placement (n = 6). Leeches in the deep suture placement group had a suture placed through the middle of the body that exited on the opposite side of the insertion point. Leeches in the superficial placement group also had a suture placed in the middle of the body, but the suture needle penetrated only the outer surface of the leech to a depth of approximately 1 mm (Fig 1). Black braided 4-0 silk sutures were used and blunt forceps were applied at both ends of each leech for control while placing sutures. All sutures were secured to the leech with nontightened knots (air knots). All leeches were subjected to the same environmental conditions. Leeches were individually placed in numbered, clear plastic containers with approximately 24 mL of water distributed from a common container. The plastic containers were covered with clear plastic wrap and secured in place with rubber bands and tape. Ventilation holes were created and the plastic containers were stored at room temperature. Leeches were examined twice a day for 7 days and assessed for leech mortality and movement in their individual containers. BODY.RESULTS: Over the course of the 7-day experiment, all leeches survived and exhibited similar activity levels in all 3 groups. On day 3, one of the leeches in the superficial suture group was observed to no longer be anchored to the suture because the suture was noted to have torn through the outer layer of the leech body. However, this leech continued to exhibit the same degree of movement as the rest of the leeches in both groups. Thus, the presence or absence of an anchoring suture did not seem to affect leech mobility and did not have an immediate impact on leech survival. BODY.CONCLUSION: This experiment was designed to examine the immediate impact of suture placement on leech survival. The results demonstrated no differences in leech survival or mobility between the suture groups (deep or superficial) or when compared with the control group. BODY.DISCUSSION: For thousands of years, the medicinal leech, Hirudo medicinalis, has been used to treat a wide array of human ailments. The practice of bloodletting in general was used by Egyptian, Greek, and Roman cultures.3 Bloodletting was accomplished through venesection or application of leeches and was thought to restore balance between the 4 humors proposed by Hippocrates. Leeches were used to treat various conditions including infectious diseases, inflammation, and even as a cure for bleeding itself. Use was commonplace until the late 19th century when overcollection of leeches threatened wild populations. During this time, the medical community also began to discredit their use. Modern medicine has reintroduced the use of medicinal leeches primarily in the realm of reconstructive surgery. During surgical construction of local and free flaps or replanted digits, venous outflow is often compromised, causing venous congestion of the surgical site which can compromise tissue viability. Leeches have been shown to alleviate local venous congestion in the postoperative period, thus allowing the body time to reestablish venous outflow.4 This is accomplished through 2 main mechanisms: direct removal of blood and introduction of leech saliva, which contains numerous chemicals including factors that provide anticoagulation, platelet inactivation, vasodilation, and local anesthesia. Hirudin, the most potent natural inhibitor of thrombin, is the primary anticoagulant that allows the leech to feed.5 The vasodilatory effects of leech saliva also contribute to increased blood flow during feeding and delayed clotting of the attachment site after the leech has fallen off its host. Hirudo medicinalis can remove up to 9 times its own body weight (5–10 mL) of blood from a host over a 20- to 30-minute period, and each feed can provide the leech with nutrients for several months.3 Postoperative use of medicinal leeches usually lasts several days, with several feeding sessions per day. For each feeding session, 1 or more leeches can be used depending on the size and severity of the congested area. Often the area of concern is not easily visualized or accessible by the patient. In these situations, the leeches often migrate to other areas of the patient body or even within the hospital room. Historically, leech migration was an issue with writings of "wandering leeches" being swallowed by patients or finding their way into other body orifices.3 More recently, reports of leeches inching their way across hospital beds or patient rooms are not uncommon. Recently, Granzow et al2 described the use of an anchoring suture to immobilize the leeches and prevent movement after feeding sessions. This group described the use of 4-0 or 5-0 sutures as "passed through the leech approximately halfway along the length of its body, 2 to 3 mm from its side." These researchers then secured the suture through the leech to a stack of surgical gauze. In addition, they noted that "[t]he application of the suture in no way seems to limit the ability of the leech to feed or move within the confines of the length of the suture." Although the authors stated that the leeches continued normal feeding and movement after the placement of an anchoring suture, the issue of leech viability after suture placement was not addressed. The standard method for applying a leech involves loading one into an empty syringe without a plunger and pressing the open end over the desired treatment area4; this can be a time-consuming activity for healthcare providers. The results of the present study suggest that it is possible to place a suture through many leeches at one time, thus making it easier to retrieve a single leech from a container and lessen the need for cumbersome forceps or empty syringes while handling them. The same anchoring suture can then be used to confine the leech to the surgical site during and after feeding. The present study supports the conclusion that a simple suture through the body of a leech does not alter short-term viability. In addition, the anchoring suture can aid in leech placement for the initiation of feeding, prevent leech migration during and after feeding, and simplify postfeeding retrieval of the leech. The primary limitations of this study are the lack of data surrounding leech feeding habits and whether an anchoring suture affects the quantity of blood removed. This information would be useful if a difference can be found because it would impact the number of leeches needed to treat a particular area of venous congestion and the time interval between feeds. As leeches continue to prove their usefulness in postsurgical management, this method can save time and increase the likelihood of a positive outcome by preventing venous congestion. Even outside the realm of surgery, leeches are becoming revered for their medicinal qualities. Their use has even been indicated in the treatment of osteoarthritis.6,7 Leeches are a valuable tool to the physician, and the present study shows that some of the limitations of leech therapy can be minimized by anchoring the leeches with a suture.

TITLE: Performance of Surgical Stress Index during Sevoflurane-Fentanyl and Isoflurane-Fentanyl Anesthesia ABSTRACT: The performance of recently introduced Surgical Stress Index (SSI), based on heart rate and photoplethysmography, was estimated during sevoflurane-fentanyl and isoflurane-fentanyl anesthesia during surgical procedures. Forty ASA I–III patients were enrolled. Anesthesia was induced with fentanyl 2 μg kg−1 and thiopentone 3–5 mg kg−1. Tracheal intubation was performed 5 minutes after fentanyl bolus. Patients were randomly allocated to receive sevoflurane (n = 20) or isoflurane (n = 20) in 30% oxygen/air. State entropy was kept at 40–60, target being 50. During surgery, fentanyl boluses 1.5 μg kg−1 were given at 30–40-minute intervals. SSI increased significantly after intubation. During surgery, the decrease of SSI after fentanyl boluses was similar in sevoflurane and isoflurane groups but SSI values were higher in sevoflurane than in isoflurane group. Tracheal intubation, skin incision, and surgical stimuli increased SSI from baseline, indicating that nociceptive stimuli increase SSI. Fentanyl boluses during surgery decreased SSI, indicating that increasing analgesia decreases SSI. BODY.1. INTRODUCTION: Estimating nociception during general anesthesia is very challenging since there have not been any direct methods to measure it. That is why clinical evaluation of nociception is based on unspecific autonomic reactions, such as blood pressure, heart rate, sweating, or tearing. Also determination of plasma concentrations of opioids (directly or estimated by pharmacokinetic models) has been used [1]. Clinical end-points, such as movement reaction in response to nociceptive stimulus, can be used as an indicator of inadequate analgesia [2], but it is not very useful during operation when patients are paralysed. Some electroencephalographic (EEG)-derived parameters, such as Entropy, are suggested to reflect also the nociceptive component of anesthesia [3–5]. Changes in skin conductivity and suppression of photoplethysmographic pulse wave amplitude (PPWA) have also been proposed as indicators of nociception [6, 7]. Recently, Surgical Stress Index (SSI), based on a sum of normalized pulse beat interval (PBI) and PPWA, was introduced for the assessment of surgical stress or nociception [8]. PBI and PPWA were combined and a simple numerical index (from 0 to 100) suitable for monitoring surgical stress was developed. Huiku et al. showed that SSI is high when noxious stimulation is high or the remifentanil concentration inadequate and that SSI is low when remifentanil concentration is high or the stimulation low [8]. SSI has shown to be a better measure of nociception than entropy parameters, heart rate, or PPWA [9]. Evaluation of SSI has been accomplished during experimental or standardized situations. Our purpose was to assess the function of SSI in clinical situation. We recorded SSI values during sevoflurane-fentanyl and isoflurane-fentanyl anesthesia with special attention to tracheal intubation, skin incision, and effect of fentanyl boluses during surgery. BODY.2. MATERIALS AND METHODS: The study was approved by the local institutional ethics committee (South Carelia Central Hospital, Lappeenranta, Finland), and written informed consent was obtained from all patients. We enrolled forty patients (Table 1), ASA I–III, scheduled for surgical procedure under general anesthesia. Exclusion criteria were known heart arrhythmia (such as chronic atrial fibrillation), neurological disorder, medication affecting central nervous system, and history of alcohol or drug abuse. The ECG, photoplethysmography, peripheral oxygen saturation (SpO2), noninvasive blood pressure (NIBP), end-tidal CO2, minimal alveolar concentrations (MAC) of sevoflurane or isoflurane, and entropy parameters [state entropy (SE) and response entropy (RE)] were monitored and collected using a data acquisition PC (Datex-Ohmeda S/5 Anesthesia Monitor, S/5 iCentral Network Workstation and S/5 iCollect data acquisition software, GE Healthcare Finland Oy, Helsinki, Finland). To collect user feedback, the SSI was visible on the data acquisition PC during the procedure. However, the SSI values used for statistical analysis were calculated offline with the algorithm described by Huiku et al. [8]. SSI is calculated as SSI = 100 − (0.33 × PBI + 0.67 × PPWA). The SSI values at baseline before induction of anesthesia are mostly 60–70, after induction of anesthesia 20–40 (without stimuli), after tracheal intubation 50–70, and during surgery 50–70 [8, 9]. Patients, premedicated with oral diazepam 0.1 mg kg−1 adjusted to the nearest 2.5 mg, received fentanyl 2 μg kg−1 and thiopentone 3–5 mg kg−1 for induction of anesthesia. Rocuronium 0.6 mg kg−1 was given to facilitate tracheal intubation. Tracheal intubation was performed 5 minutes after fentanyl bolus. Patients were randomly assigned to two groups sevoflurane (SEVO, n = 20) and isoflurane (ISOF, n = 20) in 30% oxygen in air as a maintenance inhalation agent during anesthesia. After tracheal intubation sevoflurane or isoflurane were adjusted to maintain state entropy (SE) level between 40 and 60 target being 50. During procedure, fentanyl boluses 1.5 μg kg−1 were given repeatedly at rather long 30–40-minute intervals to enhance the variability in its effects during the procedure. Reactivity of SSI to tracheal intubation, surgery, and fentanyl boluses was recorded. The average SSI response to fentanyl bolus during procedure was quantified by comparing the average SSI level 1–5 minutes before the bolus to the average SSI level 4–8 minutes after the bolus [time to peak effect for fentanyl has been reported to be 4–6 minutes [10]]. If mean arterial pressure (NIBPMAP) decreased below 60 mmHg, ethylphenylephrin 2 mg was given intravenously. Our test hypothesis was that the SSI would increase after the intubation and skin incision and that the fentanyl boluses would decrease the SSI. BODY.2. MATERIALS AND METHODS.2.1. STATISTICS: The statistical analysis was performed with the SPSS program (SPSS 14.0 for Windows, SPSS Inc., Chicago, IL, USA). Wilcoxon Signed Ranks test was used to compare changes inside groups and two-tailed Mann-Whitney U test for between groups changes; P < .05 was considered statistically significant. Values are presented as mean ± SD unless otherwise specified. BODY.3. RESULTS: The patient characteristics and surgical procedures in the two groups were similar (Table 1). The values of SSI, HR, NIBPMAP, SE, and RE at different time points are presented in Table 2. After tracheal intubation, both SSI and HR increased significantly (P < .001, Figure 1) but after skin incision only SSI increased significantly (P < .001, Figure 1). The decrease of SSI during procedure after fentanyl bolus was 7.5 ± 15.0, (P < .01). The decrease of SSI was similar in both SEVO and ISOF groups but SSI values in SEVO group were higher than in ISOF group during surgery (Figure 2). MAC and SE values were similar in SEVO and ISOF groups before fentanyl boluses [0.74 ± 0.12 versus 0.77 ± 0.17, (P = .32) and 43.5 ± 8.1 versus 45.2 ± 8.3, (P = .47), resp.]. However, HR values were significantly different in SEVO and ISOF groups 70.6 ± 13.0 versus 64.9 ± 11.8, (P < .01), respectively (Figure 2). After fentanyl boluses, SE and HR changes were similar and MAC values remained stable in both groups. BODY.4. DISCUSSION: We evaluated the performance of SSI during sevoflurane-fentanyl and isoflurane-fentanyl anesthesia in diverse surgical procedures. According to our hypothesis, the intubation and skin incision increased SSI and the fentanyl boluses decreased it. Fentanyl bolus 2 μg kg−1 given five minutes before tracheal intubation did not block the haemodynamic or SSI response to intubation. During procedure fentanyl boluses, 1.5 μg kg−1 given at 30–40-minute intervals caused equal decrease of SSI in sevoflurane and isoflurane groups but absolute SSI values were different between groups. We examined MAC, SE, RE, and haemodynamic variables and we noticed that HR values were different between groups but the other variables were similar. Heart beat interval accounts 33% of the absolute value of SSI and 67% of it consist of PPWA [8]. That is why it is logical that if anesthetics have different effects on HR then there are also different absolute values of SSI. It has been noticed in an earlier study that desflurane increases heart rate and prolongs the ECG QT interval more than sevoflurane [11]. On the other hand, Yildirim et al. [12] found that desflurane, sevoflurane, and isoflurane all prolonged QT interval but there were no between group differences. They also found that HR was higher in sevoflurane group 3 minutes after intubation compared to isoflurane and desflurane groups, but 10 minutes after reaching 1 MAC steady state concentration, there were no between group differences [12]. It has been shown that during sevoflurane or isoflurane anesthesia, there can be bradycardia episodes [13]. However, different HR values explain at least partly the different SSI values during procedures. This means that when one interprets absolute SSI values, the effect of different anesthetics on HR must be taken into consideration. Seitsonen et al. [7] showed that HR changes offer some information on adequacy of analgesia at skin incision during sevoflurane anesthesia, but suggested that multiparameter approach is required for accurate monitoring of nociception. Huiku et al. [8] developed the multivariate SSI, and in a validation study they found that SSI was accurate measurement of the nociception—antinociception balance in response to skin incision and surgery during propofol—remifentanil anesthesia. Struys et al. [9] showed that HR had some value in estimating analgesia during propofol-remifentanil anesthesia, but it was influenced by the hypnotic component of anesthesia while SSI was not. They concluded that SSI seems to be better than SE, RE, HR, or PPGA when correlating nociceptive reactions to remifentanil concentrations and that SSI might be more accurate for nociception. Our results confirm that SSI acts consistently at nociceptive situations (tracheal intubation, skin incision, surgery). Fentanyl bolus (2 μg kg−1), given five minutes before tracheal intubation, did not block the SSI response. Although we did not have any placebo group, we can assume that the reaction would have been more pronounced without fentanyl. After all, there were some patients whose reaction measured by SSI was minimal but in some other patients it was very clear. We think that this is due to interindividual difference in responsiveness and differences in opioid (or other drugs) requirements. Our results were calculated off line [8]. The SSI was also continuously calculated and displayed on line during the study in a data acquisition PC using basically the same algorithm with slight modifications needed for the PC version. This version of SSI showed some increases with no obvious exceptionally nociceptive stimulation. However, it is rather difficult to evaluate the exact level of nociception. For example, during laparothomy it cannot always be seen when the gut is distended or not by surgeon. In further studies, one solution for this could be video camera; then you can see all the time what the surgeon is doing. Especially in older patients we sometimes, before the procedure was started or if there was a break in surgery, observed inconsistency between blood pressure and SSI: while NIBPMAP decreased below 60 mmHg, SSI reported poor signal logically and increased. When ethylphenylephrin 2 mg was given iv., NIBPMAP increased and SSI decreased. In some younger patients, SSI was stable and the signal was good even when NIBPMAP was below 60 mmHg. Therefore, at least in older patients when NIBPMAP decreased below 60 mmHg, SSI acted like increased nociception and after ethylphenylephrin it acted like decreased nociception. In two neck-surgery patients, SSI increased remarkably when surgeon palpated the thyroid gland area before skin incision. This can be due to sensitive baroreflex reaction or maybe the palpation moved also tracheal tube and then the stimulus can be rather strong. BODY.5. CONCLUSIONS: Fentanyl bolus (2.0 μg kg−1), given five minutes before tracheal intubation, did not block totally the increase of SSI. Fentanyl boluses during procedure decreased SSI, indicating that increasing analgesia decreases SSI. Tracheal intubation, skin incision, and surgical stimuli during the procedure increased SSI from baseline, indicating that nociceptive stimuli increase SSI. We noticed some problems in this version of SSI but it seems that SSI is a valuable tool in estimating nociception-antinociception balance during anesthesia. However, more studies are needed to evaluate its final value.

TITLE: Text Message Feedback to Support Mindfulness Practice in People With Depressive Symptoms: A Pilot Randomized Controlled Trial ABSTRACT.BACKGROUND: It has been shown that mindfulness practice can be helpful in preventing relapse from depression. However, practicing mindfulness regularly at home is often a challenge for people with depression. Mobile phone text messaging (short message service, SMS) may be a feasible approach to assist regular mindfulness home practice. ABSTRACT.OBJECTIVE: The aim of this study was to evaluate the feasibility of text message–based feedback to support mindfulness practice in people with depressive symptoms after inpatient psychiatric treatment. ABSTRACT.METHODS: Participants received a manualized group introduction to three mindfulness exercises during inpatient treatment and were randomized at hospital discharge. All participants were asked to practice the exercises daily during the 4-month follow-up period. Only participants allocated to the intervention group received reinforcing feedback via mobile phone text messages after reporting their mindfulness practice via text message. Participation rates and satisfaction with the interventions were evaluated, and effects on relevant outcomes were explored. ABSTRACT.RESULTS: Of the 176 eligible inpatients invited to participate, 65.9% (116/176) attended the introductory mindfulness group at least once, 33.0% (58/176) were willing to participate in the study, and 41 were randomized. The majority 85% (35/41) of these participants completed the study. Among the participants allocated to the intervention group (n=21), 81% (17/21) used the text message support at least once. The average number of text messages sent during the intervention period was 14 (SD 21, range 0-91). Satisfaction rates were high. Preliminary analyses of the effects of the intervention yielded mixed results. ABSTRACT.CONCLUSIONS: Findings indicate that text messaging following inpatient treatment is feasible for some, but not for all people with depressive symptoms. Modest use of the text messaging intervention and its mixed effects imply that dose and ingredients of the intervention should be increased for this group of patients in a future full-size RCT. Such a larger study should also include a process evaluation to investigate moderators of the effect of mindfulness practice and text message feedback on clinical outcome. ABSTRACT.TRIAL REGISTRATION: International Standard Randomized Controlled Trial Number (ISRCTN): 58808893; http://www.controlled-trials.com/ISRCTN58808893 (Archived by Webcite at http://www.webcitation.org/6pmrDRnGt) BODY.INTRODUCTION: A recent meta-analysis showed that mindfulness-based interventions contribute to reducing depressive symptoms (d=0.30 at 2 months; d=0.23 at 3-6 months follow-up [1]) and may be beneficial in the treatment of outpatients with acute depression [2]. Although some studies evaluated mindfulness-based interventions for inpatients with psychosis or borderline personality disorder (eg, [3,4]), to our knowledge, only one study has evaluated mindfulness techniques in depressed inpatients [5]. In this uncontrolled pilot study, an 8-session mindfulness program yielded significant pre-post changes in depression and mindfulness. However, attrition was considerable with only half of the participants completing the intervention. The continuous practice of mindfulness exercises is a key component of mindfulness-based interventions. Time spent on formal mindfulness practices, such as body scan and mindfulness on breathing, is positively associated with outcome [6]. However, with a large share (38%) of people with depression who received mindfulness-based cognitive therapy (MBCT) not regularly practicing at home, lack of homework compliance is a frequent problem [7]. Mobile health (mHealth) apps might increase the effectiveness of mindfulness-based interventions by improving homework compliance. Being simple and efficient, the mobile phone short message service (SMS) is increasingly used to assist the delivery of mental health care [8]. A growing number of studies show that various apps of texting (monitoring, feedback, communication, homework reminders) contribute to improving uptake and outcome of mental health care [9,10]. Pilot studies have evaluated the use of texting to support homework assignments for people with depression receiving cognitive behavioral therapy [11], and as part of a Web-based monitoring and feedback intervention with a focus on mindfulness and acceptance [12]. Moreover, although recent studies have evaluated the effectiveness of Web-based and mobile phone apps, some of them also with a focus on mindfulness [13-18], there is a lack of research on the feasibility of texting interventions to support mindfulness practice in people with depression. Thus, we developed a low-intensity program using texting to support postdischarge mindfulness practice in people with depressive symptoms receiving inpatient treatment. This randomized controlled pilot study investigated (1) participation rates during the different stages of the study and the intervention (recruitment, introductory group, randomization, texting, mindfulness exercises during follow-up, return rates of follow-up questionnaires); (2) satisfaction with the mindfulness training and the text message intervention; and (3) feasibility of the outcome measures. BODY.METHODS.DESIGN: Recruitment for the study "An SMS-Assisted Mindfulness-based Intervention for Relapse Prevention in Depression" (MIND-S, ISRCTN58808893) took place between September 2013 and June 2014 at Ulm University's Department of Psychiatry II in Günzburg, Germany. The hospital provides acute and long-term inpatient mental health care for a catchment area of about 671,000 inhabitants in rural Bavaria. MIND-S was a pilot two-arm randomized clinical trial. Participants were invited to attend mindfulness group sessions at the hospital before discharge. Randomization took place shortly before discharge, with participants allocated to the intervention group receiving a simple texting intervention to support mindfulness exercises at home. Data was collected at three measurement points: (1) baseline (after giving informed consent, before or shortly after the first mindfulness group session), (2) prerandomization (shortly before discharge), and (3) follow-up (4 months after discharge). The study was approved by Ulm University's Ethics Committee. BODY.METHODS.PARTICIPANTS: Participants were included if they were inpatients or day patients aged 18-75 years, and showed symptoms of depression according to the clinical judgment of their inpatient therapist. Exclusion criteria were the presence of psychotic symptoms or a history of schizophrenia, current manic state, risk of a dissociative crisis, severe cognitive impairment, persistent severe substance abuse, suicidality or risk of self-harm during the current illness episode, insufficient command of the German language, and lack of a mobile phone. To identify eligible patients, therapists were informed about the study and the hospital database was regularly monitored. Therapists of possibly eligible patients were asked to give a more detailed account regarding inclusion and exclusion criteria. Eligible patients were contacted personally by the first author SK and provided with oral and written information about the study, asked to give informed consent and to complete the baseline questionnaire, and invited to attend the introductory mindfulness group. As the mindfulness group was open to all inpatients, several participants asked to visit the group once, before they gave their informed consent, and completed the baseline questionnaire shortly (maximum 1 day) afterwards. BODY.METHODS.RANDOMIZATION: After participants returned the prerandomization questionnaire, they were randomly allocated at a 1:1 ratio to the intervention or the control group. Randomization was based on a centralized procedure, which was coordinated by the Heidelberg site independent of the recruiting clinical site (Günzburg) using computer-generated random numbers. BODY.METHODS.INTERVENTION.PART 1: MINDFULNESS GROUP TRAINING DURING INPATIENT TREATMENT: The mindfulness instructions used in this study were based on the exercises suggested by Segal and colleagues [19] on mindful breathing, mindful walking, and the "body scan." This program ("MBCT") was originally developed for formerly depressed outpatients. Since participants were acutely depressed, all exercises were shortened to a maximum duration of 10 min and guided throughout. Following a general introduction on the principles of mindfulness and self-compassion in the context of depression, each of the following guided exercises was practiced in the group for 5-10 min: (1) mindful breathing, (2) mindful walking, and (3) mindfulness of the body ("body scan," with a focus on feet and legs). Each participant received a short written description of the exercises to take home after discharge from hospital, and it was recommended to practice one or more of the exercises about once a day for at least 5 min. The mindfulness group training was provided by the first author, who is a licensed cognitive behavioral psychotherapist with 7 years of experience in both inpatient group therapy and mindfulness practice, and who completed introductory courses in mindfulness-based treatments (but did not receive a full training in mindfulness-based stress reduction or MBCT). The 60-min sessions took place weekly, with the manualized contents described above being repeated every time. New participants could join the group at any time. The group was offered as a part of the standard clinical treatment and was open also to patients who were not participating in the study. However, therapists were asked to only send patients who match the study criteria. Study participants were required to attend at least once in order to continue the study. BODY.METHODS.INTERVENTION.PART 2: TEXTING AFTER HOSPITAL DISCHARGE: Participants allocated to the intervention group were asked to send a text message via their mobile phones to the study center whenever they practiced one or more of the mindfulness exercises. The text message should contain information on kind (A for "Atmen," ie, mindful breathing; G for "Gehen," ie, mindful walking; K for "Körper;" ie, mindfulness of the body) and duration of the exercise (minutes exercised), resulting in a short alphanumeric code (eg, A10 for a 10-min practice of mindful breathing, or G5K5 for 5 min of mindful walking followed by a 5-min body scan). As a reminder, each participant received a brief pocket guide explaining the code translations to be sent via text message. After sending a text message, the participant received an automated reply, which consisted of (1) a brief positive reinforcing feedback that was drawn from a pool of 86 messages (eg, "Great! Try to be kind to yourself while practicing."), which were formulated in advance by the study team based on the MBCT literature and randomly assigned by a computer program; and (2) the total time (in minutes) the participant had practiced since the beginning of the text message intervention (eg, "You have already practiced 25 min so far"). If a participant's text message did not match the required format, he or she received a message on how to use the program. A reminder was sent if no text messages were received for more than 1 week. Reminders were continuously sent every week until the end of the intervention in case of persistent nonresponse. We decided to send reminders weekly instead of daily because the main function of the messages was to gently reinforce training behavior (self-management) rather than merely reminding patients to practice the mindfulness exercises. SK introduced participants to the text message intervention, which started immediately after discharge from hospital and lasted 4 months. The few participants with no texting experience received instructions on how to send and receive text messages. The intervention manual is available from the authors upon request. Participants of the control group attended the mindfulness training group and were asked to regularly practice the exercises at home. They did not receive text message assistance during follow-up. The intervention was an add-on to treatment as usual. There were no constraints for participants to utilize any other treatment during the study period. All participants received €25 after returning the follow-up questionnaire. Additionally, participants in the intervention group received €10 at hospital discharge to cover their costs for sending text messages. BODY.METHODS.MEASURES: Outcomes of the intervention to be expected include broader effects on depression (severity of depressive symptoms and perseverative thinking) and on aspects more closely related to the nature of the intervention (mindfulness and self-compassion). Severity of depressive symptoms was measured with the German version of the Brief Patient Health Questionnaire-9 Item (PHQ-9 [20]), which assesses the DSM-IV criteria for major depression via patient self-report with 9 items on a 4-point Likert scale ranging from 0 ("not at all") to 3 ("nearly every day"). The sum score ranges from 0 to 27 with higher scores indicating more severe depression. The German version of the Perseverative Thinking Questionnaire (PTQ [21]) assesses repetitive negative thinking without referring to depressive symptoms in the item formulation. This self-report questionnaire consists of 15 items answered on a 5-point Likert scale ranging from 0 ("never") to 4 ("almost always"), which describe how participants typically think about negative experiences or problems (eg, "My thoughts repeat themselves"). The total score ranges from 0 to 60, with higher scores indicating more repetitive thinking. Mindfulness was assessed with the 14-item short form of the Freiburg Mindfulness Inventory (FMI; German: "Freiburger Fragebogen zur Achtsamkeit," FFA-14 [22]). Items are rated on a 4-point Likert scale ranging from 1 ("rarely") to 4 ("almost always"). The sum score ranging from 14 to 56 was used, with higher scores indicating more mindfulness. Self-compassion was assessed with the German version (SCS-D [23]) of the short form of the Self-Compassion Scale (SCS-SF [24]). The 12 items (eg, "I'm disapproving and judgmental about my own flaws and inadequacies") are rated on 5-point Likert scale ranging from 1 ("very rarely") to 5 ("very often"). Higher mean total scores indicate more self-compassion. A number of instruments measured feasibility and acceptance of the intervention at the various stages of the trial. First, mindfulness practice was measured via a short self-constructed questionnaire at follow-up. Participants were asked how often they practiced each of the three mindfulness exercises during each of the 4 months of the intervention period, yielding total number of exercises. Also at follow-up, participants were asked how long on average they practiced each of the three exercises in each of the 4 months. The mean duration per exercise was multiplied with the number of times this exercise was practiced in a given month, and this information was summarized for the three exercises over 4 months, yielding total duration of home practice per exercise. If participants reported an exercise without specifying the time spent on exercising, missing data was replaced by the grand means based on available data for each type of exercise (9 min for mindfulness breathing, 11 min for mindfulness walking, and 14 min for mindfulness of the body). Extreme outliers were omitted (3 participants in the control group who reported to have practiced over 200 times). Second, at baseline , willingness to send text messages ("Do you think you would send a text message after practicing?") and previous experience with the texting and mindfulness exercises were assessed with 1-item questions each. Third, satisfaction with the text message–based intervention was assessed at follow-up with a 20-item adaptation of a questionnaire developed and used in earlier research of our group [25], asking on a 5-point rating scale ("I do not agree," "I rather not agree," "I somewhat agree," "I fairly agree," and "I totally agree") for general satisfaction, satisfaction with the text message feedback, and technical problems. Fourth, satisfaction with the mindfulness intervention was assessed via a self-constructed 10-item questionnaire at discharge and follow-up, asking on the same 5-point rating scale about satisfaction with the mindfulness introduction, as well as about potential problems with and subjective effects of the mindfulness exercises. Fifth, at follow-up, participants of both groups were asked to rate the usefulness of the intervention as a whole ("yes, it helped me a lot," "yes, it helped me somewhat," "it neither helped nor harmed me," "no, it rather harmed me," and "no, it harmed me a lot"), including the option to add open-ended comments and suggestions. Sociodemographic information was assessed at baseline. Furthermore, inpatient therapists were asked to document the diagnoses of their participating patients at study intake according to ICD-10. BODY.METHODS.DATA ANALYSIS: According to recommendations for reporting results of pilot studies [26,27], reporting of results focuses on descriptive statistics, that is, mean and standard deviation for continuous variables, and frequencies and percentages for categorical variables. Feasibility indicators include numbers of patients eligible, willing to participate in the study, and to be randomized; numbers of participants lost to follow-up; intended, and actual use of the text message intervention; the number of text messages sent during the intervention; as well as frequency and duration of home practice. Chi-square and t-tests were used to analyze differences of feasibility indicators by allocation. For the total scores of PHQ-9, PTQ, FMI, and SCS-D group by time interaction effects were tested using repeated measures analysis of variance. Additionally, effect sizes d (standardized mean between-group differences corrected for prerandomization differences) were calculated for all outcome measures. BODY.RESULTS.SAMPLE: Socioeconomic status was assessed at baseline (see Table 1). Participants were 44-years-old on average, most of them were female, married, or living together with a partner (see Table 1). Level of education was predominantly low, and most were working at least part-time. Mean duration since the first occurrence of depressive symptoms was 11 years. According to their PHQ-9 score at baseline, more than three quarters (81.1%) of the patients showed substantial symptoms of depression (PHQ-9 ≥10). At baseline, participants who were later allocated to the intervention group were less depressed and showed more self-compassion than participants of the control group. The majority of the participants had at least some experience with texting. Regarding mindfulness exercises, about half had tried mindfulness practice once, a few were practicing regularly for up to 5 years. BODY.RESULTS.PARTICIPATION IN THE INTRODUCTORY GROUP AND THE STUDY: It was found that 54.8% (176/321) of the screened patients were judged as eligible and invited to participate in the study. Of these, 65.9% (116/176) visited the introductory group at least once. The mean number of group visits of all participants (later randomized or not) was 2.4 (SD 1.8, range 1-9), the number of group attendees varied between 1 and 19 (mean 6.9, SD 3.5). In total, 33.0% (58/176) of the invited patients consented to participate in the study and completed baseline measures. Of the latter, 71% (41/58) completed prerandomization measures and were randomized. Randomized participants took part in the introductory group 3.4 times (SD 2.0) on average (intervention: mean 3.3, SD 2.0; control: mean 3.5, SD 2.1). Follow-up data were available for 85% (35/41) of the randomized participants, with no differences by allocation. Figure 1 depicts the (simplified) flow of participants through the stages of the trial. Most common reasons to decline study participation (not systematically assessed) were not wanting to complete questionnaires (5%; 3/57) or using the mobile phone (4%; 2/57), a lack of interest in mindfulness practice (5%; 3/57), and feeling that participation in the study would be "too much" (5%; 3/57). Most patients (70%; 40/57) did not give any reason. Still, of the 32.4% (57/176) of patients who were not interested in study participation, 54% (31/57) attended the mindfulness training group at least once. Table 1 Characteristics of study participants. Characteristics IG a (n=21) CG b (n=20) Differences Age (in years), mean (SD c ) 43.4 (12.7) 44.5 (13.5) t 39 =−0.26; P =.80 Gender (female), n (%) 13 (61.9) 15 (75.0) χ 2 1 =0.8; P =.37 Level of education Qualification for university entrance, n (%) 3 (14.3) 4 (20.0) χ 2 1 =0.2; P =.63 Lower qualification, n (%) 18 (85.7) 16 (80.0) Marital status Married or living together with partner, n (%) 10 (47.6) 11 (45.0) χ² 2 =0.4; P =.84 Single, n (%) 7 (33.3) 5 (25.0) Other, n (%) 4 (19.0) 4 (20.0) Employment status Full-time, n (%) 8 (38.1) 6 (30.0) χ² 3 =0.5; P =.93 Part-time, n (%) 5 (23.8) 5 (25.0) Unemployed, n (%) 6 (28.6) 6 (30.0) Other, n (%) 2 (9.5) 3 (15.0) Primary diagnosis Major depression d , n (%) 16 (76.2) 19 (95.0) χ 2 1 =2.9; P =.09 Other e , n (%) 5 (23.8) 1 (5.0) Illness duration (years), mean (SD) 10.8 (12.2) 11.7 (8.4) t 38 =−0.29; P =.78 PHQ-9 f (depressive symptoms), mean (SD) 12.74 (5.69) 18.61 (4.86) t 35 =−3.37; P =.002 PTQ g (perseverative thinking), mean (SD) 38.50 (10.97) 45.32 (9.95) t 35 =−1.98; P =.06 FMI h (mindfulness), mean (SD) 30.56 (5.53) 27.16 (4.91) t 35 =1.98; P =.06 SCS-D i (self-compassion), mean (SD) 2.53 (0.76) 2.05 (0.59) t 36 =2.15; P =.039 Experience with mindfulness exercises None, n (%) 7 (35.0) 10 (50.0) χ² 2 =1.7; P =.44 Tried once, n (%) 12 (60.0) 8 (40.0) Experienced, n (%) 1 (5.0) 2 (10.0) Experience with texting None n (%) 5 (23.8) 3 (15.0) χ² 2 =3.2; P =.20 Some experience, n (%) 4 (19.0) 9 (45.0) Very experienced, n (%) 12 (57.1) 8 (40.0) a IG: intervention group. b CG: control group. c SD: standard deviation. d ICD-10=F32.1, F32.2, F33.1, or F33.2. e ICD-10=F31.4, F40.01, F41.0, F43.2, or F61.0. f PHQ-9: Patient Health Questionnaire. g PTQ: Perseverative Thinking Questionnaire. h FMI: Freiburg Mindfulness Inventory. i SCS-D: Self-Compassion Scale; Missing values: illness duration: N=1 (IG), PHQ-9: N=2 (each IG and CG); PTQ, FMI: N=3 (IG), N=1 (CG); SCS-D: N=2 (IG), N=1 (CG). Figure 1Participant flow through the stages of the trial. BODY.RESULTS.PARTICIPATION IN THE TEXT MESSAGE FEEDBACK INTERVENTION: Before randomization, 59% (24/41) of the study participants expressed their intent to send text messages after practicing a mindfulness exercise if randomized into the intervention group. It was found that 20% (8/41) of the participants indicated that they would not and 22% (9/41) stated that they would "rather not" use the text message assistance. Of the 24 participants expressing intent to send text messages, 50% (12/24) were later assigned to the intervention group. At follow-up, about two-third of the participants in the intervention group reported that they had not (18%) or not always (47%) sent a text message after mindfulness practice, whereas about one-third reported that they had done so always (12%) or most of the time (24%). During the intervention period, participants sent 294 text messages reporting 395 exercises. Of the 21 participants, 81% (17/21) in the intervention group sent at least one text message and 67% (14/21) of participants texted more than once. Two participants sent at least one message a week. On average, participants sent 14.00 (SD 21.00, median 9, range 0 - 91) messages, reporting 18.81 (SD 5.44, median 12) exercises during 4 months, that is, about one exercise per week. Correlations of the number of text messages with several parameters are reported elsewhere [28]. The mean duration per exercise reported via text message was 9.94 min (SD 5.38) with mindfulness of the body exercises showing the longest duration (mean 13.84, SD 4.13, N=103), followed by mindful breathing (mean 8.95, SD 5.48, N=211), and mindful walking (mean 7.54, SD 3.70, N=81). BODY.RESULTS.SATISFACTION WITH THE INTERVENTIONS: Findings about satisfaction with the text message intervention are shown in Figure 2. Overall, participants showed high satisfaction. Specifically, the program met the expectations of most of the participants, more than half reported that it helped them to practice the mindfulness exercises regularly, and most felt generally supported by the text message feedback. Two-third reported that they would use the program again, and the majority indicated that they would recommend it to a friend. However, about two-third of the participants stated that they found it difficult to send messages on a regular basis, and some were concerned about data privacy. Figure 2Satisfaction with the text message intervention. At follow-up, participants of both groups were asked to evaluate the mindfulness exercises. The introduction into the mindfulness exercises at the hospital appeared appropriate and clear to all (see Figure 3). While the majority of the intervention group found that they were able to practice mindfulness in everyday life, this was the case for less than half of the participants of the control group. Furthermore, in comparison to the control group, more participants in the intervention group perceived the mindfulness exercises helpful in relationships and in coping with rumination and negative feelings. Participants in the control group reported more difficulties in practicing the mindfulness exercises alone. At follow-up, 67% (35/53) of the participants in both intervention and control groups reported that taking part in the study helped them to some degree, the rest said it neither helped nor harmed them. The rate of agreement was higher in the intervention group (78%) than in the control group (53%). Of the 11 participants who provided written feedback at follow-up, 2 stated that they often forgot to send a text message, another 2 wrote that the weekly text message reminders were perceived as helpful, and 1 recommended further reminders in larger intervals. One participant stated that practicing in the group was easier than alone, and another one suggested to provide audio recordings of the exercises, and finally one participant recommended to offer the inpatient group sessions more frequently. Figure 3Satisfaction with the mindfulness introduction and exercises at follow-up. Table 2 Effect of the intervention on mindfulness practice and outcomes (N=35). Outcome measures Prerandomization 4-month follow-up Test statistics P value Effect size (95% CI) IG a , mean (SD b ) CG c , mean (SD) IG, mean (SD) CG, mean (SD) Number of exercises practiced 7.88 (4.92) 10.06 (6.80) 62.67 (61.64) 48.71 (44.28) t 30 =−0.72 .48 0.25 (−0.45 to 0.96) Total time practiced (min) 23.75 (13.11) 35.13 (16.55) 642.17 (815.26) 579.50 (784.28) t 30 =−0.22 .83 0.08 (−0.62 to 0.78) PHQ-9 d (depressive symptoms) 37.47 (6.49) 32.00 (6.83) 8.94 (6.61) 12.06 (7.24) F 1,31 =0.17 .68 0.14 (−0.53 to 0.82) PTQ e (perseverative thinking) 3.10 (0.77) 2.61 (0.85) 26.25 (19.28) 33.19 (16.33) F 1,30 =0.85 .37 −0.26 (−0.84 to 0.31) FMI f (mindfulness) 36.40 (8.72) 32.53 (6.40) F 1,28 =0.44 .51 0.25 (−0.49 to 0.99) SCS-D g (self-compassion) 3.11 (1.06) 2.63 (0.71) F 1,29 =0.01 .94 0.02 (−0.59 to 0.63) a IG: intervention group. b SD: standard deviation. c CG: control group. d PHQ-9: Patient Health Questionnaire-9 Item. e PTQ: Perseverative Thinking Questionnaire. f FMI: Freiburg Mindfulness Inventory. g SCS-D: Self-Compassion Scale; Missing values: PHQ-9: N=1 (each IG and CG); PTQ: N=2 (IG), N=1 (CG); FMI: N=3 (IG), N=2 (CG); SCS-D: N=2 (each IG and CG). BODY.RESULTS.FEASIBILITY OF THE OUTCOME MEASURES: The effects of the post-hoc assessment of mindfulness home practice (number and duration of exercises practiced) and the four selected outcome questionnaires (PHQ-9, PTQ, FFA, and SCS-D) yielded near zero to small, nonsignificant effect sizes (see Table 2). Note that, due to different scoring, a positive effect size on the symptom measures (PTQ, PHQ) indicates an outcome in favor of the intervention group, whereas on the FMI and the SCS-D, a positive effect size indicates a result in favor of the control group. The largest but still small differences were found on perseverative thinking, self-reported mindfulness, and the number of reported exercises. Effects for the former two measures were against expectations because the control group scored lower on perseverative thinking (d=−0.26) and higher on mindfulness (d=0.25) than the intervention group. The number of exercises practiced tended to be higher in the intervention group (d=0.25), and there was small effect on the PHQ-9 in favor of the intervention group. BODY.DISCUSSION.PRINCIPAL FINDINGS: To our knowledge, this is the first study to evaluate the feasibility of a simple, low-intensity texting app to support mindfulness practice in people with depressive symptoms after inpatient psychiatric treatment. The study showed that a considerable proportion of people with depressive symptoms receiving in inpatient treatment are interested in a mHealth mindfulness program, and that most participants were satisfied and experienced the intervention as helpful. BODY.DISCUSSION.PARTICIPATION IN THE INTRODUCTORY GROUP AND THE STUDY: According to introductory group participation rates, the mindfulness exercises seemed to interest about two-third of the target population of psychiatric inpatients with depressive symptoms. Only half of them could be motivated to participate in the study (eg, fill out questionnaires and participate in the texting intervention). However, once they decided to participate in the study, most patients stayed in the trial until the end. Retention rates were 71% for the first (prerandomization) and 85% for the second (postrandomization) part of the study, falling within the upper range of other mHealth studies (43-100% [29]). Similar, sometimes higher, attrition rates were found in studies evaluating mindfulness-based interventions in depressed patients delivered face-to-face (49% [5]; 8-38% [2]) or via the Internet or mobile phone (57% [14]; 38% [15]). Good retention rates speak to the feasibility of the study design and committed study staff. However, the possibility of a selection bias toward including rather motivated and compliant patients cannot be ruled out. BODY.DISCUSSION.PARTICIPATION IN THE TEXT MESSAGE FEEDBACK INTERVENTION: On average, participants sent about one text message per week which falls behind expectations, as daily practice was recommended. However, this result is not surprising, as before the beginning of the intervention, less than 60% of the randomized participants indicated that they intended to text. Comparing the postintervention self-report with the information from the text messages, participants texted only about every third time after a mindfulness exercise. Likewise, about two-third of the participants reported that they had not or not always sent a text message after they had practiced, and that they had difficulties texting regularly. However, the possibility of a combined effect of a social desirability bias and a 4-month recall bias should be taken into account. Nevertheless, participation patterns are comparable with another study which examined text-messaging support in the treatment of people with depression (65% response rate to text message reminders [11]), indicating that expectations might have been unrealistic. Another explanation could be that a considerable number of participants did not feel the need to be supported every time they practiced. Taken together, these findings suggest a need to enhance mode of delivery and content of text messages including tailoring feedback to increase subjective meaning. This might also be achieved by sending daily queries inquiring about type and duration of practice. BODY.DISCUSSION.SATISFACTION WITH THE INTERVENTIONS: Participants of the intervention group were predominantly satisfied with the texting intervention. Most felt supported and encouraged by the messages, and over 80% would further recommend the program. Although use of the texting intervention was moderate, in absolute values, considerably more patients in the intervention than in the control group perceived the mindfulness exercises as helpful, especially regarding coping with rumination and negative feelings. More patients in the intervention group than in the control group stated that they had practiced in daily life. Furthermore, compared with the control group, in the intervention group, fewer patients at least fairly agreed that they had problems practicing alone. Although these are no significant results and should be regarded with caution, they might indicate that the text message feedback supports mindfulness home practice and could increase the effects of mindfulness exercises as intended [30]. BODY.DISCUSSION.FEASIBILITY OF THE OUTCOME MEASURES: Although the main outcome measures (amount of home practice, depressive symptomatology) depict a small effect of the intervention, the additional measures (rumination, mindfulness, self-compassion) yielded zero or small effects in favor of the control group. Due to the small sample size, no final conclusion can be drawn. However, these results indicate that either the questionnaires were insensitive to changes, the dosage of intervention was not sufficient, or the observation period was too short to detect changes. It could be hypothesized that mindfulness and self-compassion are rather trait-like, less susceptible to change, and thus, need a longer and more intense intervention to change. BODY.DISCUSSION.LIMITATIONS: This study has a number of limitations. First, the introduction to the mindfulness exercises and the text message intervention was provided by the first author, who also was involved in data collection and analysis. Second, the intervention was only implemented at one hospital, suggesting limited generalizability of findings. Third, there could have been a selection bias, as the inpatient therapists could recommend a patient to participate or not to participate in the study due to their subjective judgment. Fourth, the mindfulness introduction was a shortened and adopted version of other mindfulness-based programs, and has not been validated in this population. Fifth, there might be a recall bias regarding the post hoc assessment of the mindfulness exercises practiced in the follow-up period, combined with a social desirability bias. There might have been a tendency to over-report the mindfulness practice in the intervention group. Finally, due to the small sample size, all results of this pilot study should be interpreted with caution. BODY.DISCUSSION.CONCLUSIONS: Taken together, the positive evaluations of the MIND-S program by the participants indicate that, in general, mindfulness practice augmented by text message feedback is a feasible intervention. However, the moderate use of the texting intervention and the mixed effects imply that dose and ingredients of the intervention should be increased for this group of patients in a future, full-scale RCT. Additional components might include expert or peer support such as regular mindfulness group visits after discharge, audio or video material, more frequent reminders, or individual expert support via texting, email or chat-groups. To minimize recall bias, frequency and duration of mindfulness practice should be assessed more frequently during the follow-up period. Furthermore, the intervention should also be tested in other populations (such as previously depressed outpatients) to assess the differential indication. A larger study will also allow to investigate the effects of the intervention, as well as moderators of the effect of mindfulness practice and text message feedback on clinical outcome.

TITLE: Use of Remifentanil and Alfentanil in Endotracheal Intubation: A Comparative Study ABSTRACT.BACKGROUND:: Opioids, such as alfentanil, are used to facilitate endotracheal intubation without the use of neuromuscular blocking agents in patients undergoing elective surgery. ABSTRACT.OBJECTIVES:: The goal of this study was to evaluate the endotracheal intubation conditions when remifentanil or alfentanil was used with propofol without the application of neuromuscular blocking agents. ABSTRACT.PATIENTS AND METHODS:: One hundred American Society of Anesthesiologists (ASA) grade I patients scheduled for elective surgery were enrolled in this prospective, randomized, triple-blinded study. The patients were randomized to group A (alfentanil) or R (remifentanil). In group A, alfentanil (50 mcg/kg) was intravenously injected over 10 seconds, and after 45 seconds or at the occurrence of apnea, propofol (2 mg/kg) was intravenously injected over 5 seconds. Thirty seconds after the administration of propofol, laryngoscopy and endotracheal intubation were attempted. In group R, remifentanil (5 mcg/kg) was administered instead of alfentanil. Intubation conditions, including ease of laryngoscopy, patency of the vocal cords, jaw relaxation, limb movement (1-4 score), and also, demographic data were evaluated. ABSTRACT.RESULTS:: There were no demographic data differences between groups (age, weight, and sex). Further, laryngoscopy, jaw relaxation, and limb movement scores were similar in the R and A groups and there were no significant differences, but vocal cords were significantly more patent in group R than those in group A (P = 0. 028). ABSTRACT.CONCLUSIONS:: The results of this study showed that remifentanil, similar to alfentanil, provided excellent conditions for endotracheal intubation when used with propofol for the induction of anesthesia; however, remifentanil improved the patency of the vocal cords to a greater extent than alfentanil. BODY.1. BACKGROUND: Endotracheal intubation is one of the measures conducted to maintain the airway while inducing anesthesia. Non-depolarizing muscle relaxants are generally administered to facilitate this procedure. However, in some situations, e. g. , a full stomach, difficult intubation, and certain neuromuscular diseases, the administration of these agents is considered controversial. Therefore, other agents and methods, such as opioid agents, intravenous (propofol instead of thiopental) or inhaled (sevofluran) hypnotics can be usedin such situations to facilitate endotracheal intubation (1-4). Opioids are agents that suppress respiration in addition to providing appropriate conditions for endotracheal intubation; they are therefore good substitutes for muscle relaxants when used along with intravenous or inhaled hypnotics during intubation (1, 2). Previous studies have examined the use of alfentanil for endotracheal intubation in children and adults without the use of muscle relaxants (5-10). Remifentanil is an ultra-short-acting opioid resulting in short-term complications (11-13). BODY.2. OBJECTIVES: The aim of this study was to compare endotracheal intubation conditions using remifentanil (5 mcg/kg) with those using alfentanil (50 mcg/kg) while administrating propofol (2mg/kg) to induce anesthesia without using muscle relaxants in adult patients with normal airways (Mallampati grade I). BODY.3. PATIENTS AND METHODS: After approval of the Medical Ethics Committee and obtaining written informed consent, 100 adult ASA I patients (age, 20–50 years) scheduled to undergo elective surgery were enrolled in this prospective, triple-blinded study. The patients were randomly (using a computer-based randomizing table) assigned to group A (alfentanil) or group R (remifentanil) (50 in each group). Exclusion criteria were as follows: Drug or alcohol abuse and smoking,Airway assessed as Mallampati grade greater than I,Full stomach To achieve triple-blinding, the identity of the administered opioid was withheld from the physicians involved in drug administration, endotracheal intubation, and patient evaluation, and the person in charge of analyzing the data received them in an encrypted form. After placing the patients on the operation bed and completing the physical examination, an intravenous (IV) line was set up, and 500 mL of ringer solution was infused. The patients were monitored for cardiac activity and heart rate by lead II electrocardiography, non-invasive blood pressure, heart rate and pulse oximetry. The dose of the opioid agent (500 mcg/mL alfentanil or 50 mcg/mL remifentanil in a 10 mL syringe) was prepared by an anesthesiologist blinded to study. Intravenous midazolam (1 mg) was injected before the administration of the other drug and 100% oxygen was given for 3 minutes. Then, IV alfentanil (50 mcg/kg) was slowly administered within 10 seconds. After 45 seconds or at the occurrence of apnea, propofol (2 mg/kg) was injected within 5 seconds, and after 30 seconds, laryngoscopy and endotracheal intubation was performed by another anesthesiologist (unaware of the opioid agent administered). After successful intubation, the drug necessary for maintaining anesthesia (propofol 100 and alfentanil 0. 5 mcg/kg/min) was administered. In group R, iv remifentanil 5 mcg/kg was administered instead of alfentanil during induction, and the rest of the procedure was the same as in control group. The following conditions of endotracheal intubation were assessed (Table 1): ease of laryngoscopy,patency of vocal cords,jaw relaxation, andlimb movement, based on 1 to 4 scoring. Table 1. Evaluation of the Quality of Endotracheal Intubation Score 1 2 3 4 Possibility of laryngoscopy Simple Fairly simple Difficult Impossible Patency of vocal cords Patent With movement Closing Blocked Jaw relaxation Fully relaxed Fairly relaxed Moderate relaxation Locked Limb movement Not moving Little moving Moderately moving Entirely moving BODY.3. PATIENTS AND METHODS.3. 1. STATISTICAL ANALYSIS: Normal distribution of quantitative variables (age and weight) was evaluated by the Kolmogorov–Smirnov test. If the variables were normally distributed, the data were analyzed using a t-test. The collected data were expressed as the mean ± standard deviation. The qualitative (nominal) and ordinal variables (sex and intubation score) were assessed by the chi-square test, Mann-Whitney test, and Kendall's Tau-b non-parametric correlations. Analytical studies were conducted using SPSS ver. 12 software. P > 0.05 were considered statistically significant. BODY.4. RESULTS: The distribution of age and weight variables was normal. Demographic data (age, weight, and sex) were not significantly different between the two groups (Table 2). As shown in Table 2, group A and group R did not differ significantly in mean age (32. 5 ± 8. 7 vs. 35 ± 9. 5 years; P = 0. 165) and mean weight (66 ± 9. 7 vs. 67. 6 ± 7. 3 kg; P = 0. 352). Male patients comprised 46% of group A and 58% of group R, indicating that the two groups did not differ significantly in this regard (P = 0. 230). As shown in Table 3, the scores for ease of laryngoscopy (A), jaw relaxation (C), and limb movement (D) did not differ significantly between the two groups (Mann-Whitney test), but vocal cord patency (B) in group A was significantly higher than that in remifentanil group (P = 0. 028). This means that the probability of movement of the vocal cords and their closing in group A was higher than that in group R. Table 2. Demographic Data in the Studied Groups Alfentanil Remifentanil P value Age ,y a 32. 5 ± 8. 7 35 ± 9. 5 0. 165 Sex (M/F) a 23/27 29/21 0. 23 Weight, Kg, Mean ± SD 66 ± 9. 7 67. 6 ± 7. 3 0. 352 a No significant difference Table 3. Comparison of Endotracheal Intubation Scores Alfentanil (%) Remifentanil (%) P value Laryngoscopy score a 0. 277 1 74 82 2 20 18 3 6 0 4 0 0 Vocal cords’ patency score b 0. 028 1 64 84 2 26 10 3 8 6 4 2 0 Jaw relaxation score a 0. 911 1 76 76 2 18 22 3 6 2 4 0 0 Limb movement score a 0. 865 1 68 70 2 24 20 3 4 10 4 4 0 a No significant difference b With significant difference BODY.5. DISCUSSION: In our study, following administration of remifentanil and propofol there was quite a suitable condition for endotracheal intubation in most of the patients. Endotracheal intubation following administration of remifentanil led to the same conditions as those using alfentanil, with better patency of the vocal cords. Previous studies have shown that endotracheal intubation is possible without the use of muscle relaxants (1-4). Propofol administration alone can enable intubation in 60% of cases (4), and the addition of alfentanil increases the possibility to 86% in adults (3). Klemola's study showed that remifentanil (at 3 and 4 mcg/kg) in combination with propofol afforded excellent intubation conditions (in comparison with alfentanil 30 mcg/kg) with success rates of only 55% and 20%, respectively, but the most suitable condition was created in 93% (14). Various studies have indicated different success rates of intubation under desirable conditions, and these differences can be attributed to differences in the study design, scoring system for the intubation condition, the criteria defined for scoring, the dosage of the drugs, the order of administration of drugs, agents administered before the main drugs, the speed at which the drugs are injected (slow injection using an injection pump or fast injection), the duration of induction (90 seconds to 4 minutes), and the age of the patients (from infancy to adulthood). In a study by Alexander et al. , remifentanil (2 mcg/kg), alfentanil (50 mcg/kg), and succinylcholine (1 mg/kg) were administered after propofol, and excellent conditions for intubation were obtained in 35%, 85%, and 100% of the cases, respectively (15). The authors concluded that remifentanil is not as suitable for intubation as alfentanil and succinylcholine. However, in another study by the same group, remifentanil (3, 4, or 5 mcg/kg) was administered in combination with propofol and midazolam (injected before the other drugs), and excellent conditions were obtained in 60%, 95%, and 95% of the cases, respectively (16). Tracheal intubation has also been performed with the use of intravenous hypnotics (propofol, thiopental, and etomidate) in some studies. These studies have revealed that propofol, in combination with remifentanil, is a suitable intravenous hypnotic for the induction of anesthesia and provides better intubation conditions compared to other intravenous hypnotics (1, 17). A study on the pharmacokinetic properties of remifentanil showed that propofol reduced the need for remifentanil required to suppress the patient's reaction to laryngoscopy, thereby indicating a synergistic action between the two drugs (18). Similarly, remifentanil reduces the need for propofol during the induction of anesthesia (19). Collectively, these findings indicate that a combination of these two agents may be useful in inducing anesthesia for intubation. Remifentanil is 20- to 25-fold more potent than alfentanil, but has a shorter duration of action. This limited period of action is another advantage of remifentanil over alfentanil, which has the disadvantage of causing prolonged respiratory arrest when used in short surgeries (8). The combination of remifentanil with propofol may also be advantageous in cases of long and difficult intubation, wherein it may not only be possible to inspect the airway with the laryngoscope but also assess whether the procedure can be continued or aborted in order to preclude the complications of prolonged respiratory arrest during anesthesia. This short period of respiratory arrest afforded with remifentanil is also beneficial in the case of infants, with the duration of respiratory arrest with the use of remifentanil (2 mcg/kg) and propofol (4 mg/kg) being similar to that with the use of succinylcholine (20). This combination is also useful in conditions when endotracheal intubation is necessary but muscle relaxation for surgery is not required, such as cases in which non-depolarizing muscle relaxants are contraindicated (e. g. , myopathy) and those in which succinylcholine is contraindicated despite the need for a fast endotracheal intubation (e. g. , hyperkalemia, burns, choline-esterase enzyme deficiency, or susceptibility to malignant hyperthermia). In addition, complications of depolarizing and non-depolarizing muscle relaxants and their antagonists and the resultant prolongation of the recovery period can be averted by avoiding their administration. A combination of remifentanil and propofol has been used for endotracheal intubation in patients of at different ages (children and adults) (1, 21-23), and the doses of 3 mcg/kg and 3 mg/kg for remifentanil and propofol, respectively, were found to provide desirable conditions for intubation (21). In adult patients who received iv midazolam and lidocaine before remifentanil (2 mcg/kg) and propofol (2 mg/kg) had better intubation conditions compared to those receiving thiopental (5 mg/kg) after 2. 5 minutes of induction: 85% of the propofol group and 50% of thiopental group had excellent conditions for intubation (23). Our study was conducted on healthy young patients, and the outcome may be different in other population groups. The problems with this method and its limitations, as cited in a few studies, include its unsuitability for the elderly, decrease in intravascular volume, and its limitations in patients with cardiovascular or cerebrovascular diseases (24); since decrease in blood pressure induced in these patients might not be well tolerated. Further, intubation in such patients without the use of muscle relaxants might sometimes be dangerous; for example, if laryngoscopy and endotracheal intubation are conducted under inadequate conditions, they may cause injury to the airway or compromise ventilation. Thus, this method is best avoided in patients with high Mallampati grades or airway difficulties. Remifentanil administration (1 to 3 mcg/kg) without propofol can lead to muscle stiffness (i. e. , difficult ventilation with a mask) depending on the drug dosage and the speed of administration (12, 13), but if administered in combination with propofol, muscle stiffness will rarely occur (20). Similarly, alfentanil (40 mcg/kg) in combination with propofol does not lead to significant muscle stiffness (8, 9). Though high doses of short-acting opioids (such as remifentanil and alfentanil) may be avoided in outpatient procedures in order to prevent anesthetic-induced complications and enable earlier discharge, they can be used efficiently in hospitalized patients requiring prolonged surgery and in whom the use of muscle relaxants is considered controversial. On the other hand, due to its pharmacokinetic properties and the possibility of faster recovery (compared to alfentanil) and spontaneous return of respiratory functions (25), remifentanil appears to be the best choice opioid in such conditions. Notably, the use of this combination may be limited in elderly patients and subjects with low intravascular volume, because in such cases, high dosages of opioids may lead to bradycardia or a severe drop in blood pressure level. The combination may also be avoided in patients with Mallampati grades higher than I, i. e. , in those with airway difficulties, and those requiring short surgeries in whom the use of high opioid doses (especially alfentanil) delays discharge. In summary, we conclude that remifentanil in combination with propofol can be used for laryngoscopy and endotracheal intubation in young healthy patients to avoid the use of muscle relaxants.

TITLE: Critical reappraisal of embryo quality as a predictive parameter for pregnancy outcome: a pilot study ABSTRACT: Aim of the study: Pilot study to analyse the efficacy and embryo morphology using a new human embryo culture medium (GM501) versus the conventional used medium (ISM1). Methods: Over a four-month period, all patients at the Leuven Institute of Fertility and Embryology (LIFE) were randomly allocated to have their embryos cultured in either the standard sequential culture medium ISM1 (control) or in a new universal medium (GM501) (study group). Primary outcome parameters were clinical pregnancy and live birth rate. The secondary outcome parameter was the correlation of embryo fragmentation rate with pregnancy outcome. Results: We did not observe any differences between the ISM1 control group and GM501 study group with regard to fertilization, pregnancy, implantation rates, ongoing pregnancy, and babies born. The number of embryos with a minimal fragmentation rate (less than 30%) was significantly higher in the GM501 study group. Conclusion: Although a significant higher embryo fragmentation rate was seen in In vitro culture of embryos in GM501, pregnancy outcome results were comparable to those of embryos cultured in ISM1. According to our results the value of embryo morphological criteria as a parameter for pregnancy outcome should be examined and discussed again. BODY.INTRODUCTION: In vitro, embryos are exposed to stress that can compromise their physiology, gene expression, and development (Gardner et al., 2005). In particular, the culture medium is an important determinant of successful in vitro interactions between gametes and subsequent embryo development (Gardner et al., 1997). Most in vitro fertilization (IVF) centers use commercially available culture media, but the formulations of these media are seldom disclosed. In general, manufacturers follow either the "back to nature" or the "let the embryo choose" philosophies (Summers et al., 2003). "Back to nature" philosophy attempts to mimic the different biochemical environments that gametes and embryo encounter during the natural reproductive process and provide different media in a sequential order (two-step protocol). The "let the embryo choose" philosophy has led to a family of media in which all of the substances necessary to early embryological development are provided, and there is no need for a media change (one-step protocol). There is no scientific evidence showing any advantage to one philosophy over the other. Biggers et al. (2002) demonstrated the efficacy of a universal medium for human blastocyst development. In a prospective randomized study, no differences were observed for culture and development of human blastocysts, implantation, or pregnancy rates when either a universal or a sequential medium (Macklon et al., 2002). Gynemed GM501 is an optimized variant of KSOMAA medium; it is a novel medium and no comparative data about its use in embryo culture is available. Therefore, we performed a prospective randomized comparison between this medium and the standard sequential medium ISM1 in terms of pregnancy outcome. Our secondary aim was to investigate the predictive value of embryo fragmentation on pregnancy outcome. BODY.METHODS.POPULATION: In a 4-month period, the embryos of all of the patients entering the Leuven Institute of Fertility and Embryology (LIFE) IVF/intracytoplasmic sperm injection (ICSI) program were randomly allocated to either the GM501 culture medium study group or the ISM1 culture medium control group. Blastocyst culture, preimplantation genetic diagnosis (PGD) patients, testicular sperm extraction (TESE), and egg donation procedures were excluded from the study. Randomization was blinded by providing envelopes filled with an equal amount of adhesive labels printed with either "GM501" or "ISM1." In accordance with Belgian embryo protection law (Gordts et al., 2005), the embryo transfer procedures differ depending upon the age of the patient. Therefore, we performed a separate randomization for the < 36 y age group, and the ≥ 36 y age group; the envelopes were contained in different boxes. When the IVF lab forms were prepared, the age of the female patient was determined and an envelope was removed from the appropriate box. The adhesive label taken from the envelope was attached to the lab form, and this determined the culture medium used for all of the embryos from that particular patient. BODY.METHODS.CULTURE MEDIA: ISM1 (MediCult, Jyllinge, Denmark) was used as the control medium, and Gynemed GM501 (Gynemed GmbH & Co. KG, Lensahn, Germany) was used as the study medium. Both media were supplemented with penicillin and streptomycin. Media were pre-equilibrated for at least 4 h in 5% CO2 at 37°C prior to use. BODY.METHODS.OVARIAN STIMULATION AND OOCYTE COLLECTION: Patients were treated with a long or short gonadotrophin-releasing hormone agonist (GnRHa) protocol using buserelin (nasal spray, 9 × 100 μg/day; Suprefact, Hoechst, Frankfurt, Germany) starting in the mid-luteal phase of the cycle preceding the IVF attempt for a period of at least 2 wk in the long protocol, or starting on the first day of menstruation following the intake of a contraceptive pill for 2-3 wk in the short protocol. Hormonal stimulation was performed with urinary gonadotrophins (Menopur; Ferring, Limhamn, Sweden) or recombinant follicle-stimulating hormone (FSH, Puregon; Organon, Oss, The Netherlands). When follicles reached a diameter of 18 mm, 10,000 IU of human chorionic gonadotropin (hCG, Pregnyl; Organon) was administrated and ultrasound-guided oocyte aspiration was performed 35 h later. Oocyte collection was performed through ultrasound-guided transvaginal aspiration of the follicles under sedation and local anaesthesia. Oocytes were collected in an Earle's Balanced Salt Solution (EBSS) with phenol red (Lonza, Verviers, Belgium) and penicillin (50 U/ml) and streptomycin (50 μg/ml). After collection, oocytes were rinsed with Universal IVF medium (MediCult) and placed in an in vitro fertilization four-well plate (Nunclon Surface; Nunc, Roskilde, Denmark). A maximum of four oocytes were placed per well. BODY.METHODS.SPERM PREPARATION: The sperm sample was obtained through masturbation in a sterile container (the day of oocyte collection: day 0), and immediately placed on a warm plate at 37°C. Sperm concentration and motility were determined before the procedure. Sperm morphology was determined according to Kruger strict morphology criteria. For sperm preparation, a swim-up technique was used: approximately 0.5 ml of the sample was placed at the bottom of 5 ml polystyrene round-bottom tubes (Falcon; Becton Dickinson Labware Europe, Meylan, France) containing pre-equilibrated Universal IVF medium with 5 mM glucose, penicillin, and streptomycin (MediCult; the medium did not contain phenol red). The tubes were incubated for 45 min in a humidified atmosphere of 5% CO2 at 37°C. Afterwards, supernatants were collected and placed in 15 ml polystyrene conical tubes, which were centrifuged for 10 min at 0.5 × g. The pellet was resuspended in 1 ml of pre-equilibrated Universal IVF culture medium and the sperm concentration and motility were recounted. BODY.METHODS.FERTILIZATION: Approximately 4 h after oocyte collection, insemination was performed (day 0) by standard IVF or ICSI procedures. For conventional IVF, a maximum of four oocytes/well in a four-well plate with Universal IVF medium were inseminated with one million capacitated spermatozoa and returned to the incubator for 1 h. For ICSI, oocytes were denuded enzymatically with 40 IU/ml hyaluronidase (SynVitro; Hyadase, MediCult), and mechanically with a glass Pasteur pipette. Afterwards, they were rinsed with Universal IVF medium (MediCult). After IVF or ICSI, the inseminated oocytes were placed in microdrops under light mineral oil (Irvine Scientific, Santa Ana, California) consisting of pre-equilibrated media: either the control medium (ISM1) or GM501 medium depending on the randomization protocol. Because fertilization techniques can affect pregnancy outcomes, it was important to have the same percentages in both groups. Therefore, either 100% ICSI or IVF was performed, and in some patients 50% IVF–50% ICSI was performed (mixed cycle). Fertilization was checked the morning after IVF or ICSI (day 1) under an inverse microscope at 200× magnification. An oocyte with two pronuclei (2PN) present was considered to be fertilized. BODY.METHODS.EMBRYO TRANSFER: Conventional embryo transfer was performed on day 2 or 3. For the purposes of our study, a predefined organizational scheme for conventional transfers was implemented. The day of puncture determined the day of transfer and the length of the in vitro embryo culture. Patients were not able to choose between day 2 or day 3 transfer. On the day of the transfer, the embryo morphology was scored and the number of embryos to be transferred was determined in accordance with Belgian legislation (Gordts et al., 2005). For the transfer, pre-equilibrated Universal transfer medium (UTM) with phenol red (MediCult) was used. The transfer was performed with a Semtrac soft catheter (#2000 Semtrac C; Gynétics Medical Products N.V., Hamont-Achel, Belgium), double catheter (#4219 Emtrac Set; Gynétics), or Soft-Trans Embryo Catheter (K-Soft 5100; Cook, Brisbane, Australia). Transfer catheter policy is standardised in the local working protocol in such a way that an equal distribution of the used catheters is provided for both groups. Furthermore all catheters have received the same in house embryo toxicity tests before use In all cycles, the luteal phase was supported with 3 × 200 mg of micronized natural progesterone (Utrogestan; Besins International, Brussels, Belgium), administered vaginally. BODY.METHODS.FOLLOW-UP: Following embryo transfer, blood samples were taken for analysis of serum estradiol, progesterone, and hCG concentrations on day 12. A cycle was considered to be a conception cycle when hCG values of > 10 mIU/ml were obtained on day 12 following embryo transfer and any day thereafter, indicating the beginning of implantation. A clinical pregnancy was defined as a conception cycle if there was at least one fetal sac with a positive heartbeat at ultrasound. BODY.METHODS.OUTPUT PARAMETERS: The main outcome parameters were fertilization and implantation rates, pregnancy outcome, and babies born rates. Fertilization rate was defined as the number of 2PN/number of oocytes (%); pregnancy rate as the number of patients who were hCG+/number of transfers (%); implantation rate as the number of gestational sacs/number of embryos transferred (%); clinical pregnancy rate as the number of patients who were hCG+ with at least one gestational sac/number of transfers (%); pregnancy loss rate as the number of pregnancy losses/number of patients who were hCG+ (%); ongoing pregnancy rate as the number of patients who were hCG+ with at least one gestational sac with fetal heart rate (FHR) after 12 wk/number of patients who were hCG+ or as the number of patients who were hCG+ with at least one gestational sac with FHR after 12 wk /number of transfers (%); and babies born as the number of babies born/number of transfers (%). For the analysis, the total biochemical pregnancies, miscarriage, and ectopic pregnancies were grouped under "pregnancy losses." BODY.METHODS.EMBRYO MORPHOLOGICAL SCORING SYSTEM: For embryo scoring, we took into account the number of blastomeres, the cell similarity, and the fragmentation rate. The fragmentation rate was classified into three groups: no fragmentation, less than 30% fragmentation, and more than 30% fragmentation. Embryos were scored by an experienced embryologist on the day of transfer. Multinucleated (MN) embryos where discharged and allocated to the group of no transferable embryo's (no fertilization, degeneration and MN). BODY.METHODS.STATISTICS: Data were analyzed using the GraphPad Instat program, version 3.06 for Windows. Fisher's exact test was performed. P < 0.05 was considered statistically significant. BODY.RESULTS: Patient and cycle characteristics are summarized in Table 1. A total of 172 patients were randomized: 87 for ISM1 and 85 for GM501. Five patients (four in the ISM1 group and one in the GM501 group) were excluded from the study because no transfer was performed; therefore, 83 and 84 transfers were performed for ISM1 and GM501 groups, respectively. There were no significant differences (P > 0.05) with regard to age, number of oocytes per cycle, number of embryos per transfer, and technique used for fertilization between the study group and the control group. Table I. Patient and cycle characteristics. Parameter ISM1 GM501 Age (mean ± SD) No. of cycles No. of oocytes No. of oocytes/ cycle (mean ± SD) 100% IVF (%) 100% ICSI (%) Mixed IVF/ICSI (%) 34.7 ± 5.0 87 652 7.5 ± 4.7 24.1 61.7 13.3 34.5 ± 5.1 85 595 7.0 ± 3.9 21.4 61.9 16.6 No. of fresh embryos transferred No. of embryos cryopreserved No. of transfers No. of embryos/transfer (mean ± SD) 124 177 83 1.5 ± 0.8 130 181 84 1.7 ± 0.8 Because no significant differences were found between the two age groups for any of the outcomes, we presented the information from both groups together. There were no differences between the two groups with regard to any of the outcomes of this study, i.e. fertilization, implantation and clinical pregnancy rates, pregnancy losses, ongoing pregnancies, and live birth rate (Table 2). When the pregnancy outcomes were analyzed depending on the day of the transfer, day 2 or 3, no significant differences between GM501 and the control medium were observed (Table 3). Table II. Patient and cycle characteristics. ISM1 GM501 No. of two pronuclei (2PN) Fertilization rate 446 68.4% (446/652) 433 72.8% (433/595 No. of hCG+ patients Pregnancy rate 27 32.5% (27/83) 23 27.4% (23/84 No. of gestational sacs Implantation rate 22 17.8% (22/124) 26 20% (22/130) No. of patients with at least one gestational sac with FHR Clinical pregnancy rate 22 26.5% (22/83) 19 22.6% (19/84) Number of pregnancy losses Pregnancy loss rate 8 29.6% (8/27) 5 21.7% (5/23) No. of ongoing pregnancies Ongoing pregnancy rate per hCG+ Ongoing pregnancy rate/transfer 19 70.4% (19/27) 22.9% (19/83) 18 78.3% (18/23) 21.4% (18/84) No. of children born 23 27.7% (23/83) 23 27.4% (23/84) Table III. Pregnancy, pregnancy loss, and ongoing pregnancy rates of embryos transferred on days 2 or 3. Day 2 Day 3 ISM1 GM501 ISM1 GM501 Age 33.6 ± 4.7 34.4 ± 5.2 35.3 ± 4.8 34.3 ± 4.8 No. of transfers 53 56 30 28 No. of patients hCG+ Pregnancy rate 19 35.8% (19/53) 17 30.4% (17/56) 8 26.7% (8/30) 6 21.4% (6/28) No. of pregnancy losses Pregnancy loss rate 7 36.8% (7/19) 4 23.5% (4/17) 1 12.5% (1/8) 1 16.7% (1/6) No. of ongoing pregnancies Ongoing pregnancy rate/transfer 12 22.6% (12/53) 13 23.2% (13/56) 7 23.3% (7/30) 5 17.9% (6/28) On the day of transfer, the best-looking embryos were chosen and the fragmentation rate was recorded (Table 4). No differences in the number and symmetry of the blastomeres were found between the embryos transferred in the two groups. However, fewer nonfragmented embryos (25.4% vs. 49.2%, p < 0.0001) and a higher percentage of minimally fragmented embryos, i.e., < 30% fragmentation rate, (70% vs. 47.6%, p = 0.0003) were available for transfer when GM501 was used. Table IV. Fragmentation rate of transferred embryos. Fragmentation rate ISM1 GM501 0% Fragmentation (% embryos) 61 (49.2%) 33 (25.4%)* < 30% Fragmentation (% embryos) 59 (47.6%) 91 (70%)* > 30% Fragmentation (% embryos) 4 (3.2%) 6 (4.6%) Total 124 130 *p < 0.05 vs. control group (Fisher’s exact test). BODY.DISCUSSION: Optimal IVF and embryo culture conditions depend upon several parameters, including temperature, stable environment, humidity, oxygen and carbon dioxide concentrations, medium pH, and composition of the culture media. The ideal culture medium must be designed to mimic the natural environment of the embryo; thus, its composition is a balanced blend of physiological components. Following the "back to nature" philosophy, media were developed based on the different environments in the fallopian tubes and uterus. To do this, these fluids were collected from these regions and their composition was analyzed. However, there are at least two confounding factors. First, there is fluid exchange between the fallopian tubes and the uterus that is not taken into consideration. As a consequence, the composition of the embryo's microenvironment cannot be clearly defined and most probably varies dynamically. Second, the collection of these fluids and the stabilization of the components until analysis are technically problematic. The "let the embryo choose" principle is based on the development of "computer-optimized media," which uses a mathematical model to optimize both the combination and concentration of various components. For example, KSOM medium is a variant of Simplex Optimization Medium (SOM), in which potassium chloride is added (Biggers, 1998). Later, essential and nonessential amino acids were added to KSOM to produce KSOMAA, which was successfully used in a one-step protocol to culture human zygotes to the blastocyst stage (Biggers et al., 2002). Gynemed GM501 is an optimized variant of KSOMAA medium; it is a novel medium, and thus no comparative data about its use in embryo culture was available. In this study, we found that good results were obtained using GM501 medium. Equal pregnancy rate, pregnancy losses, clinical pregnancies, ongoing pregnancy, and babies born rates were observed when GM 501 medium was used in comparison with ISM1. The pregnancy outcome was also evaluated depending on the day of transfer (day 2 or 3), and no differences were found between the media. Embryo selection based on morphological criteria provides a good correlation with embryo viability and pregnancy rates (Scott et al., 2008). Gordts et al. (2005) emphasized the importance of embryo selection under the restrictions of the Belgian legislation on embryo protection. These authors found that despite an increase in single embryo transfers (from 14% to 49%), consistently good pregnancy rates could be reached (36% vs. 37% for multiple and single embryo transfers, respectively). Thus, Belgian legislation on embryo protection, with its restrictive policy regarding embryo transfer, has not led to lower pregnancy rates. The conclusion that can be drawn from this is that the widely used morphological and biological criteria are sufficient to provide a prognosis for the developmental potential of embryos. The two parameters most critical to good-quality embryos are cleavage speed and fragmentation level (Cummins et al., 1986; Claman et al., 1987). That is, an embryo with the best prognosis would have four blastomeres the second day of development and eight cells in the morning of the third day, and not more than 20% of the embryo would be fragmented. Cytoplasmatic pitting can also have a negative influence on implantation outcome (Ebner et al., 2005). In our trial, better embryo morphology was observed with ISM1, because a higher percentage of non- or low-fragmented embryos were observed in this medium. A possible explanation for this may be differences in the media composition. In addition, ISM1 medium contains phenol red and GM501 does not; it was demonstrated previously that phenol red has estrogenic activity (Ortmann et al., 1990; Berthois et al., 1986). Whether phenol red might improve embryos morphology must be investigated in future studies. Although there is general agreement that a positive relationship exists between embryo quality and pregnancy rate, in our study, the pregnancy rate was the same for both media, although more fragmented embryos were transferred using GM501. These results are consistent with those of Aoki et al. (2005), who also demonstrated that regardless of the superior embryo morphology achieved with one of the media that they tested, there were no significant differences in implantation rates. There are at least two explanations that could account for the observation that equal pregnancy outcomes were achieved regardless of the differences in fragmentation rates at least up to 30%. First, the number of cells is more important than the fragmentation rate (Giorgetti et al., 1995; Ebner et al., 2003). Second, embryo fragmentation could be only a momentary state, because spontaneous lyses and resorption has been well documented (Hardarson et al., 2001; Van Blerkom et al., 2001). There is some debate as to whether, in addition to light microscopy, other non-invasive methods for judging embryo vitality should be used. Recently, several noninvasive techniques have been discussed, such as measurement of the usage or production of different metabolites, the respiratory activity of the embryo, and the kinetic events during the first cell division steps (Brison et al., 2004; Lopes et al., 2007). Recently, proteomics and metabolomics have also been used to assess embryo quality and developmental potential (Nagy et al., 2008). The use of image analysis systems (time-lapse recordings) to assess embryo developmental timing has also clearly demonstrated differences in the developmental potential of fast- and slow-cleaving embryos with the same cell number and morphological quality (Ramsing et al., 2007; Lemmen et al., 2008). Several of previous studies demonstrated that levels of glucose (Sallam et al., 2006) and oxygen consumption (Lopes et al., 2007), amino acid turnover (Brison et al., 2004), protein expression (Dominguez et al., 2008), metabolomic profiles (Nagy et al., 2008), and kinetics (Lemmen et al., 2008) of human embryos are significantly correlated with clinical pregnancy and live birth. Moreover, other non-invasive methods for judging the sperm should be used. Gianaroli et al. analysed the pattern of birefringence in the sperm during the ICSI to selectively inject acrosome-reacted and acrosome-nonreacted spermatozoa and they demonstarted that, althought there was no effect on the fertilizing rate and embryo development, the implantation rate was higher in oocytes injected with reacted spermatozoa in comparison with those injected with nonreacted spermatozoa (Gianaroli et al., 2010). It will be some time before these new technologies can be used as routine procedures; nevertheless, our data and that of previous studies indicate that embryo should be judged less by their morphology than by their content. BODY.CONCLUSION: in vitro culture of embryos in Gynemed GM501 media yielded pregnancy outcome results comparable to those of embryos cultured in ISM1, in spite of the higher embryo fragmentation rate in GM501. Because embryo culture in GM501 is a one-step procedure, and the medium has a long shelf life of 6 months, this product could represent a more practical and convenient medium for embryo culture in IVF laboratories. Scientific evidence is provided that the implementation of non invasive alternative methods to judge the embryo quality seems to contribute to a more accurate selection of the best embryo.

TITLE: A comparison between subpleural patient-controlled analgesia by bupivacaine and intermittent analgesia in post-operative thoracotomy: A double-blind randomized clinical trial ABSTRACT.BACKGROUND:: The efficacy of subpleural analgesia to reduce postoperative pain intensity in patients after lateral thoracotomy is controversial. In this study, we demonstrated the efficacy of two types of subpleural analgesia. ABSTRACT.METHODS:: This prospective, controlled, randomized, double-blind trial was performed in Department of Thoracic Surgery of Alzahra Hospital associated with Isfahan University of Medical Sciences from June 2009 until August 2010. After posterolateral thoracotomy and admission to the ICU, patients were randomly assigned into two groups of subpleural patient-controlled analgesia (SPCA) (0.02 cc/kg/h of 0.5% bupivacaine) and subpleural intermittent analgesia (SIA) (0.1cc/kg/6h of 0.5% bupivacaine). The data regarding age, sex, visual analog scale (VAS) (at 8, 16 and 24 hours after initiation of analgesia), morphine consumption, systemic adverse effects, length of ICU and hospital stay, complications, public health service (PHS) criteria, and cost was recorded. Data was analyzed by Mann-Whitney U-test, repeated measured test, chi-square test and the Fisher's exact test. A p < 0.05 was considered significant. ABSTRACT.RESULTS:: The study population consisted of 90 patients. There were no significant differences in sex, age, weight, intraoperative analgesics, duration of one-lung ventilation, and adverse effects between the SPCA and SIA groups. Although pain scores were significantly reduced at 16 hours after the first subpleural instillation of bupivacaine 0.5% with patient-controlled analgesia, comparison between mean pain scores in the two groups at 8 and 24 hours after the first subpleural instillation of bupivacaine 0.5% revealed no significant difference. In addition, no significant difference was found in VAS scores at the three evaluated times (p < 0.05). ABSTRACT.CONCLUSIONS:: Optimal use of SPCA bupivacaine for postoperative pain treatment is more effective in pain reduction than SIA bupivacaine. The consumption rate of opioid and bupivacaine was also decreased in SPCA group. BODY: Postoperative pain after thoracic surgery is the most important factor responsible for ineffective ventilation, ineffective cough, and impaired ability to sigh and breathe deeply. This may contribute to pulmonary atelectasis leading to ventilation/ perfusion abnormalities and hypoxemia, as well as infection, after thoracotomy. Although analgesia has the potential to reduce pulmonary morbidity, most analgesic techniques carry the risk of concomitant complications associated with systemic drug administration or placement of catheters (e.g., epidural catheterization). Therefore, the choice of pain therapy in thoracotomized patients is complex and controversial.123 Subpleural (SP) analgesia is induced by placing local anesthetic into the SP space which lies between the parietal pleurae and costal department. subpleural analgesia can produce regional analgesia of the chest wall and is used for pain therapy of different indications which include breast, renal, gall bladder and thoracic surgery, and chronic pain like intrapleural analgesia.4 In patients undergoing lateral thoracotomy, this technique has the advantage of intraoperative catheter placement under direct vision with a low risk for complications caused by catheterization. However, there is a controversy about its efficacy for pain relief after thoracotomy for pulmonary surgery.5–7 We compared subpleurally patient-controlled analgesia(SPCA) bupivacaine for postoperative pain treatment with subpleurally intermittent analgesia (SIA). Our objective was to determine whether SPCA bupivacaine affects postoperative pain relief, morbidity, and hospital stay. Furthermore, we analyzed the effects of sex and anterolateral or posterolateral approaches on postoperative pain. BODY.METHODS: This study was a double blind randomized clinical trial which approved by the institutional review board of the Isfahan University of Medical Science. Sampling method was done by two mean comparison sample size formula with type I error α < 0.05 and power 80% (Type II error β < 20%). Ninty Patients were included whom had indication for thoracic surgery. Patients with an allergy to bupivacaine, with postoperative air leak through the chest tubes, undergoing pleurectomy, having repeat thoracotomy, having full rib resection, or unable to cooperate were excluded from the study. Written, informed consent for participation was obtained. After premedication with midozolam, anesthesia was induced IV with 3 μg/kg fentanyl, 5 mg/kg thiopantal and muscle relaxation with 0.5 mg/kg atracurium. The tracheas of all patients were intubated with a double-lumen endobronchial tube. Anesthesia was maintained with 50% nitrous oxide in oxygen and isoflurane supplementation as required. During one-lung ventilation, inspired oxygen concentration was increased to 100%. Intraoperative systemic analgesia was limited to fentanyl in unrestricted doses. Pulmonary operations were performed at the fifth or sixth intercostal space through a standard posterolateral thoracotomy, respectively. Posterolateral thoracotomy extended from the anterior axillary line to a point midway between the vertebral spines and the vertebral border of the scapula.8 At the end of the surgical procedure, just before chest closure, the surgeon released parietal pleural from the chest wall interval between the second intercostal space and the ninth intercostal space and posteriorly on the paravertebral line and inserted an SP catheter percutaneously in this space. Then the margin of parietal pleurae was sutured to chest wall to close this space. After surgery, patients were ventilated with pressure support; all patients were tracheally extubated after rewarming, during the first 4 h after admission to the intensive care unit (ICU). After admission to the ICU, every participants had a code and based of this code, they were allocated to two groups randomly.. Just after, arrival patients in the SPCA group received 0.02 cc/kg/h of 0.5% bupivacaine subpleurally patient-controlled analgesia, whereas patients in the SIA group received 0.1 cc/kg/6h 0.5% bupivacaine subpleurally intermittent analgesia in 60 seconds. In the SPCA group, bupivacaine was administered via a patient-controlled analgesia (PCA) device (model 1000 Korea). The PCA device was programmed to provide a bolus of 0.005 cc/kg bupivacaine; the lockout time was 15 min. The contents of the syringes were prepared immediately before injection by a nurse who was not further involved in this investigation. Patients were kept in a supine position before injection of the study solution into the SP catheter. When the patients were awake, they were encouraged to take supplementary doses of morphine as much as the patient needed and the surgeon postulated. In both groups 2cc bupivacaine was subpleuraly administered at the end of operation. Data collection was commenced at 8, 16 and 24h after initiation of analgesia. They were asked to assess the intensity of the chest pain, at rest and when coughing, using a visual analog scale (VAS) (0–100 mm; 0 = complete pain relief and 100 = unbearable pain). At the same time, the morphine demand within the elapsed 8 h was recorded. During the study period, patients were evaluated for systemic adverse effects (i.e., drowsiness, confusion, dizziness, nausea, vomiting, hallucinations and drug sensitivity). We further recorded length of ICU and hospital stay, complications, such as pneumonia and atelectasis. All evaluations was done by different investigator which could perform blindness. It was a double-blind study. Statistical analysis: Data were analyzed by SPSS 15 software (SPSS Chicago, IL). The VAS scores of each patient were summarized as three VAS mean scores: Mean VAS at 8,16 and 24h after first injection of the study solution and mean of all. The dose of analgesics administered was compared between two groups, male and female patients using the Mann-Whitney U-test. Differences in the mean VAS scores within the groups after the administration of bupivacaine at 8, 16 and 24h after first injection of the study solution, were evaluated by using repeated measure of ANOVA. Differences between the groups for sex and postoperative complications were calculated by using the chi square test and the Fisher's exact test. Differences in numeric demographic data (e.g., age, and body weight), ICU and hospital stay were analyzed by using the Mann-Whitney U-test. A P < 0.05 was considered significant. BODY.RESULTS: 90 patients participated in the study. There were no differences in sex, age, weight, intraoperative analgesics, and duration of one-lung ventilation between SPCA and SIA groups (Table 1). Table 1 Patient Characteristics, Operation Data, and Postoperative Complications With regard to the outcome data, there were no significant differences between the SP-treatment groups comparing the incidence of adverse effects, such as pneumonia, atelectasis, bronchoscopic interventions, reintubations, hypersensitivity and postoperative ICU and hospital stay (Table 1) Reintubation of two patients (Table 1) was necessary after the study was terminated. One patient was reintubated in the SPCA group, with suspected pulmonary embolism, and one patient with pneumonia was reintubated in the SIA group. Mean pain scores were significantly reduced at 16 h after the first SP instillation of bupivacaine 0.5% with patient-controlled analgesia. However, there was no difference between the groups when comparing mean pain scores at 8 and 24h the first SP instillation of bupivacaine 0.5% (Figure 1). When VAS scores were analyzed duration 24h, there were also no differences between the groups. There weren't any differences between sex and VAS scores at three times. (p<0.05) Figure 1Visual analog scale (VAS) mean scores at 8,16 and 24 h after the first SP instillation of bupivacaine 0.5% as patient-controlled analgesia (SPCA group) and intermittent analgesia (SIA group) respectively. Treatment significantly reduced mean pain scores in both groups, however, no differences in pain scores were observed between Groups SPCA and SIA duration 24h. VAS mean score at 16 h after the first SP instillation of bupivacaine 0.5% in SPCA group was significantly reduced to SIA group. Supplemental opioid demand, PHS criteria and bupivacaine consumption were statistically significant, as, rate of IV opioid consumption, PHS criteria and bupivacaine consumption in SPCA group was significantly lower than in SIA group. (Table 2). Table 2 Morphine consumption, initial consumption, PHS criteria, cost and bupivacaine consumption in two groups BODY.DISCUSSION: The aim of postoperative pain management is to provide good subjective comfort and to contribute to early recovery and a good outcome after surgery. After thoracotomy, pain therapy with systemic opioids has the potential for a good pain relief at rest with a lack of effective pain reduction when coughing or breathing deeply. However, effective coughing is necessary for a sufficient bronchial clearance to prevent atelectasis and bronchopulmonary infection. Therefore, many attempts have been made to combine systemic drug administration with different kinds of regional anesthesia to improve postthoracotomy pain control.910 Intrapleural analgesia has studied more than subpleural analgesia but Silomon suggested that Interpleural analgesia does not influence postthoracotomy pain.11 Subpleural analgesia use has increased follow publication of its excellent effect on postthoracotomy pain.912 The mechanism of action appears to be through a multilevel intercostal nerve blockade.12 If the catheter is placed intraoperatively under direct vision, this technique is safe and easy. Despite its wide use, systemic absorption and toxicity from local anesthetics have not been substantiated in clinical studies that assayed plasma levels, even when using larger amounts of bupivacaine than we administered.13 in comparison to intrapleuraly use that may lead to blood levels in the toxic range.2 Furthermore, Richardson et al.14 suggested a chest tube loss of bupivacaine after intrapleural(IP) administration, and IP local anesthetics may actively induce diaphragmatic and abdominal muscle weakness and cause impairment of respiratory function.1415 There are two methods for installation of drug subpleuraly, subpleurally patient-controlled analgesia (SPCA) and subpleurally intermittent analgesia (SIA).we compare these two methods about their effects. The current study indicated a significant pain reduction 16 hours after subpleural injection of bupivacaine 0.5% in SPCA method in comparison to SIA method. Furthermore, we could not show any difference in mean VAS scores after 8 and 24 hours.VAS scoring was shown to be a valid measure of pain in the early postoperative period.16 Chung showed that Subpleural block with 20 ml 0.5% bupivacaine can be an alternative approach of regional anesthesia in patients with multiple rib fractures.4 Koehler recommended that continuous administration of local anesthetics directly in the subpleural plane, posterior to the intercostal incision, provides excellent pain control.17 The cause of various effects of analgesia in SPCA group possibility related to various dose of bupivacaine that use by patients. Since interval 8h until 16 h after the first SP instillation of bupivacaine, the patients used bupivacaine optimal and duration first 8h and after 16h because of incompletely consciousness and sleeping they didn't use bupivacaine optimal. We showed that bupivacaine consumption was significantly lower in SPCA than SIA group. Furthermore, supplemental analgesia was significantly reduced in SPCA group and patients received opioid lower than SIA group at their request. Like other study, there are a few studies that support effectiveness of the postoperative pain management via SP analgesia after thoracotomy. A dilution of the local anesthetics by pleural exudation doesn't appear to play a subordinate role in SP analgesia that seen in IP analgesia.14 In the current study the posterolateral thoracotomy was shown to be the more painful approach that may be explained by greater damage to the latissimus dorsi muscle18 and more stress on the costovertebral joints, with wide rib retraction near the spinal column. These anatomical factors may contribute to the pain intensity at rest; however, they appear to have less influence during the dynamic process of coughing. The effect of sex on postoperative pain intensity was opposite to recent studies showing sex to be an important variable in recovery from general anesthesia.19 The possible mechanisms leading to higher pain scores after surgery in women as shown are not fully understood.20 Because pain during coughing and therefore, the ability to cough, was not influenced by sex, a clinical relevance of sex differences appears to be questionable. This conclusion is supported by the outcome data showing no differences between male and female patients. However, further studies dealing with postoperative pain measurement should pay special attention to the distribution of sex within subsamples. Many studies have shown post-thoracotomy pain relief enhanced quality of life of patients and reduced morbidities.21–23 Patient-control analgesia after thoracic surgery has been consider because of safety, patients ' satisfaction.2425 It has been shown that Bupivacaine and the similar drugs are appropriate for post operative analgesia. In our study we could find that subplural patient-control analgesia was better clinically not statistically in patients with post-thoracotomy pain relief. Other studies have supported our result approximately.2126–28 Additionaly we can reduce the consumption of morphine in SPCA method. Thus we can run SPCA as a safe method in our clinical practices. BODY.AUTHORS’ CONTRIBUTIONS: VG designed the study and drafted this manuscript, SAT, SMH, GRM Carrried out the study and drafted the manuscript, MAR, ZME, FSH, helped in Statistical analysis, revising the manuscript procedures.

TITLE: Promoting sexual abstinence intention among female university students: A quasi-experimental study ABSTRACT.BACKGROUND:: The effectiveness of a theory-based educational intervention on intension for sexual abstinence among female university students was evaluated. ABSTRACT.MATERIALS AND METHODS:: Female students were recruited from humanity sciences department through cluster sampling. Educational intervention was applied for four 90-min sessions and by application of cognitive theories during 4 weeks. ABSTRACT.RESULTS:: One hundred and nine female students with mean age of 20.74 ± 1.57 years took part in the study. Despite the similarity of two groups of intervention (n = 53) and control (n = 59) at baseline, there were significant differences between the two groups in mean scores of the variables, knowledge (4.62 ± 1.38 vs. 3.53 ± 1.61), perceived susceptibility (14.05 ± 1.51 vs. 12.37 ± 2.11), and perceived benefits (28.41 ± 2.14 vs. 27.51 ± 3.05), at follow-up time after 3 months (P < 0.05). Additionally, these variables were observed with improvement over 3 months in the intervention group (P < 0.05). However, this study showed no significant effect on the behavior intention and self-efficacy. ABSTRACT.CONCLUSION:: This study showed that educational intervention could improve knowledge, perceived benefits, and self-efficacy of the female students regarding HIV/AIDS. BODY.INTRODUCTION: Throughout the history of human beings, HIV/AIDS has been one of the greatest challenges that have made the world unable to find an effective cure.[1] Since its discovery, over 25 million lives have been lost in just 30 years.[2] Thus, the mortality and morbidity rate of this disease remains high, especially in developing world.[3] The first case of HIV infection in Iran was reported in 1986 which was a child diagnosed with hemophilia. However, the spread of the HIV epidemic in Iran has been ever increasing since that time.[4] According to the center of disease management of Iran, the incidence of newly reported case of HIV was found to be highly increasing.[5] According to the world reports, about 50% of the recent HIV infections are amongst the youth of age range 15-24 years.[6] Iran, a country with mostly young population, is at risk of increased HIV rate. Reports from the Center of Disease Control of the Ministry of Health and Medical Education of Iran showed that the rates of risky behaviors, which could lead to HIV/AIDS infections, are increasing rapidly. The study conducted in 2006 indicated that 28% of teenage boys living in Tehran verified their unsafe sexual behaviors.[7] Attempts to control the disease have concentrated on the preventive behaviors, so education and preventive strategies to change unhealthy behaviors have been considered as the first priorities.[8910] Preventive strategies toward HIV/AIDS are directed by some healthy behavior changes based on theories and models.[11] Most of the previous educational interventions were based on theories such as social cognitive theory (SCT), theory of reasoned action (TRA), and health belief model (HBM).[12] Most theories of healthy behavior regard cognitive determinants of the HIV risk such as knowledge, attitude, and behavioral intention to address preventive strategies.[13] Previous studies revealed that educational interventions could improve knowledge, self-efficacy, attitude, behavioral intention, and also behavior of condom using.[14151617] However, Walker's study showed education could not improve condom using behavior.[18] Educational and inspirational interventions suitable to the youth's cultural values had been the focus of the previous study.[19] For instance, in the study conducted in Iran, defining sexual health program based on specific needs of Iranian women due to their information and concerns was verified.[20] On the other hand, since in Iran due to some cultural restrictions (considerations), the majority of health improvement intervening studies regarding HIV/AIDS have been focused on male students rather than female, in this study, the female students were selected as the target group. On the other hand, evidences show that female gender is more physiologically vulnerable to be infected with HIV/AIDS. Thus, women are at higher risk to the consequences of HIV/AIDS, compared to men.[21] Furthermore, it has been documented while the educational interventions are in consistence with the culture and being designed to meet needs of a target population, it would be more likely to get positive results.[22] As previous evidences reported, more than half of new AIDS/HIV infection cases are in the age range of 15-24 years,[6] and this age group is mostly found in universities. However, due to its centrality and being regarded as the mother university, the University of Tehran was chosen as the place of the study. Moreover, as all the students of the University of Tehran were studying in three majors of humanities, engineering, and fundamental sciences, and also 60% of them were studying in humanity science at the time of the study, the subjects of this study were selected from this faculty. This study was designed to investigate the effects of the educational intervention based on social cognitive theory on knowledge, self-efficacy, perceived benefits, perceived susceptibility, sexual abstinence intention, and refusal intention of female students of Tehran university. BODY.MATERIALS AND METHODS.STUDY SETTING: This study was a quasi-experimental research that was conducted among female students of Tehran University in Tehran, Iran, from 2009 to 2010. To be eligible for the study, the students had to be aged between 15 and 25 years, single, living in Tehran, and also be willing to take part in the study. Students who reported to be suffering from any disabilities or infectious disease were excluded from the study. The study was approved by the ethics committee of Tehran University and ethical principles were adhered to throughout the study with research project number 181. Participants provided informed consent, confirmed in writing, after they were explained the purpose and procedures of the study. Using cluster sampling, among all faculties of humanity sciences, two faculties of law and literature were randomly selected; then from these two faculties, two classes, one class from law faculty and the other class from literature faculty, were selected randomly. Finally, all female students of the classes who met the inclusion criteria and were willing to enter the study were chosen. In this study, the participants from law faculty were considered as experimental group and the other students were labeled as control group. The interviewer who collected data and the statistical analyst were masked to the group assignment, while the participants were instructed to say nothing about their group assignment to the interviewer. Because of the nature of the intervention, full masking of participants was impractical. A total of 109 participants were enrolled in the study as intervention group (n = 53) who received educational intervention plus routine education or control group (n = 56) who received just routine education. BODY.MATERIALS AND METHODS.DATA COLLECTION: A basic demographic questionnaire as well as a questionnaire generated based on the previous studies[232425] were administered to both groups at two points of time as baseline and follow-up. To check the validity of the questionnaire, a group of 20 experts who were specialists in the fields of nursing, health education, infection science, and behavioral psychology rated the questionnaire. Their answers were "essential," "useful," or "not necessary" for each item. The number of "essential" ratings for the items was calculated. The formula CVR = [(E − (N/2))/(N/2)], using the total number of experts (N) and the number who rated the items as essential (E), was applied to determine the content validity ratio (CVR). Of all primary questions of the questionnaire, three self-efficacy questions, three perceived susceptibility questions, nine perceived benefit questions, and three behavioral intention questions were eliminated due to their low CVR of <0.42. CVR for the 50-item questionnaire was calculated as 0.84. The final version of knowledge scale was provided with three questions rated through 3-option Likert system of yes, no, and not sure. Each option of yes or no was scored as 2 and not sure scored as 1. Thus, the whole knowledge scale was scored in the range of 0-6, in which 0 was the worst and 6 was the best score. Other scales used 5-option Likert system which were scored from 1 for strongly disagree to 5 for strongly agree. In this scoring, behavioral intention questionnaire with four questions was scored from 4 to 20, self-efficacy with three questions was scored from 3 to 15, perceived benefits with six questions was scored from 6 to 30, and perceived susceptibility was scored from 5 to 25. The assigned time for completing the final 50-item questionnaire was 6-7 min that was confirmed by the researcher and the experts who reviewed the questionnaire. To determine reliability, a test–retest with a time interval of 2 weeks was administered to knowledge, self-efficacy, and behavioral intention questions, and the results were satisfactory (r = 0.73, r = 0.79, and r = 0.87, respectively). Cronbach's alpha or internal consistency was 0.87 for perceived benefits, 0.77 for behavioral intention, 0.85 for self-efficacy, and 0.70 for perceived susceptibility, which confirmed the scale. The questionnaires were consciously and willingly administered to the anonymous participants. BODY.MATERIALS AND METHODS.INTERVENTION: Firstly, the students in both groups completed the self-administrated questionnaires. Then an educational program based on a course plan for teaching about HIV/AIDS[26] in accordance with gender issues of the female students was educated by the first author. As it is documented, intervention for HIV/AIDS prevention targeted on females should be gender based and appropriate with respect to the subjects' sex.[27] The aim of the educational intervention was to increase the intended behavior of sexual abstinence and refusing risky offers through increasing subjects' knowledge, perceived benefits, and susceptibility. Educational intervention was applied through four 90-min sessions during 4 weeks, based on the selected mentioned variables and by applying the designed educational intervention. The educational methods included lectures with power points, question and response sessions, group discussion, and team work. In order to have the ultimate effects on subjects, the intervention was student based and involved the students' participation actively. The teaching materials were on the basis of female students' needs. In the first session, the learning strategies regarding knowledge awareness promotion about HIV/AIDS and risky behavior were applied. By asking the key question "Have you ever thought about HIV/AIDS?" at the beginning of the first session, the students were asked to think about AIDS. Then, they were taught about HIV/AIDS epidemiology, its global statistics, and ways of identifying the disease, using audiovisual equipments. Afterward, the students were given a paper and asked to write the ways of HIV/AIDS transmission and prevention in two separate columns. In the second session, educational strategies about improving attitude toward abstinence and refusing risky offers were discussed. The students in groups of four to five persons discussed the task. At the end of the group discussion, representatives of each group presented the achieved results to the rest of the students, and then the researcher explained the vague points and answered the students' questions. Also, the ways of transmission and disease cycle were presented and their misconceptions were corrected. For instance, there were some misconceptions like "anal sex does not lead to HIV/AIDS," "by having sex once, a person will not be infected," or "only pervert people become infected"; therefore, some explanations were given. Furthermore, in the second session, through the prevention ways, the students were asked to talk about their feelings toward sexual abstinence. For providing the students with clear and more precise prevention techniques, extracts from the Holy Quran verses and religious literature were used, which brought about a kind of religious support. Also, the address of the behavior consulting center and its services was presented. During the third session, discussion about perceived susceptibility and perceived risk was offered and it was also discussed that women and girls are more vulnerable to the infection. They were asked to think about the reasons of this potentiality and write their opinions on a paper. Early prevention interventions that have positive effects on behavior, intention, attitude, and beliefs include self-efficacy increasing programs.[28] Therefore, in the fourth session, the focus was on the construct of self-efficacy and teaching materials concentrating on self-efficacy, refusal skills, assertiveness, avoiding risky situations, and abstinence; also, there was an elaborate speech on the techniques of how to say "no." As Bandura discussed, perceiving self-efficacy increases through four ways: Success in action, persuasive discourse, replacement experience, and physiologic or emotional states.[29] In the educational sessions, discourse intriguing and making the students sure about possessing expose skills and decisiveness were employed frequently. Religious and cultural, worthiness of being virtuous were discussed repeatedly. Also, the skill of self-efficacy was emphasized by narrating a scenario. The scenario was about a girl student who suggests her friend to go to a party, but she refuses with decisive answers. Afterward, the participants were asked to write a short story about a girl who was experiencing a high-risk situation, and to analyze how she could avoid such situation. This task provides them with replacement experience. At the end, a multimedia educational package (CD) and a book with the name "Healthy Life" were distributed among the participants. In addition, the educational movie "Born with HIV/AIDS" was shown for them, which focused on several guiding points for girls. Three months after applying the educational program, the same questionnaires except for demographic questions were administered to the intervention and control groups. The final analysis through comparing the two groups was done to determine how effective the educational intervention was on the desired variables. The control group did not undergo any intervention in their regular curricula. However, due to ethical considerations, the materials were presented to the control group at the end of the study. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSES: The results were analyzed by SPSS 14, t-test, paired t-test, Mann–Whitney U test, and Wilcoxon signed-rank test, with a significance level of 5%. BODY.RESULTS: The mean age of the subjects was 20.74 ± 1.57 years; among them 35.7% (n = 41) were Tehran dwellers. First, the variables' distribution was investigated by Kolmogorov–Smirnov test, where it was found that variables of age and perceived susceptibility construct had a normal distribution while the other variables did not. As the findings demonstrated, the students' fathers' educational background in 41.5% (n = 21) of the experimental group and 42.9% (n = 24) of the control group were above high school diploma, while the value were 18.9% (n = 10) and 23.6% (n = 13) for mothers of participants in the experimental and control groups, respectively. The Mann–Whitney test did not show any significant difference here. The majority of the participants reported their family income to be in a stable level and there was no significant difference between the groups regarding this variable. In regards to familial dimension, Mann–Whitney did not demonstrate any significant difference either. Therefore, both groups were similar in these background variables. Furthermore, with respect to pre-test variables level, the two groups did not have any significant difference and a considerable number of participants admitted that their information about HIV/AIDS was at an average level [Table 1]. Table 1 Comparison of mean scores of constructs in intervention and control groups pre-test and post-test separately using t -test and Mann–Whitney U test In order to make a comparison between the intervention and control groups, they need to be compared with regard to their similarities. No significant statistical differences existed between the two groups with regards to mean scores of independent study variables before the intervention (P > 0.05). In fact, both the groups were consistent with regard to all variables. Table 2 shows a significant difference in the intervention group between two time points of before and after intervention with regard to the variables of knowledge, perceived susceptibility, and perceived benefit (P < 0.05). Table 2 Comparison of mean scores of constructs in intervention and control groups pre-test and post-test using Wilcoxon signed-rank test According to Table 2, the results of the data in two stages of before and after intervention showed no significant difference in the control group. BODY.DISCUSSION: The prevalence rate of HIV/AIDS is increasing,[30] and this increase has attracted the researches' attention to examine the knowledge and attitude of individuals and community groups.[31] The findings of this research indicated that knowledge score in the pre-test for both groups was at an intermediate level, which reached to a desirable level after the intervention. In both groups, the pre-test scores of perceived benefits were above half of the questionnaire's obtainable scores, which were at a favorite level. The scores of perceived susceptibility in both groups were at a moderate level. Behavioral intention and perceived benefits' scores were higher than the others. The results of this study showed improvement of intervention group regarding knowledge, perceived susceptibility, and perceived benefit after educational intervention, which is an indicator of the positive effects of the gender-based and culturally based educational intervention. As these differences were not found in the control group, it is highly likely that educational intervention was the reason for these effects. Also, the results indicated that HIV/AIDS education can result in an increase of HIV knowledge of the female students in the intervention group, whereas in the control group, no such increases were found. Previous school-based interventions showed these increases in the knowledge of study populations as well.[1232] However, knowledge cannot predict a healthy sexual behavior on its own. For instance, in a study by Davis et al. (2007) on African-American students, it was found that more knowledge, lower age, sexual contacts with more than one partner, positive attitude toward condom use, more personal behavioral skills, and male sexuality were the main predictors of condom using behavior.[33] Results of the present study showed that perceived benefits before the educational intervention in both groups were at favorite levels, and designed education was successful in having a positive and significant effect on the perceived benefits of sexual abstinence and avoidance of risky situation. In some other theory-based studies, the same results were obtained.[343536] One study carried out in Iran by Eshrati et al. (2008)[37] also confirmed this finding. In a study conducted on prisoners which was aimed to investigate the effects of the risk reduction education according to the HBM, it was revealed that the only model construct correlating with decrease of high-risk behavior was the perceived benefit. As a result, when the prisoners were convinced of the effectiveness of risk reduction strategies, they decreased their high-risk behaviors. Another objective of the present study was to determine and compare the self-efficacy of sexual abstinence and avoidance of high-risk situations in female students before and after the intervention in the intervention and control groups. In the pre-test of both groups, self-efficacy scores were above half of the questionnaire's obtainable score, which was at a favorable level. After the education, both groups did not show a significant difference in the self-efficacy scale, which contradicts some other study results. For example, an educational intervention based on SCT was carried out in HIV-positive patients in the State of Georgia of USA to reduce their high-risk behaviors. In that study, education had a significant effect on reduction of unprotected sex, condom use behavior, and self-efficacy of suggesting using condom to a new sexual partner.[38] Furthermore, in a study on pregnant African-American adolescents, negotiating and refusal skills had significant effects on self-efficacy in avoiding high-risk sexual behaviors; the author regards this effect as a result of the cultural context of this group.[39] Also, in a randomized control trial (RCT) carried out on couples in California, which was done in three sessions according to Fishbein's integrated behavior change model and Information, Motivation, Behavior Skills(IMB) model, education had positive effects on self-efficiency of condom use.[40] Perhaps this difference is due to the fact that education can have different effects in different cultural contexts. Students claimed that being constantly told to say "No" and creating a sense of fear of the opposite sex might have been the reasons why the researcher's education was unable to increase self-efficacy. Besides, it can be argued that educational interventions to increase self-efficacy which are combined with behavioral skills should most likely be carried out over a long period of time and continuously. With regard to the behavioral intention, in the pre-test of both groups, its scores were above half of the questionnaire's obtainable score, which was at a high favorite level; thus, education had no significant effects on its increase in the control group. When people are asked about behaviors which are not along with common ethics of society, the answers get close to what is approved by the society; this is true among girls since our community has passed more strict rules and customs for them compared to the other gender. Also, this behavioral intention was high at the time of pre-intervention and concordantly one must not expect a significant increase. There are similar results about intention in other overseas studies. For instance, in a school-based study in Ukraine, no increase was observed in intention to refuse sexual behaviors among students after a six-session intervention.[41] Also, in the study conducted by Morrison-Beedy (2005), HIV/AIDS risk reduction group-based education based on the IMB model had no significant effect on the behavioral intention of HIV/AIDS risk reduction (i.e., using condoms, not taking drugs and alcohol before sexual activity) among teenage girls. Nevertheless, like the present study, it had a significant effect on knowledge, motivation, and reducing negative aspect of using condoms, which emphasizes the importance of prerequisites to behavior change. Crepaz, et al. concluded in their meta-analysis that successful interventions would be theory-based ones and those which were based on ethnographic research and focused on building skills. They also revealed the intervention should be in accordance with the cultural context.[1] There are some limitations in this study, such as small sample size and specific participants among Islamic culture should be considered in interpreting of the results. In the present study, the impact of the intervention was investigated after 3 months, though more precise long-term investigations for 6-12 months are required to indicate the effect of such educational intervention. BODY.CONCLUSION: This study has shown that educational intervention could improve knowledge, perceived benefits, and self-efficacy of the female students regarding HIV/AIDS.

TITLE: Popliteal sciatic nerve block versus spinal anesthesia in hallux valgus surgery ABSTRACT.BACKGROUND: We compared clinical properties and patient satisfaction between spinal anesthesia and popliteal sciatic nerve block (PSNB) for hallux valgus surgery. ABSTRACT.METHODS: Forty patients undergoing hallux valgus surgery were divided into spinal group (spinal anesthesia with 2.5 ml of 0.5% bupivacaine [n = 20]) and PSNB group (PSNB with 30 ml of 0.75% ropivacaine mixed with 10 ml of normal saline solution using a nerve stimulator [n = 20]). The PSNB group used a patient-controlled-analgesia (PCA) pump for postoperative pain control. The quality and side effects were compared between the two groups. A questionnaire was used to evaluate patient satisfaction with the use of anesthetic techniques and postoperative pain control in the PSNB group. This study was assessed 3 days postoperatively by a blinded observer. ABSTRACT.RESULTS: Procedure time and time from anesthesia until start of sugery were significantly shorter in the spinal group than those in the PSNB group (P < 0.01). Anesthesia-related complications such as hypotension, bradycardia, shivering, nausea/vomitting, post-dural puncture headache (PDPH) and urinary retension were observed in 15%, 10%, 5%, 5%, 10%, and 20% of patients in the spinal group, respectively. PSNB was not associated with these complications. Patient satisfaction was slightly higher for PSNB than for spinal anesthesia. In the PSNB group, patient satisfaction with postoperative pain-control was 95% above ordinary satisfaction. ABSTRACT.CONCLUSIONS: Despite the long duration of the procedure, PSNB is relatively safe, provides an adequate level of anesthesia, effectively controls postoperative pain and reduces side effects. Therefore, PSNB could be a potential anesthetic technique for hallux valgus surgery. BODY.INTRODUCTION: Hallux valgus is one of the most common foot deformities. The condition manifests with the hallux presenting with the lateral valgus of the first metatarsophalangeal joint, and the first metatarsal bone is tilted toward the anterior of the second metatarsal bone. The incidence of hallux valgus is highest in females between the ages 40 and 50 [1]. Surgical treatment is performed for patients with severe pain and symptoms. Several anesthetic methods can be chosen, including local anesthesia, nerve block, regional anesthesia, general anesthesia, and, most commonly, spinal anesthesia. Since McCutcheon's [2] report on the use of regional anesthesia for foot surgery in 1965, regional anesthesia has been going through various improvements. In 1992, Myerson et al. [3] reported improved outcomes with anesthesia and found that about 70% of foot and ankle joint surgeries can be carried out with nerve block. It has been reported that peripheral nerve blocks reduce the cardiovascular side effects associated with regional or general anesthesia [4,5]. Among various nerve blocks, popliteal sciatic nerve block (PSNB) has the disadvantage of causing a minor effect on the saphenous nerve territory. However, the area subjected to anesthesia for hallux valgus surgery is mainly affected by the common peroneal nerve territory. In recent years, PSNB has been frequently used for hallux valgus surgery. In this study, the authors aimed to determine the difference in clinical significance and patient satisfaction between PSNB and spinal anesthesia in prospective hallux valgus surgery. BODY.MATERIALS AND METHODS: Patient consent was obtained following authorization from the institutional review board, and 40 patients were selected as the study subjects. The subjects were aged from 20 to 65 years, grade 1 or 2 according to the American Society of Anesthesiologists physical status classification, and due for elective orthopedic surgery. The following patients were excluded from the study: those with spinal malformation, nerve system conditions, regular administration of medication due to medical or surgical disease, hemostatic disorders or infection at the anesthetic injection site. Prior to surgery, an 18 G venous catheter was placed in the left arm to provide fluid during surgery. Prior to administration of anesthesia, a non-invasive blood pressure monitor was placed around the patient's right brachium, and an electrocardiogram and a pulse oximetery were applied continuously. After 5 minutes of rest in the supine position, the blood pressure and heart rate were measured and set as baseline values. The spinal anesthesia group (n = 20) was administered with 2.5 ml of 0.5% hyperbaric bupivacaine (Marcain Spinal 0.5% Heavy®, AstraZeneca, Sweden), and the PSNB group was administered with a mixture of 10 ml of normal saline and 30 ml of 0.75% ropivacine (Ropiva Inj 7.5 mg/ml, HanLim Pharm, Seoul, Korea). Spinal puncture was carried out in the spinal anesthesia group. A 26 G Quincke needle (Hakko Co. Ltd, Chikuma, Japan) was used to puncture the location at the central line of the third and fourth lumbar in the lateral decubitus position, and 2.5 ml of 0.5% hyperbaric bupivacaine was slowly administered after making sure that clear cerebrospinal fluid had been drawn out. The patient was then changed to the supine position, and the sensory block was confirmed through the pin-prick method with a 26 G syringe needle. After confirmation, surgery ensued. The chosen needle insertion site for the PSNB group was the point 60 mm upward from the popliteal fossa crease and 10 mm to the left of the central line while the patient was in the prone position (Fig. 1A). An 18 G 100 mm needle coated with an insulator (Contiplex® set, B. Braun, Melsungen, Germany) was used with a nerve stimulator (Digistim 3 plus, Neurotechnology Inc, USA) in the stimulating mode. The popliteal area was disinfected with Betadine and alcohol, and the needle was inserted after preconditioning with local anesthetics. The nerve stimulation mode was activated, followed by connection of the nerve stimulator. Stimulation intensity started at 1.0 mA. While checking foot inversion or dorsiflexion, the intensity was gradually reduced to access the needle around the nerve area. When the muscle maintained minimal contraction under the intensity of 0.5 mA or less, this value was set as the minimum stimulation intensity (Fig. 1B). The anesthesia syringe was reversed to ensure there was no backflow of blood, and then the anesthetic was slowly injected. A catheter was inserted through the previously inserted needle and moved 3 cm forward from the end of the needle. The needle was then slowly removed while exercising care to maintain the position of the catheter. A catheter connector and filter were attached, and a sterile bandage was placed at the insertion site on the skin to fix the catheter so that it does not get removed. Sensory loss was confirmed through the pin-prick test, and surgery proceeded after placement of the calf tourniquet. The duration of the spinal anesthesia procedure was measured from the moment of needle insertion for spinal puncture to completion of administration of the spinal anesthetic. In the PSNB group, the time was measured from the point of local anesthetic infiltration in the skin until fixation of the perineural catheter. Thereafter, the time taken for the block was measured from the moment of administration of spinal or regional anesthesia. For postoperative pain control, a PCA pump (Accumate 1000, Wooyoung Medical, Seoul, Korea) was connected (basal rate: 10 ml/h, bolus: 2 ml, lock out interval: 30 min) to administer a mixture of 50 ml mepivacaine (Mevan Inj, HanLim, Seoul, Korea) and 150 ml of normal saline solution. The PCA provided pain block for up to 1 day after surgery, and it was not refilled when all the medication was used. Extra analgesics and sedatives were administered relative to the degree of the block. About 50-100 μg of fentanyl was administered IV bolus if the patient complained of pain, and 2 mg of midazolam was administered intravenously if the patient complained of anxiety. During surgery, 5-10 mg of ephedrine was administered IV bolus when the systolic blood pressure fell below 100 mmHg, and 0.5 mg of atropine was administered intravenously when bradycardia occurred (heart rate < 50 BPM). A survey was carried out 3 days post-surgery by a physician who was not involved in this study to allow for objective assessment. The survey questions (yes/no) included unpleasant feeling during surgery, anxiety during surgery, lumbodynia or pain at the popliteal needle insertion site, and pain under the discission. The satisfaction level regarding the anesthesia procedure was surveyed with 5 classifications: very satisfied, satisfied, average, dissatisfied, very dissatisfied. The satisfaction level regarding PCA, which was used in the PSNB group for up to 1 day post-surgery, was also surveyed with 5 classifications: very satisfied, satisfied, average, dissatisfied, very dissatisfied. The sample size calculation was based on the difference in the incidence of side effects. In the pilot study with 20 patients, the spinal anesthesia group had a total of 3 patients (30%) who experienced hypotension, nausea and vomiting, and postdural puncture headache (PDPH), respectively. No side effects were reported in the PSNB group. The difference in the incidence of side effects in each group was 30%. The sample size for each group was calculated as 17 patients when the alpha value was 0.05 and the power 80%. When the elimination rate was set up as 20%, the valid sample size was calculated to be 20 patients per group. SPSS version 12 version was used for statistical procedures. Student's t-test and the χ2 test were used to analysis statistical validation in each group. Statistical significance was defined as a P value of less than 0.05. BODY.RESULTS: There were no differences in age, sex, weight, and height between the 2 groups (Table 1). The duration of the procedure, time from anesthesia induction until start of surgery, and surgery time were shorter in the spinal anesthesia group than in the PSNB group (P < 0.01). The anesthesia-related side effects in the spinal anesthesia group were hypotension, bradycardia, shivering, vomiting, postoperative headache, and urinary retension, occurring in 15%, 10%, 5%, 5%, 10%, and 20% of patients, respectively. There was no incidence of side effects in the PSNB group, but it was not statistically significant. Fentanyl, which was used as an extra analgesic, was used in the PSNB group, but not in the spinal anesthesia group. The use of midazolam was more frequent in the spinal anesthesia group than in the PSNB group, but there was no statistical significance (Table 2). According to the survey, unpleasant feelings during surgery and anxiety were higher in the spinal anesthesia group, but there was no statistical significance. Back pain was present in 25% (5 cases) of patients in the spinal anesthesia group, and pain at the popliteal needle insertion site occurred in 10% (2 cases) of patients in the PSNB group. Incisional pain was not reported in the spinal anesthesia group but was reported by 15% (3 cases) of patients in the PSNB group; however, these figures were not statistically significant (Table 3). In the satisfaction survey, 60% (12 cases) of the spinal anesthesia group and 85% (17 cases) of the PSNB group responded that they were satisfied with the anesthesia procedure (including those who responded 'average'), although the results were not statistically significant (Table 4). The satisfaction survey regarding pain control after the catheter insertion procedure in the sciatic nerve in the PSNB group showed the following results: satisfaction 50% (10 cases), average 45% (9 cases), and dissatisfaction 5% (1 case) (Table 5). BODY.DISCUSSION: This study, which was conducted on patients who underwent hallux valgus surgery, resulted in a longer duration for application of anesthesia and longer surgery time in the ropivacaine-administered PSNB group relative to the spinal anesthesia group. However, the PSNB group was associated with a low incidence of anesthesia-related complications, relatively high patient satisfaction with the anesthesia procedure, and effective post-surgical pain control. The PSNB procedure was first described by Labet in 1923, but it has not been commonly practiced due to limits in the area of application of the block, high incidence rates of side effects including dysesthesia, and problems with the reliability of the block on sciatic nerves or its branches [6]. However, recently, PSNB has been attempted quite often as regional anesthesia for surgeries or post-surgical pain control because its value and reliability are being reassessed in accordance with improvements in insulator needles and nerve stimulators [7]. Moreover, the time taken for the complete block of PSNB has been shortened, and the success rate has been increased due to the use of nerve stimulators or ultrasound. Also, there are several reports with regard to the success rate of PSNB associated with exercise reaction with the use of a nerve stimulator. Sukhani et al. [8] reported that inversion and plantar flexion require a shorter duration to reach complete block than do eversion and dorsiflexion. Taboada et al. [9] reported that plantar flexion reduces the time taken to achieve anesthesia and increases the success rate compared with dorsiflexion, Borgeat et al. [10] reported that inversion and plantar flexion increase the success rate to more than 97%. There are several ways to perform a sciatic nerve block, such as the front approach, lateral approach, posterior approach, and popliteal approach. In particular, the popliteal approach is the method with which most patients are satisfied because it is easy and the homonymous hamstring muscle can be reserved even after sciatic nerve block, which allows early post-surgical mobilization. In addition, this approach involves less uncomfortable feeling and does not induce pain from the calf tourniquet [7,11,12]. Also, a sympathetic nervous system block can be expected with PSNB given that the sciatic nerve has sensory nerves and sympathetic nerve fibers in the lower limbs; it can be applicable not only to pain control but also to control post-traumatic foot pain or burning pain [13]. The time taken for complete PSNB varied widely between the patient group. More specifically, 3 patients did not complain of pain when the pin-prick test was done but complained of mild pain under incision, so fentanyl and midazolam were administered. Following this, the surgery proceeded without complaints of pain, and patients did not complain of pain or respiratory disturbance until the surgery was completed. The reason for these differences appears to be related to the dose of regional anesthesia or the time difference between incomplete block and complete block due to the position of the needle during the PSNB procedure. For those patients with incomplete block at the beginning of surgery became a complete block with time. A number of cadaver studies reported that the site where the tibial nerve and the common peroneal nerve branch out from the sciatic nerve at the upper part of popliteal fossa crease is anatomically varied. Vloka et al. [14] reported that branching out was observed 60.0 ± 27.0 mm to the upper part of the popliteal fossa crease, and Saleh et al. [15] reported that the site is somewhere between 50.0 mm to 180.0 mm. For this reason, delivery of an anesthetic can be limited when regional anesthesia is administered at the inferior site rather than the branching-out site. In some cases, the distance between the tibial nerve and the commom peroneal nerve is several centimeters apart, and the regional anesthesia cannot cover this whole distance. Moreover, the fat layer and connective tissues within the popliteal fossa space interfere with the flow of the anesthetic and delay the time of action. This can induce individual time differences to the complete block or cause incomplete block. Therefore, many methods, such as the double-injection technique, [16,17] are used to identify the tibial nerve and the commom peroneal nerve separately and administer at each point to increase the success rate of PSNB. The needle should be positioned 10 cm above the popliteal fossa crease where the sciatic nerve branches out, as was reported by Paqueron et al. [17], but these theories require verification through clinical studies involving large numbers of patients. Peripheral nerve block can limit the side effects of general anesthesia, such as nausea and vomiting or cardiovascular adverse reactions. Also, it is known to reduce several side effects induced by regional anesthesia. In the present study, there was no incidence of hypotension, bradycardia, shivering, postoperative urinary retention, and PDPH relative to spinal anesthesia. In addition, surgery could be carried out in hemodynamically unstable patients who were on on anticoagulation therapy without facing a huge risk. It can also be an appropriate anesthetic procedure in geriatric patients who have anatomical issues due to degenerative changes [18]. The incidence of PDPH is higher in females than males and among younger patients [19], which was in accordance with this study showing higher incidence in female patients in their 30s and 50s. In addition, hallux valgus generally occurs in females, which encompasses the high-risk age group for PDPH. Even with the above advantages, the side effects of peripheral nerve block surgery limit the usefulness of the procedure. The common side effects are: incomplete block, direct nerve damage, localized hematoma and consequent ischemic damage, infection, and the risk of intravenous administration of local anesthetic [18]. There were no side effects in the PSNB group in this study, but Hajek et al. [20] reported superficial peroneal nerve and sural nerve damage in 3 patients (1.91%) out of 157 patients who were treated with continuous peripheral nerve block (CPNB) at the popliteal. Possible causes were exposed peroneal nerve and sural nerve damage, nerve toxicity of the local anesthesia, direct nerve damage and ischemia, and blockage of the local anesthetic flow in relation to the tourniquet. Despite the small chance, patients should be informed about such side effects. Those who require an appropriate anesthetic procedure need close monitoring for the incidence of side effects, and more investigation is warranted to decide on the ideal anesthetic procedure. In the patient satisfaction survey, PSNB was shown to have relatively high satisfaction compared with spinal anesthesia. This can be explained by the incidence of back pain from spinal anesthesia and various side effects during and after surgery, as these played important roles in reducing patients' satisfaction. Aside from 2 patients who developed PDPH, 5 patients (25%) indicated they were "very dissatisfied" due to back pain. In the PSNB group, satisfaction was low among patients who experienced pain at the needle insertion site, a longer procedural time, and pain under discission. These differences in the satisfaction levels would be greater if comparisons were made with a 26 G needle, a thicker needle, in the spinal anesthesia group or with a higher amount of spinal anesthesia. The satisfaction survey about postoperative pain control after sciatic nerve catheterization could not be used for the comparative study because patients with the spinal anesthetic rejected the use of intravenous patient-controlled analgesia (IV PCA) or the use of various methods of post-surgical pain control. However, in PSNB patients who used a PCA due to catheter insertion around the sciatic nerve, there was no incidence of side effects such as nausea and vomiting, which were common in those who recieved IV PCA or oral administration. Furthermore, 95% of PSNB patients showed satisfaction, including "average" satisfaction. Elliot et al. [21] reported that continuous nerve block due to sciatic nerve catheterization after foot and ankle joint surgery can reduce the need for extra pain relief and pain after surgery. Navas et al. [22] reported that this procedure increases pain relief after surgery, thus improving patient satisfaction. Although the study was carried out prospectively, the limitation of the study was that there was no random sampling and no blinding, and the comparisons of satisfaction scores and the levels of pain relief were not done in a double-blinded fashion. To validate the significance of the difference in side effects and patient satisfaction, a further study with a larger patient population is warranted. In summary, this study demonstrated that in hallux valgus surgery patients, the time taken for the procedure and nerve block in the PSNB group with 0.5% ropivacaine administration was longer than that taken for the spinal anesthesia group with 2.5 ml of 0.5% bupivacaine administration. There were individual variations in the time to complete the nerve block, but PSNB was determined as relatively safe, provided an appropriate level of anesthesia, reduced possible side effects from the spinal anesthesia, and showed excellent results on postoperative pain control. The authors suggest that PSNB be considered as the anesthetic procedure for hallux valgus surgery anesthesia and for postoperative pain control, especially in patients for whom spinal anesthesia is not feasible.

TITLE: Alterations of consciousness and mystical-type experiences after acute LSD in humans ABSTRACT.RATIONALE: Lysergic acid diethylamide (LSD) is used recreationally and in clinical research. Acute mystical-type experiences that are acutely induced by hallucinogens are thought to contribute to their potential therapeutic effects. However, no data have been reported on LSD-induced mystical experiences and their relationship to alterations of consciousness. Additionally, LSD dose- and concentration-response functions with regard to alterations of consciousness are lacking. ABSTRACT.METHODS: We conducted two placebo-controlled, double-blind, cross-over studies using oral administration of 100 and 200 μg LSD in 24 and 16 subjects, respectively. Acute effects of LSD were assessed using the 5 Dimensions of Altered States of Consciousness (5D-ASC) scale after both doses and the Mystical Experience Questionnaire (MEQ) after 200 μg. ABSTRACT.RESULTS: On the MEQ, 200 μg LSD induced mystical experiences that were comparable to those in patients who underwent LSD-assisted psychotherapy but were fewer than those reported for psilocybin in healthy subjects or patients. On the 5D-ASC scale, LSD produced higher ratings of blissful state, insightfulness, and changed meaning of percepts after 200 μg compared with 100 μg. Plasma levels of LSD were not positively correlated with its effects, with the exception of ego dissolution at 100 μg. ABSTRACT.CONCLUSIONS: Mystical-type experiences were infrequent after LSD, possibly because of the set and setting used in the present study. LSD may produce greater or different alterations of consciousness at 200 μg (i.e., a dose that is currently used in psychotherapy in Switzerland) compared with 100 μg (i.e., a dose used in imaging studies). Ego dissolution may reflect plasma levels of LSD, whereas more robustly induced effects of LSD may not result in such associations. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00213-016-4453-0) contains supplementary material, which is available to authorized users. BODY.INTRODUCTION: Lysergic acid diethylamide (LSD) is the prototypical hallucinogen (Nichols 2016; Passie et al. 2008). LSD became famous, with a high cultural influence, in the 1960s. LSD continues to be used for recreational and personal purposes (Krebs and Johansen 2013). Additionally, there is much interest in its therapeutic potential (Baumeister et al. 2014; Davenport 2016; Gasser et al. 2014; Gasser et al. 2015; Krebs and Johansen 2012; Kupferschmidt 2014). Only one modern study has tested the therapeutic effects of LSD in patients (Gasser et al. 2014), whereas several clinical trials have recently evaluated the therapeutic potential of psilocybin (Bogenschutz et al. 2015; Carhart-Harris et al. 2016a; Garcia-Romeu et al. 2015; Griffiths 2016; Grob et al. 2011; Guss 2016), a similar serotonergic hallucinogen (Rickli et al. 2016). A series of studies showed that psilocybin acutely induced mystical experiences in healthy subjects and patients (Garcia-Romeu et al. 2015; Griffiths et al. 2008; Griffiths et al. 2011; Griffiths et al. 2006; MacLean et al. 2011). Additionally, greater acute effects of psilocybin on the Mystical Experience Questionnaire (MEQ; Barrett et al. 2015; Griffiths et al. 2006; MacLean et al. 2012) were associated with positive long-term effects on mood and personality in healthy subjects (Griffiths et al. 2008; Griffiths et al. 2011; Griffiths et al. 2006; MacLean et al. 2011) and better therapeutic outcomes in patients with anxiety, depression, and substance use disorder (Garcia-Romeu et al. 2015; Griffiths 2016; Griffiths et al. 2008; Griffiths et al. 2011; Griffiths et al. 2006; MacLean et al. 2011). Early studies reported on mystical experiences after experimental administration of LSD, but methodological details are missing (Turek et al. 1974). Whether and the extent to which LSD produces mystical-type effects in the MEQ are currently unknown. Therefore, we characterized the effects of 200 μg LSD on the MEQ and evaluated the way in which mystical experiences are related to LSD-induced increases in 5 Dimensions of Altered States of Consciousness (5D-ASC) scale scores and plasma levels of LSD. Clinical experimental research with LSD has recently seen a resurgence (Carhart-Harris et al. 2016b; Carhart-Harris et al. 2015; Carhart-Harris et al. 2016c; Dolder et al. 2015b; Dolder et al. 2016; Kaelen et al. 2015; Kaelen et al. 2016; Lebedev et al. 2016; Roseman et al. 2016; Schmid et al. 2015; Speth et al. 2016; Strajhar et al. 2016; Tagliazucchi et al. 2016; Terhune et al. 2016). An increasing amount of data has been generated on the effects of LSD (75 μg) on various neuronal correlates of brain activation (Carhart-Harris et al. 2016c; Kaelen et al. 2016; Lebedev et al. 2016; Roseman et al. 2016). Researchers have correlated subjective drug effects with brain functional magnetic resonance imaging (fMRI) data (Carhart-Harris et al. 2016c; Kaelen et al. 2016; Lebedev et al. 2016; Roseman et al. 2016). This approach likely produces significant findings for subjective effects that show large between-subject variance but not for subjective effects of the substance that are consistently present in all subjects. Lower doses of LSD may also result in more variable responses across subjects compared with higher doses. Furthermore, higher doses of LSD (e.g., 200 μg) that are currently used therapeutically (Gasser et al. 2014) may produce more pronounced but also qualitatively different subjective effects (Dolder et al. 2016). Importantly, plasma concentrations of LSD have not been determined in any of the published LSD fMRI studies to date; therefore, unclear is the way in which LSD exposure in the body is linked to subjective effects in these studies. Therefore, a second goal of the present study was to describe the subjective peak effects of two doses of LSD (100 and 200 μg) using the 5D-ASC scale (Studerus et al. 2010). The 5D-ASC scale has been used in all of the recent experimental studies with LSD (Carhart-Harris et al. 2016b; Carhart-Harris et al. 2016c; Schmid et al. 2015; Tagliazucchi et al. 2016) and with many other psychedelics, providing an opportunity to compare findings between studies and across substances and research groups. Thus, the present study assessed LSD dose- and plasma concentration-response functions using the 5D-ASC scale in 40 subjects (Dolder et al. 2015b; Dolder et al. 2016; Schmid et al. 2015), thus allowing comparisons with other studies that used the 5D-ASC scale but did not determine plasma LSD concentrations (Carhart-Harris et al. 2016b; Carhart-Harris et al. 2016c; Kaelen et al. 2016; Lebedev et al. 2016; Roseman et al. 2016; Speth et al. 2016; Tagliazucchi et al. 2016; Terhune et al. 2016). A third goal of the present study was to assess associations across subjects between the peak and total plasma exposure to LSD and its effects on 5D-ASC scale scores (Studerus et al. 2010). The effects of 100 μg LSD on 5D-ASC scale scores are reported for the first time in the present study, whereas the effects of 200 μg have been previously published (Schmid et al. 2015). However, the latter study did not evaluate dose- or concentration-response functions. Other data that were generated in the present study have been previously reported including acute and subacute adverse effects (Dolder et al. 2015b; Dolder et al. 2016; Schmid et al. 2015; Strajhar et al. 2016). BODY.MATERIAL AND METHODS.STUDY DESIGN: We performed two similar studies using double-blind, placebo-controlled, cross-over designs with two experimental test sessions (LSD and placebo) in a balanced order. Study 1 used a dose of 100 μg LSD and placebo in 24 subjects. Study 2 used 200 μg LSD and placebo in 16 subjects. The washout periods between sessions were at least 7 days. The studies were conducted in accordance with the Declaration of Helsinki and approved by the local ethics committee. The administration of LSD to healthy subjects was authorized by the Swiss Federal Office for Public Health, Bern, Switzerland. All of the subjects provided written consent before participating in either of the studies, and they were paid for their participation. The studies were registered at ClinicalTrials.gov (NCT02308969, NCT01878942). BODY.MATERIAL AND METHODS.PARTICIPANTS: Forty healthy participants were recruited from the University of Basel campus via online advertisement. Twenty-four subjects (12 men, 12 women; 33 ± 11 years old [mean ± SD]; range, 25–60 years) participated in study 1, and 16 subjects (8 men, 8 women; 29 ± 6 years old; range, 25–51 years) participated in study 2. The inclusion and exclusion criteria were identical for both studies. Subjects younger than 25 years of age were excluded from participating in the study. Additional exclusion criteria were age >65 years, pregnancy (urine pregnancy test at screening and before each test session), personal or family (first-degree relative) history of major psychiatric disorders (assessed by the semi-structured clinical interview for Diagnostic and Statistical Manual of Mental Disorders, 4th edition, Axis I disorders by the study physician and an additional interview by a trained psychiatrist), use of medications that may interfere with the study medication, chronic or acute physical illness (abnormal physical exam, electrocardiogram, or hematological and chemical blood analyses), tobacco smoking (>10 cigarettes/day), lifetime prevalence of illicit drug use >10 times (except for tetrahydrocannabinol), illicit drug use within the last 2 months, and illicit drug use during the study (determined by urine drug tests). The subjects were asked to abstain from excessive alcohol consumption between test sessions and particularly limit their use to one standard drink on the day before the test sessions. Additionally, the participants were not allowed to drink xanthine-containing liquids after midnight before the study day. Eleven subjects had used a hallucinogen, including LSD (six participants), one to three times, and most of the subjects (29) were hallucinogen-naive. We performed urine drug tests at screening and before each test session, and no substances were detected during the study. BODY.MATERIAL AND METHODS.STUDY PROCEDURES: Each study included a screening visit, a psychiatric interview, two 25-h experimental sessions, and an end-of-study visit. The experimental sessions were conducted in a quiet standard hospital patient room. The participants were resting in hospital beds except when going to the restroom. Only one research subject and one or two investigators were present during the experimental sessions. The participants could interact with the investigator, rest quietly, and/or listen to music via headphones, but no other entertainment was provided. LSD or placebo was administered at 9:00 AM. The subjects were never alone during the first 12 h after drug administration, and the investigator was in a room next to the subject for up to 24 h while the subjects were asleep (mostly from 1:00 AM to 8:00 AM). BODY.MATERIAL AND METHODS.STUDY DRUG: LSD (d-LSD hydrate, HPLC purity >99 %, Lipomed AG, Arlesheim, Switzerland) was administered in single oral doses of 100 or 200 μg as gelatin capsules. Note that these LSD hydrate doses correspond to LSD tartrate doses of 123 and 246 μg, respectively. In the 1960–1970s, small doses of LSD tartrate of 25–150 μg were typically used in "psycholytic therapy" and higher doses of >200 μg in "psychedelic" therapy (Pahnke et al. 1970). The dose used in a recent LSD-assisted psychotherapy study was 200 μg LSD hydrate (Gasser et al. 2014). Both doses used in the present study were within the range of doses that are taken for recreational purposes (Passie et al. 2008). Corresponding placebo capsules were used. BODY.MATERIAL AND METHODS.MEASURES.MYSTICAL-TYPE EXPERIENCES: In study 2, mystical experiences were assessed using a German version (Supplementary Appendix 1) of the 43-item MEQ (Griffiths et al. 2006; MacLean et al. 2012; Pahnke 1969) embedded in the 100-item States of Consciousness Questionnaire (SOCQ; (Griffiths et al. 2006). The original English questionnaire was independently forward-translated into German by two translators with German as their mother tongue. Discrepancies between the two forward-translated versions and a previous German version were then discussed and selected items backtranslated. The version was then pretested for comprehension by persons with previous LSD or MDMA use. The MEQ has been used in numerous experimental and therapeutic trials with psilocybin (Garcia-Romeu et al. 2015; Griffiths et al. 2008; Griffiths et al. 2011; Griffiths et al. 2006; MacLean et al. 2011). The MEQ items provide scale scores for each of seven domains of mystical experiences: internal unity, external unity, sacredness, noetic quality (as real as or more real than everyday reality), deeply felt positive mood, transcendence of time and space, and ineffability/paradoxicality (difficulty describing the experience in words). The total of all scale scores was used as an overall measure of the mystical-type experience. We also derived the four scale scores of the newly validated revised 30-item MEQ: mystical, positive mood, transcendence of time and space, and ineffability (Barrett et al. 2015). A complete mystical experience was defined as scores ≥60 % on all MEQ30 factors (Barrett et al. 2015). The MEQ was administered 24 h after drug administration, and the participants were asked to retrospectively rate drug effects during peak drug effects. For comparison, we included MEQ ratings that were obtained 6 h after administration of 3,4-methylenedioxymethamphetamine (MDMA) and methylphenidate in another study using a similar research setting (Schmid et al. 2014). Additionally, we included MEQ ratings from patients who were treated with 200 μg LSD for anxiety related to life-threatening illness in another study (Diesch 2015; Gasser et al. 2014; Gasser et al. 2015). All of these additional MEQ findings have not been previously published in scientific journals and were obtained in studies that were previously described in detail (Diesch 2015; Gasser et al. 2014; Gasser et al. 2015; Schmid et al. 2014). BODY.MATERIAL AND METHODS.MEASURES.ALTERATIONS OF CONSCIOUSNESS: The 5D-ASC scale was used in both studies to assess the overall peak alterations of consciousness. The 5D-ASC scale measures altered states of consciousness and contains 94 items (visual analog scales). The instrument consists of five subscales/dimensions (Dittrich 1998) and 11 lower-order scales (Studerus et al. 2010). The 5D-ASC dimension "Oceanic Boundlessness" (27 items) measures derealization and depersonalization associated with positive emotional states, ranging from heightened mood to euphoric exaltation. The corresponding lower-order scales include "experience of unity," "spiritual experience," "blissful state," and "insightfulness." The dimension "Anxious Ego Dissolution" (21 items) summarizes ego disintegration and loss of self-control phenomena associated with anxiety. The corresponding lower-order scales include "disembodiment," "impaired control of cognition," and "anxiety." The dimension "Visionary Restructuralization" (18 items) consists of the lower-order scales "complex imagery," "elementary imagery," "audio-visual synesthesia," and "changed meaning of percepts." Two additional dimensions describe "Auditory Alterations" (15 items) and "Reduction of Vigilance" (12 items). The scale is well-validated and widely used to characterize the subjective effects of various psychedelic drugs (Carhart-Harris et al. 2016b; Hasler et al. 2004; Hysek et al. 2011; Schmid et al. 2015; Vollenweider et al. 2007; Vollenweider and Kometer 2010). In addition to the subscale analyses, we also analyzed the effects on ego dissolution item 71 (the boundaries between myself and my surroundings seemed to blur) because the concept of ego dissolution was often used in recent imaging studies (Tagliazucchi et al. 2016). The 5D-ASC scale was administered 24 h after drug administration, and the participants were asked to retrospectively rate the drug effects. 5D-ASC ratings were also performed at 3 and 10 h in study 1. BODY.MATERIAL AND METHODS.ANALYSIS OF PLASMA LSD CONCENTRATIONS: Blood was collected into lithium heparin tubes before and 0.5, 1, 1.5, 2.5, 3, 4, 6, 8, 10, 12, 16, and 24 h after LSD administration. The 0.5, 1.5, and 2.5 h samples were not collected in study 1. Blood samples were immediately centrifuged, and the plasma was rapidly stored at −20 °C and later analyzed using liquid-chromatography-tandem mass-spectrometry as previously reported (Dolder et al. 2015a; Steuer et al. 2016). Maximal plasma concentrations (C max) and total exposure (area under the plasma concentration-time curve [AUC]) were estimated using compartmental modeling in Phoenix WinNonlin 6.4 (Certara, Princeton, NJ, USA). A one-compartment model was used with first-order input, first-order elimination, and no lag time. BODY.MATERIAL AND METHODS.STATISTICAL ANALYSES: The data analysis was performed using Statistica 12 software (StatSoft, Tulsa, OK, USA). Differences between LSD and placebo or between the 100 and 200 μg doses of LSD were compared using dependent or independent t tests, respectively. Associations between outcome measures were assessed using Pearson correlations. Significance was assumed at p < 0.05. BODY.RESULTS.MYSTICAL-TYPE EXPERIENCES: LSD (200 μg) significantly increased all MEQ scores compared with placebo (Fig. 1a, Table 1). The effects of MDMA and methylphenidate on MEQ scores are included for comparison (Fig. 1a). The effects of LSD (200 μg) and placebo on MEQ scores in 11 patients during LSD-assisted psychotherapy (Gasser et al. 2014) are also shown in Fig. 1b. LSD-induced mystical experiences were comparable in healthy subjects in the laboratory setting in the present study and in patients in the therapeutic setting (Fig. 1b). Only two subjects in each of the studies had a complete mystical experience. The MEQ30 total scores were <5 % in both settings after placebo administration (Fig. 1b).Fig. 1Effects of LSD on the Mystical Experience Questionnaire (MEQ). a In the present study in healthy subjects, LSD (200 μg) significantly increased scores on all scales of the MEQ43 and MEQ30 compared with placebo (Table 1). The data are expressed as the mean ± SEM in 16 subjects. For comparison, 3,4-methylenedioxymethamphetamine (MDMA; 75 mg) and methylphenidate (40 mg) produced small increases in MEQ ratings in 30 different participants in another study in the same research setting (Schmid et al. 2014). b Effects of LSD on the MEQ in patients with anxiety in the context of life-threatening illness. The data were analyzed identically to the data that were obtained in the present study. The study and patient characteristics have been previously published in detail (Diesch 2015; Gasser et al. 2014; Gasser et al. 2015; Schmid et al. 2014). Similar to the present study, the MEQ was administered on the day after LSD (200 μg) or active placebo (25 μg LSD) administration and was embedded into the larger 100-item States of Consciousness Questionnaire (SOCQ; Griffiths et al. 2006). The patient data are expressed as the mean ± SEM in 11 subjects for LSD (200 μg, same formulation as in the present study) and four subjects for placebo. On the 43- and 30-item versions of the MEQ, LSD (200 μg) increased MEQ rating scores in the patients in the therapeutic setting (b) to a similar extent as in the healthy subjects in the present study (a). Notably, the placebo response (a very low dose of LSD of 25 μg was used as the active placebo) in the patients was small (b), which was also similar to the response in healthy subjects in the present study (a) Table 1Statistics for the effects of LSD in the 5D-ASC and MEQLSD 100 μg T test vs. placeboLSD 200 μg T test vs. placeboLSD 100 vs. 200 μg T test T= P= T= P= T= P= 5 Dimensions Altered States of Consciousness (ASC) scale Total ASC score9.72<0.00110.02<0.0012.23<0.05 Oceanic boundlessness8.44<0.0019.61<0.0011.89NS Anxious ego dissolution6.43<0.0014.01<0.0011.50NS Visionary restructuralization9.79<0.00115.32<0.0012.34<0.05 Auditory alterations3.72<0.015.87<0.0010.42NS Reductions of vigilance7.44<0.0015.93<0.0010.79NS Experience of unity6.85<0.0017.77<0.0010.68NS Spiritual experience4.31<0.0013.91<0.0011.10NS Blissful state6.56<0.0018.27<0.0013.00<0.01 Insightfulness4.11<0.0015.81<0.0012.28<0.05 Disembodiment6.93<0.0015.87<0.0010.13NS Impaired control and cognition7.01<0.0015.04<0.0010.86NS Anxiety3.02<0.0012.04NS1.37NS Complex imagery7.10<0.0017.48<0.0010.31NS Elementary imagery9.96<0.00111.12<0.0010.57NS Audio-visual synsthesia9.19<0.00112.52<0.0011.96NS Changed meaning of percepts6.25<0.0019.66<0.0013.39<0.01 Ego dissolution (item 71)7.63<0.0015.32<0.0010.36NSMystical Effects Questionnaire (MEC43) Internal unityNANA6.22<0.001NANA External unityNANA6.08<0.001NANA SacrednessNANA6.80<0.001NANA Noetic qualityNANA5.71<0.001NANA Deeply felt positive moodNANA11.43<0.001NANA Transcendence of time/spaceNANA10.63<0.001NANA IneffabilityNANA16.22<0.001NANAMystical Effects Questionnaire (MEQ30) MysticalNANA5.99<0.001NANA Positive moodNANA13.13<0.001NANA Transcendence of time/spaceNANA11.12<0.001NANA IneffabilityNANA25.14<0.001NANA MEC30 total scoreNANA14.91<0.001NANASixteen subjects participated in the high-dose study (200 μg) and 24 subjects in the moderate-dose study (100 μg). Dependent T tests were performed to assess differences from placebo, and independent T tests were performed to assess differences between doses of LSD NA not assessed BODY.RESULTS.ALTERATIONS OF CONSCIOUSNESS: LSD induced pronounced peak alterations of waking consciousness, with significant increases in all dimensions and subscales of the 5D-ASC scale (Fig. 2). The 200 μg dose of LSD produced significantly greater scores on the overall ASC scale, the dimension of visionary restructuralization, and the blissful state, insightfulness, and changed meaning of percepts subscales compared with the 100 μg dose (Fig. 2, Table 1). The mean ± SEM ego dissolution (item 71) scores were 49 ± 6 and 53 ± 10 after the 100 and 200 μg doses, respectively (Table 1). There were only minimal differences between the 5D-ASC ratings at 3, 10, and 24 h (supplementary Fig. S1 online).Fig. 2Effects of LSD on the 5 Dimensions of Altered States of Consciousness (5D-ASC) scale. LSD mainly increased ratings of oceanic boundlessness (OB) and visionary restructuralization (VR), with significantly higher ratings for the ASC total score and VR dimension at 200 μg compared with 100 μg. LSD-induced increases in anxious ego dissolution (AED) and auditory alterations (AA) were relatively small. LSD also produced vigilance reduction (VIR). LSD-induced changes on the 5D-ASC scale were significant compared with placebo for both doses and all of the scales, with the exception of the effects of the 200 μg dose on anxiety (Table 1). At 200 μg, LSD produced significant and relevantly higher ratings of blissful state, insightfulness, and changed meaning of percepts compared with 100 μg (one asterisk p < 0.05, two asterisks p < 0.01, t tests). The data are expressed as the mean ± SEM in 24 subjects and 16 subjects for the 100 and 200 μg doses of LSD, respectively BODY.RESULTS.PLASMA LSD CONCENTRATIONS: Plasma concentrations varied between subjects, especially at the lower 100 μg dose. The median (range) C max values were 1.4 ng/ml (0.32–3.7) and 3.2 ng/ml (1.9–7.1) for the 100 and 200 μg doses, respectively. The corresponding AUC values were 8.5 ng × h/ml (1–19) and 20.7 ng × h/ml (11–39). BODY.RESULTS.ASSOCIATIONS BETWEEN ALTERATIONS OF CONSCIOUSNESS AND MYSTICAL-TYPE EXPERIENCES: Table 2 shows the cross-tabulation of all correlations between the 5D-ASC scale and MEQ30 subscale ratings. LSD-induced alterations of consciousness (ASC total score) were significantly correlated with ratings of mystical experience (MEQ30 total score) on the MEQ (R p = 0.87, p < 0.001, n = 16; Fig. 3). Scores on the MEQ positive mood scale were strongly associated with scores on the ASC experience of unity and blissful state scales (R p = 0.85 and 0.80, respectively; both p < 0.001, n = 16; Table 2).Table 2Associations between LSD-induced alterations in consciousness (5D-ASC) and mystical experiences (MEQ30)Mystical Effects Questionnaire (MEQ30)MEQ30 total scoreMysticalPositive moodTranscendence of time/spaceIneffability5D-ASC scale Total ASC score 0.87 0.73 0.65 0.82 0.57 Oceanic boundlessness 0.93 0.88 0.83 0.74 0.45 Anxious ego dissolution 0.60 0.390.35 0.68 0.55 Visionary restructuralization 0.65 0.54 0.38 0.68 0.45 Auditory alterations0.300.140.020.490.38 Reductions of vigilance 0.61 0.410.47 0.64 0.47 Experience of unity 0.82 0.86 0.85 0.56 0.25 Spiritual experience 0.79 0.76 0.76 0.60 0.33 Blissful State 0.80 0.77 0.80 0.72 0.16 Insightfulness 0.77 0.79 0.68 0.52 0.42 Disembodiment 0.71 0.53 0.62 0.71 0.41 Impaired control and cognition 0.63 0.370.45 0.79 0.46 Anxiety0.450.320.190.47 0.51 Complex imagery0.480.310.32 0.69 0.19 Elementary imagery0.360.370.080.290.42 Audio-visual synesthesia0.230.070.220.45−0.01 Changed meaning of percepts 0.80 0.67 0.59 0.70 0.63 Ego dissolution (item 71) 0.74 0.73 0.74 0.65 0.12Values are Pearson correlation coefficients in 16 subjects describing correlations between %5D-ASC and %MEQ30 scores. Bold values for P < 0.05, italic values for P < 0.001 Fig. 3LSD-induced alterations of consciousness are significantly associated with the LSD-induced mystical experience. The data are expressed as a percentage of ASC total scores on the 5D-ASC scale and a percentage of total scores on the MEQ30 for each of 16 participants after administration of 200 μg LSD. The lines indicate the regression and 95 % confidence intervals (R p = 0.87, p < 0.001) BODY.RESULTS.CORRELATIONS BETWEEN PLASMA LSD CONCENTRATIONS AND LSD-INDUCED ALTERATIONS OF CONSCIOUSNESS AND MYSTICAL-TYPE EXPERIENCES: The C max and AUC values for LSD were not positively correlated with ratings of peak subjective effects on the 5D-ASC scale or MEQ across subjects or within dose groups (Table 3). For example, LSD induced consistently high ratings of audio-visual synesthesia in almost all of the subjects at the high dose (200 μg), resulting in little within-subject variance and no association with plasma exposure to LSD (Table 3, Fig. 4a). One exception was ego dissolution (item 71) at the lower dose of LSD (100 μg; Table 3, Fig. 4b). The ratings showed high interindividual variance, and there was a significant positive correlation with the LSD AUC value in the 100 μg dose group (R p = 0.51, p < 0.05, n = 16; Table 3, Fig. 4b). At the 200 μg dose, there were significant negative correlations between C max values for LSD and subjective effects on the 5D-ASC scale including visionary restructuralization, elementary imagery, and changed meaning of percepts.Table 3Associations between predicted maximal LSD plasma concentrations (C max) and LSD exposure (AUC) and alterations in consciousness (SD-ASC) and mystical experiences (MEQ30) N = 24 N = 16100 μg200 μg C max AUC C max AUC5D-ASC scale ASC total score0.190.21−0.350.15 Oceanic boundlessness0.240.26−0.350.10 Anxious ego dissolution0.040.07−0.100.32 Visionary restructuralization0.120.15−0.59 −0.16 Auditory alterations0.020.12−0.180.08 Reductions of vigilance−0.010.13−0.100.38 Experience of unity0.340.33−0.030.33 Spiritual experience−0.020.06−0.32−0.03 Blissful state0.250.14−0.230.03 Insightfulness0.240.20−0.370.12 Disembodiment−0.040.08−0.230.08 Impaired control and cognition−0.010.01−0.200.18 Anxiety0.220.300.010.38 Complex imagery0.060.14−0.28−0.04 Elementary imagery−0.13−0.03−0.53 −0.15 Audio-visual synesthesia0.230.26−0.010.00 Changed meaning of percepts−0.03−0.06−0.62 −0.10 Ego dissolution (item 71)0.40 0.51 −0.27−0.14MEQ30 MEC30 total scoreNA−0.300.17 MysticalNA−0.250.13 Positive moodNA−0.080.21 Transcendence of time/spaceNA−0.230.10 IneffabilityNA−0.490.13Values are Pearson correlation coefficients describing correlations, the peak concentrations of LSD predicted by the one-compartment model, and LSD-induced %5D-ASC and %MEQ30 scores. Bold values for P < 0.05C max maximal LSD plasma concentration predicted by the one-compartment pharmacokinetic model, AUC area under the LSD concentration-time curve predicted by the model Fig. 4Correlations between plasma LSD concentrations and subjective peak effects. a At 200 μg, LSD induced high ratings of audio-visual synesthesia in all but two of the 16 participants. There was little variance in the response and no correlation between total plasma exposure to LSD (area under the concentration-time curve [AUC]) and audio-visual synesthesia (R p = 0.0, p > 0.05, n = 16). b In contrast, ego dissolution was present to highly variable degrees across subjects after administration of 100 μg LSD. Total exposure to LSD (AUC) positively correlated with LSD-induced ego dissolution (R p = 0.51, p < 0.05, n = 24). The lines indicate the regression and 95 % confidence intervals BODY.DISCUSSION: The present study characterized LSD-induced mystical experiences using the MEQ after a dose of 200 μg and alterations of consciousness on the 5D-ASC scale after a dose of 100 μg. The study also evaluated associations between plasma LSD concentrations and these subjective effects. LSD produced mean MEQ30 total score ratings of 61 % (range 40–98 %) and a complete mystical experience in only two participants (12.5 %). The MEQ has typically been used with psilocybin, and data on MEQ30 scores are available for various doses of psilocybin, placebo, and methylphenidate (active placebo; Barrett et al. 2015). Psilocybin (at the highest studied dose of 30 mg/70 kg) produced a high mean MEQ30 total score rating of 77 % and complete mystical experiences in as many as 67 % of healthy subjects (Barrett et al. 2015). However, in this psilocybin study setting, inactive and active placebo (methylphenidate) also produced high mean MEQ30 ratings of 23 and 33 %, respectively (Barrett et al. 2015). In contrast, in the present study, placebo increased MEQ30 scores only to 1 %. Similarly, MDMA and methylphenidate produced only small increases in MEQ scores in a similar laboratory setting (Schmid et al. 2014). Another study evaluated psilocybin-assisted psychotherapy in tobacco smokers and also found complete mystical experiences in only 10 of 26 sessions (38 %) that were conducted in 14 patients with high-dose psilocybin (30 mg/70 kg; Garcia-Romeu et al. 2015; Johnson et al. 2014). Accounting for the higher placebo ratings in some of the psilocybin studies compared with our study, LSD increased MEQ30 score differences from placebo overall more than psilocybin and produced greater ineffability and positive mood but lower effects on the mystical subscale than psilocybin (Barrett et al. 2015). Additionally, the MEQ has been used in patients with anxiety associated with life-threatening illness who were treated with 200 μg LSD (Gasser et al. 2014; Gasser et al. 2015). In this therapeutic setting, LSD produced similar mystical experiences as in the present study and complete mystical experiences in only two of 11 patients. MEQ scores were only within the range of 3–9 % after active placebo administration (25 μg LSD) on the MEQ subscales. Altogether, these findings indicate that mainly the placebo response and/or the expectancy of a mystical experience were greater in the study setting in some psilocybin studies compared with the LSD studies. Additionally, the participants in the psilocybin studies may have been more spiritually inclined (Griffiths et al. 2006) than our study participants leading to more mystical experiences (Studerus et al. 2012). Furthermore, others may have provided more extensive preparation of the subjects and interpersonal support, contributing to mystical experiences. The present findings do not support the view that LSD produces lower overall effects than psilocybin at the doses tested. In contrast, the high dose of LSD (200 μg) produced greater placebo-adjusted positive mood ratings than psilocybin on the MEQ30 (Barrett et al. 2015) and very pronounced increases in 5D-ASC blissful state ratings and produced far greater effects than the highest doses of psilocybin or dimethyltryptamine (DMT) that were tested so far on this scale (Gouzoulis-Mayfrank et al. 2005; Hasler et al. 2004). Additionally, LSD-induced MEQ scores were highly correlated with 5D-ASC scores in the present study. One could argue that mystical and spiritual experiences are not the most prominent feature of the LSD response. Mean ratings on the spiritual experience scale of the 5D-ASC were 22 and 33 % at the 100 and 200 μg doses, respectively, in the present study and approximately 23 % after 75 μg LSD in another study (Carhart-Harris et al. 2016c). Mean ratings of "the experience had a spiritual or mystical quality" were also only approximately 28 % in an imaging study that evaluated the effects of LSD (Tagliazucchi et al. 2016). However, a direct within-subjects comparison of LSD and psilocybin in the same research setting is needed to determine possible differences in mystical-type responses between these substances. Whether mystical-type experiences (Barrett et al. 2015; Garcia-Romeu et al. 2015; MacLean et al. 2011) are critical for the therapeutic potential of substance-assisted psychotherapy requires further study. At least in the case of LSD, the mystical experiences (MEQ scores) were highly associated with other alterations of consciousness on the 5D-ASC scale, and LSD produced additional effects on emotion processing that could facilitate psychotherapeutic interventions (Dolder et al. 2016). Recent experimental studies associated the subjective effects of LSD (75 μg, intravenous) on the 5D-ASC scale with fMRI data but in the absence of data on plasma LSD levels (Carhart-Harris et al. 2016c; Kaelen et al. 2016; Lebedev et al. 2016; Roseman et al. 2016). Assuming high oral bioavailability of LSD of 70–100 % (Dolder et al. 2015b), similar plasma exposure (AUC) can be assumed after oral administration of 100 μg LSD (present study I) or intravenous administration of 75 μg LSD (all studies by Carhart-Harris and colleagues). Supporting this assumption, the intravenous 75 μg dose of LSD produced very similar mean ratings on the 5D-ASC scale (Carhart-Harris et al. 2016b) to the present study that used an oral dose of 100 μg. In contrast, the 200 μg dose produced significantly greater ASC total scores and particularly greater 5D-ASC subscale scores of blissful state, insightfulness, and changed meaning of percepts. As previously reported, the 200 μg dose of LSD also produced greater feelings of closeness to others, happiness, openness, and trust than the 100 μg dose (Dolder et al. 2016). Altogether, the data indicate that the 200 μg dose produces overall greater effects and particularly more positive and MDMA-like effects than lower doses (Dolder et al. 2016). This is relevant because the higher dose is currently being used in LSD-assisted psychotherapy (Gasser et al. 2014; Gasser et al. 2015), and the lower dose is being tested in experimental fMRI studies (Carhart-Harris et al. 2016c). The 200 μg dose of LSD also produced greater ASC scores than high doses of the serotonergic hallucinogens DMT and psilocybin (Gouzoulis-Mayfrank et al. 2005; Hasler et al. 2004; Vollenweider and Kometer 2010), ketamine (Gouzoulis-Mayfrank et al. 2005; Studerus et al. 2010), and MDMA (Hysek et al. 2011), although direct comparisons within the same studies and subjects are missing. The present analyses showed no positive correlations between LSD levels and effects across subjects, possibly because of the relatively high levels of LSD and generally consistently high subjective response ratings in most subjects. Thus, if relatively high and similar doses of LSD are used that result in plasma levels clearly above the EC50 of a particular response measure, then it is unlikely that the response varies relevantly across subjects because responses are close to maximal. This would typically also be the case with measures with a maximal effect limit such as VAS ratings and some physiological effects like pupil size (Hysek and Liechti 2012). In fact, responses to MDMA or LSD or other drugs in a standardized experimental setting may vary only if the response is not induced consistently in all subjects (e.g., at the beginning of the response) and are mostly attributable to individual differences in drug absorption/distribution (Hysek and Liechti 2012) or when a response is evaluated that is not robustly induced or when a lower dose is used. Specifically, correlations of plasma levels with the subjective and cardiovascular effects of MDMA across subjects are only weak during the peak response but stronger at onset (Hysek and Liechti 2012). This is an important consideration. For example, LSD-induced subjective ego dissolution was recently shown to be associated with specific brain activation patterns in a study that administered a relatively low dose of LSD of 75 μg intravenously (Tagliazucchi et al. 2016). Interestingly, LSD-induced ego dissolution correlated with plasma LSD levels after administration of an equivalent oral dose of 100 μg in the present study, and this was the only pharmacodynamic effect of LSD for which a positive association with plasma levels could be demonstrated across subjects. This finding needs to be kept in mind when interpreting associations between ego dissolution and fMRI parameters because the fMRI findings may also reflect other processes that are related to the plasma levels of LSD. Furthermore, the likelihood of detecting correlations within a dose group increases for effects that are not robustly induced in all subjects and thus for effects that are not typically present in all subjects after LSD administration. Finally, unclear is the extent to which a full LSD response was induced in the imaging studies that have been conducted to date because all of these studies used relatively low 75 or 100 μg doses. In the present study, the 200 μg dose of LSD produced particularly marked increases in visionary restructuralization including changed meaning of percepts which were significantly greater after the 200 compared with the 100 μg dose. Contrary to expectations, these perceptual alterations were greater in participants with relatively lower C max levels of LSD within the 200 μg dose group further supporting the view that higher plasma levels of LSD may not produce greater subjective alterations above a certain threshold level and if high doses of LSD are used. In conclusion, LSD (200 μg) rarely produced full mystical experiences in the present study and in patients during LSD-assisted psychotherapy compared with psilocybin in another set and setting. This raises questions regarding expectancy effects and placebo responses and the therapeutic role of mystical experiences. LSD produced significantly greater bliss, insightfulness, and changes in meaning of percepts at 200 μg compared with 100 μg, in addition to the previously reported greater empathogenic effects. This could be relevant for LSD-assisted psychotherapy (200 μg) and the interpretation of fMRI data (75–100 μg). Generally, no association was found between plasma LSD levels and its robust effects when analyzed across different subjects and within a dose group. This may have implications for studies that interrelate different effects of LSD, namely fMRI studies. BODY.ELECTRONIC SUPPLEMENTARY MATERIAL: ESM 1(DOCX 202 kb)

TITLE: Comparative effectiveness of two stabilization exercise positions on pain and functional disability of patients with low back pain ABSTRACT: The study investigated the effects of two stabilization exercise positions (prone and supine) on pain intensity (PI) and functional disability (FD) of patients with nonspecific chronic low back pain (NSCLBP). The 56 subjects that completed the study were randomly assigned into stabilization in prone (SIP) (n=19), stabilization in supine (SIS) (n=20), and prone and supine (SIPS) position (n=17) groups. Subjects in all the groups received infrared radiation for 15 min and kneading massage at the low back region. Subjects in SIP, SIS, and SIPS groups received stabilization exercise in prone lying, supine lying and combination of both positions respectively. Treatment was applied twice weekly for eight weeks. PI and FD level of each subject were measured at baseline, 4th and 8th week of the treatment sessions. Data were analyzed using descriptive and inferential statistics. The alpha level was set at P<0.05. Within-group comparison indicated that PI and FD at the 4th and 8th week were significantly reduced (P<0.001) when compared with baseline in all the three groups. However, the result showed that there was no significant difference in the PI and FD at the 8th week (P>0.05) of the treatment sessions across the three groups when compared. It can be concluded that stabilization exercises carried out in prone, supine and combination of the two positions were equally effective in managing pain and disability of patients with NSCLBP. However, no position was superior to the other. BODY.INTRODUCTION: Low back pain (LBP) had been a major public health problem all over the world in which most people had suffered series of incapacity at one time or the other (Koley and Sandhu, 2009). It is a prevalent musculoskeletal condition, and a common cause of disability especially in its chronic/recurrent state (Esther, 2012). LBP has a point prevalence of about 7% to 33% and lifetime prevalence of nearly 85% (Walker, 2000). The report of Louw et al. (2007) on LBP in Africa revealed a prevalence of 12% among adolescence and 32% among adult. Omokhodion (2002), conducted a survey in the South-Western part of Nigeria and found that 40% of the sample population had LBP in the past 12 months, whereas 33% had LBP at the time of study, indicating that LBP is a common condition among Africans that is rising and should be of global concern (Omokhodion, 2002). Nonspecific chronic low back pain (NSCLBP) is a widespread problem which limits activities in middle aged individuals with major social and economic consequences, the commonest reason for physician consultation (Manchikanti et al., 2009). NSCLBP accounts for serious job absenteeism in industrialized societies, a case that would have been similar in most parts of Africa except that there is hardly any financial compensation for sick leave, hence less report of LBP in clinics (Krismer and van Tudler, 2007). LBP is regarded as a symptom from impairments in the structures in the low back which originates from muscles, ligaments and intervertebral disc (Mense and Gerwin, 2014). Implicated muscles in LBP are the lumbar multifidi and abdominals especially the transversus abdominis (Esther, 2012). Evidences by Hides et al. (1992) supported the positive role of the lumbar multifidus muscle in segmental stabilization of the lumbar spine. Among the lumbar muscles which play substantial role of which their strengthening and coordination are necessary for proper function of low back are deep stabilizer muscles; the multifidus muscle, transversus abdominis muscle, and internal oblique abdominal muscle (McGill et al., 2003). Other muscles which serve as superficial stabilizer includes, the erector spinae muscle, rectus abdominis muscle, and external oblique abdominal muscle-play a role in lumbar segmental stability and as a basic support, and for lumbar segmental stabilization (McGill et al., 2003). Exercise therapy appears to be the most often-used physical therapy intervention in treating people with back pain (Hayden et al., 2012). The aims of exercise therapy are to abolish pain, restoring and maintaining full range of motion, improving the strength and endurance of lumbar and abdominal muscles, thereby contributing to early restoration of normal function (Saunder, 2007). Clinical application of exercise has been shown to improve strength of the muscles of the gluteus resulted in decrease LBP, disability index and increase in lumbar muscle strength and balance ability (Jeong, 2015). Exercises are commonly prescribed for LBP but only seem to be supported as an intervention by evidence for patients with chronic LBP (Esther, 2012). Liddle et al. (2004) and Lewis et al. (2008) in their systematic reviews affirmed that exercises were effective in reducing pain in people with chronic LBP. Most studies concluded that active exercises were a valuable therapeutic approach in managing LBP, despite the lack of consensus on the optimal exercise techniques, intensity or active intervention (Esther, 2012). In a study by França et al. (2010) segmental stabilization and strengthening exercises effectively reduced pain and functional disability in individuals with chronic LBP. Among the various exercises, stabilization exercises were majorly used to treat pain and dysfunctions in LBP which were reported to enhance control over the lumbar spine and the pelvis (Hodges et al., 2003) and can be performed in diverse body positions using cocontraction of abdominal and multifidus muscles (Andrusaitis et al., 2011). In practice, emphasis had been on exercises in prone and supine lying in order to strengthen different groups of the spinal muscles (Kisner and Colby, 2007). Most clinical protocols combine different exercises, techniques and position, making it difficult to isolate the efficacy of specific strategies and positions (Hayden et al., 2005). This is of great clinical importance and needs to be further clarified through research. Therefore, this study aimed at comparing the effectiveness of two stabilization exercise positions (prone and supine) on the pain intensity and functional disability of patients with NSCLBP. BODY.MATERIALS AND METHODS: Quasi pre- and postexperimental design was used in this study. BODY.MATERIALS AND METHODS.SUBJECTS: Sixty-two subjects attending the Outpatient Physiotherapy Department of the State Specialist Hospital, Asubiaro, Osogbo, Osun state, Nigeria were invited for this study but 56 participated. BODY.MATERIALS AND METHODS.INCLUSION CRITERIA: The following categories of patients were recruited into the study: Subjects referred by physician with diagnoses of mechanical LBP of not less than 3 months, with pain being provoked by activity. Subjects whose age ranges from 25 to 65 years and those without defects in the trunk, upper and lower extremities. BODY.MATERIALS AND METHODS.EXCLUSION CRITERIA: Subjects with specific pathology, such as systemic inflammatory diseases (such as systemic lupus erythematous, rheumatoid arthritis, nephritis etc.), prolapsed disc, pregnancy related, fractures (spine or extremities), tumors, infection. BODY.MATERIALS AND METHODS.SAMPLING TECHNIQUE: A purposive sampling technique was used to recruit subject for this study. BODY.MATERIALS AND METHODS.RESEARCH DESIGN: It was a randomized control trial. BODY.MATERIALS AND METHODS.INSTRUMENTS: The following instruments were used in this study: Infra-Red lamp: Floor model SM-10H. Infrared lamp microfield instrument. Made in England. Verbal Rating Scale (VRS): The patients place a check mark next to the phrase that best describes the current intensity of their pain. A response of "No Pain" is given a value of zero, 1=mild pain, 2=moderate, 3=severe, 4=very severe, 5=Worst possible pain. The VRS was validated with the visual analogue scale by Williamson and Hoggart (2005), who concluded that VRS provides a useful alternative to the visual analog scores in the assessment of chronic pain. Rolland Morris Disability Questionnaire: This 24-item questionnaire was derived from the sickness index profile by Bergner et al. (1981). It entails totaling the sum of circled items (maximum, 24), thus representing the final score. Weighing scale: A Bathroom Weighing Scale manufactured by the Hanson Company of Ireland in the year 2000 (0–120 kg) was used to measure the body weight of subjects in kilogram to the nearest 1.0 kg. Stadiometer: Made by Prestige Company in China. Calibrated from 20 to 205 cm. It was used to measure the height of the subject to the nearest 0.1 cm. Tape rule: A Standard Inextensible rule (0.7 cm wide and 150 cm long) made in China was used to measure the waist and hip circumference of the subjects. Stop watch: A quartz stop watch, made in china, was used to determine the period of exercise activity in seconds. BODY.MATERIALS AND METHODS.SAMPLE SIZE CALCULATION: N=4σ2 (Zcrit+Zpwr)2D2 where N is the total sample size, σ is the assumed standard deviation (SD) of each group (assumed to be equal for the three groups) and this is assumed to be 6 (Rosner, 2000). Zcrit is the standard normal deviate corresponding to the selected significance criterion (i.e., 0.05; 95%=1,960). Zpwr is the standard normal deviate corresponding to the selected statistical power (i.e., 0.80; 95%=0.842). D is the minimum expected difference among the three means and D=5. Therefore: N=4×(6)2 (1.96+0.842)252N=45.22. However, 63 subjects were examined for this study in order to give room for attrition, but 56 completed the study BODY.MATERIALS AND METHODS.PROCEDURE.ETHICAL APPROVAL: Ethical approval for this study, with protocol number IPH/OAU/12/492 was obtained from the Health Research and Ethics Committee of the Institute of Public Health Obafemi Awolowo University, Ile Ife before the commencement of the study. The nature and purpose of the study was explained to each subjects and informed consent was obtained. BODY.MATERIALS AND METHODS.PROCEDURE.ASSESSMENT: Each subject was screened for eligibility with the following tests according to Konin et al. (2006). They were as follow: Ely's test, Laseague test, forward flexion, backward extension, side rotation, digital pressure along the spine. If at least two of the tests provoke pain at low back, such patient is qualified for the study. X-ray of such patient was then examined to rule out any red flag signs. BODY.MATERIALS AND METHODS.PROCEDURE.RANDOMIZATION: Subjects were randomly allocated into three groups using the fish bowl technique of simple random sampling. Participants were allocated into any of the three treatment groups according to the group a participant picked from the pool of groups stabilization in prone (SIP), stabilization in supine (SIS), prone and supine (SIPS) position in the bowl (Fig. 1). BODY.MATERIALS AND METHODS.PROCEDURE.MEASUREMENTS: Before the treatment the weight, height, waist and hip circumference of each subject were measured according to Lean et al. (2013). Initial pain intensity was assessed using VRS and disability was examined with Rolland Morris Disability Questionnaire. BODY.MATERIALS AND METHODS.OUTCOME MEASURES.VERBAL RATING SCALE: This was used to measure subject's present pain intensity verbally (i.e. pain at the time of study), at the beginning, 4th and 8th weeks of the treatment sessions. BODY.MATERIALS AND METHODS.OUTCOME MEASURES.THE ROLLAND MORRIS DISABILITY QUESTIONNAIRE: It is a commonly utilized instrument for measuring spinal disability as an outcome measure. Assessment of functional disability was done at baseline, 4th and 8th weeks of intervention using RMLDQ. BODY.MATERIALS AND METHODS.INTERVENTION: After the assessment, all the subjects received infrared radiation therapy for 15 min, according. Kneading massage with Neurogesic greaseless ointment was also given as base line treatments. BODY.MATERIALS AND METHODS.SIP POSITION: Subjects in prone group received stabilization exercise in prone lying position. The following were the three exercises done in prone lying position. The exercises were done according to Sung (2013). Exercise 1: Subject position the 2 arms by the side, lifting the head and chest off the plinth for 3 to 5 sec, two sets of 15 repetitions, performed as tolerated. Exercise 2 includes the alternate arm and leg lifted off the plinth from neutral position to extension simultaneously. The position was maintained for 3 to 5 sec and then returned, two sets of 15 repetitions performed as tolerated and in the third exercises one leg was lifted off the plinth with the hip hyper extended, knee extended and both arms stretched forward on the plinth. This position was maintained for 3 to 5 sec and then returned. Two sets of 15 repetitions performed as tolerated (Figs. 2–4). BODY.MATERIALS AND METHODS.SIS POSITION: Subjects in supine group received stabilization exercise in supine lying position. The following were the three exercises done according to Sung (2013). Alternate arm and leg lifted off the plinth from neutral position with the arm extended, knee and hip flexed. This position was maintained for 3 to 5 sec and then returned, two sets of 15 repetitions performed as tolerated. Exercise 2 includes knee drag to the chest from neutral position with both arms on the plinth. The knee was held off the plinth for 3 to 5 sec and then returned, two sets of 15 repetitions were performed as tolerated. And the third exercise involved the head and trunk slightly lifted off the plinth for 3 to 5 sec, two sets of 15 repetitions performed as tolerated (Figs. 5–7). BODY.MATERIALS AND METHODS.SIPS POSITIONS: Subjects in the combined group received SIPS lying positions combined (control group). Those in this group did any two exercise protocols each from both prone and supine positions. Exercise regimens were the same as in the other groups. BODY.MATERIALS AND METHODS.DATA ANALYSIS: Data were analyzed using SPSS ver. 17 (SPSS Inc., Chicago, IL, USA). The analyses were summarized using descriptive and inferential statistics. Analysis of variance (ANOVA) was used to compare the mean values of the physical characteristics of all the subjects in the three groups. Repeated measure ANOVA was used to compare the mean values of the outcome measures within and among the groups. Post hoc least of significant difference comparison was carried out where appropriate. Alpha level of 0.05 was set as significant level. BODY.RESULTS.PHYSICAL CHARACTERISTICS OF THE SUBJECTS: Presented in Table 1 were the physical characteristics of all the subjects in the three groups. It was observed that the mean age of subjects in the SIP group was 52.18±9.99 years, SIS group was 54.36±12.81 years while SIPS group was 48.63±11.90 years and the sum total was 51.72±11.51 years. There was no significant difference in the physical characteristics, among the three groups (P>0.05). BODY.RESULTS.COMPARISON OF PAIN INTENSITY AND DISABILITY INDEX AMONG PRETREATMENT, 4TH AND 8TH WEEK IN SIP POSITION GROUP: Presented in Table 2 is the result of comparison of outcome measures of stabilization exercise in prone position group. The result revealed that there was a significant reduction between pretreatment, 4th week of PI (F=22.500, P<0.001) and DI (F= 15.582, P<0.001) and 8th week. BODY.RESULTS.COMPARISON OF PAIN INTENSITY AND DISABILITY INDEX AMONG PRETREATMENT, 4TH AND 8TH WEEK IN SIS POSITION GROUP: Presented in Table 3 is the result of comparison of outcome measures of SIS position group. The result revealed that there was a significant reduction when pretreatment, 4th week of PI (F= 13.314, P<0.001) and DI (F=15.95 P<0.001) and 8th week were compared. BODY.RESULTS.COMPARISON OF PAIN INTENSITY AND DISABILITY INDEX AMONG PRETREATMENT, 4TH AND 8TH WEEK IN SIPS POSITION GROUP: Presented in Table 4 is the result of comparison of outcome measures of stabilization exercise in both prone and supine position group. The result revealed that there was a significant reduction between pretreatment and 4th week PI (F=17.894, P< 0.001) and DI (F=17.200 P<0.000) and 8th week. Comparison of PI and DI among the SIP, SIS, and SIPS were shown in Table 5. There was no significant difference when the pretreatment (F=0.57, P>0.05), 4th week (F=0.779, P>0.468) and 8 week (F=0.000, P>1.000) pain intensity and disability index were compared across the groups. BODY.DISCUSSION: The specific objective of this study was to assess which position is more effective for administration of stabilization exercises for patients with NSCLBP. The evaluated positions are prone lying, supine lying and combination of both supine and prone lying. It was observed from this study that the physical characteristics of the subjects in the three groups were not significantly different from each other. This is an indication that subjects in the three groups were comparable. The study observed a significant reduction at the 4th and 8th week of outcome measures when compared with the baseline of the subjects that underwent exercise in prone lying position. This inferred that stabilization exercises in prone position is indicated in the treatment of NSCLBP. Smith et al. (2014) reported that stabilization or (core stability exercise) have been suggested to reduce symptoms of pain and disability and form an effective treatment in patients with LBP. The determinant of spinal stability is the strength of the muscles and osteoligamentous structures of the trunk (Arokoski et al., 2004). In a case of excessive loading of the ostoeoligamentous structures of the spine that may occur in normal daily activities; which at times leading to damage of the spine, it is the responsibility of the lumbar and abdominal muscles to provide the essential stiffness needed for maximal loading in order to prevents such injury from overload (Gardner-Morse and Stokes, 1998). Stabilization exercises improves the strength of such ligaments and hence improves the activities of the back musculature. This study also observed a significant reduction when the baselines outcome measures were compared with the 4th and 8th week measurements of subjects that underwent exercise in supine lying position. It can be inferred that stabilization exercise in supine lying is indicated in the management of NSCLBP. In patients with chronic LBP, there is impairment of stabilizers of the back especially extensors with respect to coordination and functions as a result of disuse and deconditioning associated with muscle atrophy (Mannion et al., 2000). It is hence imperative that a specific back exercise programme is purposefully directed to this group of muscles, in which stabilization exercise in supine lying is accounted for. Again our study observed a significant reduction between the baselines, 4th and 8th weeks of pain intensity and disability of subjects that underwent exercise in prone and supine lying positions combined. This result is in line with the result of other authors which reported that specific lumbar stabilizing therapy can reduce the intensity of the pain and disability in LBP and pelvic girdle pain patients when used as a single therapy or combined with other treatments (Koumantakis et al., 2005). It was also in accordance with the work of Lee et al. (2012) which concluded that lumbar rehabilitation exercise program reduced pain and disability in patients with chronic mechanical LBP. Based on the stability of the trunk, there are local and global muscle stabilizers from which multifidus, transversus abdominis, and obliquus abdominis form the local; while longissimus thoracic, rectus abdominis, and obliquus externus abdominis muscles form the global stabilizers (Bergmark, 1989). Stabilization exercises especially in both prone and supine lying were directed to strengthen those muscles. Increment of muscle strength and balance in lumbar spine and relief of pain could be achieved by stabilizing exercise, functional exercise and resistance exercise (Park and Kim, 2012). More importantly, all exercises carried out in our study were isometric in nature. Researches have documented that isometric exercises has hypoalgesic effect on the contracting body part, the contralateral and a distant body part to the contracting one (Kadetoff and Kosek, 2007). This implies that isometric exercises activate a central inhibitory pain mechanism by static muscle contraction (Kosek and Lundberg, 2003); the mechanism involves upsurge in secretion of beta-endorphins, attention mechanism, activation of diffuse inhibitory controls or interaction of systems that regulate the pain (Lannersten and Kosek, 2010). In addition, isometric exercises activates the secretion of endogenous opioid system which reduces pain perception (Stagg et al., 2011). In conclusion, stabilization exercises carried out in prone, supine or the combination of both positions were effective on pain intensity and disability of patients with NSCLBP.

TITLE: Bisoprolol in the treatment of chronic heart failure ABSTRACT: Bisoprolol fumarate is a highly selective beta-1 receptor blocker. Bisoprolol has been extensively studied in three large mortality trials in stable chronic heart failure (CHF) patients. The CIBIS trial enrolled 641 patients and demonstrated the good tolerability of bisoprolol in a large CHF population, without evidence for any harmful effect. The CIBIS-II study was the first large randomized, double-blind, placebo-controlled study demonstrating in 2647 patients a dramatic reduction in mortality with a beta-blocking agent in CHF patients. CIBIS-III demonstrated in 1010 patients the equivalence of 2 different therapeutic strategies in de novo CHF patients. There was no difference in morbidity and mortality between sub-groups of patients receiving first bisoprolol or enalapril. These three trials also demonstrated the good tolerability of bisoprolol fumarate. Other studies were either limited in number of patients or not randomized. However, these studies confirmed the good tolerability of bisoprolol in CHF patients, even in elderly population. Bisoprolol fumarate is a selective beta-1 receptor blocker that significantly reduced morbidity and mortality in stable CHF patients. Bisoprolol is well tolerated with few significant side effects in different large trials. BODY.INTRODUCTION: Chronic heart failure (CHF) represents a major health problem and is one of the leading causes of hospitalization, in particular in elderly patients. Medical treatment had significantly improved during the last decade and several studies have demonstrated that angiotensin converting enzyme inhibitors (ACEI) and beta-blocker therapy are now the cornerstone of the treatment of patients with CHF (The SOLVD Investigators, 1991, 1992; Pfeffer et al 1992; CIBIS-II investigators and Committees 1999; MERIT-HF study group 1999; Packer et al 2001; Flather et al 2005). In the 1970s, Waagstein and collaborators first reported, in uncontrolled studies, that a treatment with a beta-blocker may dramatically improve symptoms and ventricular function in patients with mild to severe heart failure due to idiopathic dilated cardiomyopathy (Waagstein et al 1975; Swedberg et al 1979). Recently, different mortality trials have clearly demonstrated the beneficial effects of beta-blocker therapy. However, other trials did not demonstrate a significant mortality reduction (The Beta-Blocker Evaluation of Survival Trial Investigators 2001) or showed different effects between beta-blockers (Poole-Wilson et al 2003), leading international guidelines to recommend only 4 beta-blockers for CHF: bisoprolol, metoprolol succinate, carvedilol, and nebivolol (Hunt et al 2005; Swedberg et al 2005). Beta-blocker agents represent a large heterogeneous family with one important difference concerning receptor selectivity. In CHF, three recommended drugs are beta-1 adrenoreceptor blockers, namely bisoprolol, metoprolol succinate, and nebivolol, and one, carvedilol, is a beta-1–beta-2 adrenoreceptor blocker with additional alpha-1 vasodilatory activity. We will not focus our review on the comparison of these different beta-blockers. This review will summarize the results of different studies with bisoprolol in stable CHF patients, and particularly the different Cardiac Insufficiency Bisoprolol Studies (CIBIS). BODY.PHARMACOKINETICS OF BISOPROLOL FUMARATE: Bisoprolol fumarate is a beta-1 receptor blocker, very freely soluble in water, with a molecular weight of 383.48 kDa (Leopold et al 1986; McGavin and Keating 2002). Bisoprolol is well absorbed after oral administration with a bioavailability of 90% and has a low plasma protein binding (30%). Food intake does not modify its biodisponibility. Bisoprolol is metabolized in the liver in inactive metabolites (50%) and eliminated (50%) via renal excretion without metabolisation. The plasma elimination half-life ranges from 10 to 12 hours. The pharmacokinetics of bisoprolol is minimally changed in patients with hepatic impairment or with a creatinine clearance between 10 and 30 mL/min. In patients with severe renal impairment (creatinine clearance <10 mL/min), the exposure to bisoprolol is increased 2-fold. The plasma elimination half-life increases to 24.2 hours in the latter case (Kirch et al 1987). There is limited information available on the pharmacokinetics of bisoprolol in patients with stable heart failure. In NYHA class III patients, receiving a chronic treatment of 10 mg/day, peak plasma concentrations were 78% higher, with a plasma elimination half-life reaching 17 hours. BODY.PHARMACOKINETICS OF BISOPROLOL FUMARATE.BETA-RECEPTOR SELECTIVITY: Different experimental studies have demonstrated that bisoprolol fumarate is one of the most selective beta-1 adrenoreceptor blockers, with a 19-fold higher affinity for the beta-1 receptor than for the beta-2 receptor (Wellstein et al 1986; Smith and Teilter 1999). Even at higher dose, there is no beta-2 blockade effect. Nebivolol is 3.5 times more beta-1 adrenoreceptor selective than bisoprolol in human myocardium and in vitro study (Bundkirchen et al 2003). In a randomized, double-blind, placebo-controlled, cross-over study in 12 patients with stable angina pectoris and non-asthmatic chronic obstructive lung disease, a single dose of 100 mg of atenolol mg was compared with 20 mg of bisoprolol. Both drugs had a similar effect on heart rate but airway resistance increased with atenolol, whereas was unchanged with bisoprolol compared with placebo (Dorow et al 1986). Similar results were found in another randomized, double-blind, placebo-controlled, cross-over study in 12 hypertensive asthmatic patients, comparing the airway resistance after the administration of 10 or 20 mg of bisoprolol, 100 mg of atenolol, or placebo (Chatterjee 1986). BODY.EFFICACY STUDIES: CIBIS TRIALS: Bisoprolol fumarate has the advantage to have been studied largely in 3 major mortality trials in stable CHF patients, demonstrating the important benefits of this beta-1 blocking agent and its good tolerability. BODY.EFFICACY STUDIES: CIBIS TRIALS.CIBIS: CIBIS was the first randomized, double-blind, placebo-controlled study with the primary objective of evaluate the impact of bisoprolol on mortality in patients with heart failure (CIBIS Investigators and Committees 1994). The inclusion criteria were ambulatory CHF patients in NYHA class III-IV, with a left ventricular ejection fraction (LVEF) <40%, receiving diuretics and vasodilator therapy and not registered on a waiting list for heart transplantation. At this time, the tolerability of a chronic beta-blocker therapy was unknown in a large population. An important prerequisite before inclusion was the need of a clinical phase of stability, without any episode of heart failure decompensation and the absence of major modification of heart failure therapy in the last 3 weeks before randomization. The initial dose of bisoprolol was 1.25 mg once daily, which could be increased 48 hours later to 2.5 mg/day and 1 month after to the maximal dose of 5 mg/day. It was not a forced titration procedure and each investigator was free to give to their patient, according to their clinical status, one of the four doses: 1.25, 2.5, 3.75, or 5 mg/day. CIBIS enrolled 641 patients with CHF, in NYHA class III in the vast majority (609, 95%). Clinical characteristics are summarized in Table 1. During a mean duration of follow-up of 1.9 ± 0.1 years, there were 120 deaths, 67 (20.9%) in the placebo group and 53 (16.6%) in the bisoprolol group (Figure 1). This difference was not statistically significant with a risk reduction of 0.80 (0.56–1.15). Figure 1Survival curves in CIBIS patients. Table 1 Clinical characteristics of the different CIBIS a trials CIBIS CIBIS II CIBIS III n 641 2647 1010 Age, years (mean) 59 61 72 Female 17% 19% 32% NYHA class III/IV 95%/5% 83%/17% 50%/0% Ischemic heart failure 55% 50% 62% Idiopathic dilated cardiomyopathy 36% 12% 10% ACEI 90% 96% – Diuretics 100% 99% 90% Heart rate, bpm 83 ± 1.5 81 ± 15 79 ± 13 Systolic blood pressure, mmHg 126 130 ± 19 134 ± 17 Diastolic blood pressure, mmHg 78 80 ± 11 80 ± 10 LVEF (%) 25 ± 0.9 27 ± 6 28.8 ± 5 Mean dose bisoprolol, mg 3.8 ± 0.2 a for CIBIS, quantitative results are presented as mean ± SEM. Abbreviations: ACEI, angiotensin converting enzyme inhibitor; CIBIS, Cardiac Insufficiency Bisoprolol Studies; LVEF, left ventricular ejection fraction. Despite this negative result, CIBIS was a very important study demonstrating the good tolerability of bisoprolol in a large CHF population, without evidence for any harmful effect of the beta-blocker therapy. Bisoprolol decreased the rate of hospitalization for worsening heart failure (107 (17%) compared with 154 (24%) in the placebo group, p < 0.001). Moreover, more patients in the bisoprolol group than in the placebo group improved their functional status. At the end of the trial, 21% of the patients receiving bisoprolol had an improvement of at least 1 class, compared with 15% in the placebo group (p < 0.03). Deterioration of at least 1 NYHA class was similar in the two sub-groups (13% vs 11%). BODY.EFFICACY STUDIES: CIBIS TRIALS.CIBIS-II: The CIBIS-II study was the first large randomized, double-blind, placebo-controlled study demonstrating a dramatic reduction in mortality with a beta-blocking agent in CHF patients (CIBIS-II investigators and Committees 1999). The equivalent study for the ACE-I was the CONSENSUS trial with enalapril (The CONSENSUS Trial Study Group 1987). CIBIS-II enrolled 2647 class III-IV stable CHF patients with a LVEF <35%, who were receiving diuretics and vasodilator therapy. In contrast to the CIBIS study, bisoprolol titration was forced to the maximal tolerated dose, with the highest possible dose of 10 mg/day. The clinical characteristics of the CIBIS-II population are summarized in Table 1. Bisoprolol induced a significant heart rate reduction (of 9.8 ± 14.7 beats/min), with a limited effect on blood pressure (systolic blood pressure reduction of 4.1 ± 16.4 mmHg with bisoprolol but of 2.3 ± 16.4 mmHg with placebo) (Lechat et al 2001). The study was prematurely stopped because of the significant mortality benefit associated with bisoprolol (Figure 2). After a mean follow-up period of 1.3 years, there were 384 deaths, with 228 (17.3%) deaths in the placebo arm and 156 (11.8%) in the bisoprolol arm (hazard ratio (HR) of 0.66 [0.54–0.81]). Bisoprolol significantly reduced cardiovascular mortality (HR: 0.71 [0.56–0.9]), sudden cardiac death (HR: 0.56 [0.39–0.80]), hospital admission (HR: 0.80 [0.71–0.91]), and hospital admission for worsening heart failure (HR: 0.64 [0.53–0.79]) compared with placebo. The magnitude of the benefit was similar in NYHA sub-classes, and independent of the etiology of heart failure. However, in patients with atrial fibrillation (n = 521, 20%), bisoprolol did not decrease total mortality when compared with placebo. This result must be taken with caution because of the limited number of patients with atrial fibrillation and the retrospective nature of the analysis (Lechat et al 2001). Although heart rate at baseline and heart rate reduction were independently associated with survival, there was no interaction with bisoprolol, suggesting that the beneficial effect of bisoprolol was not influenced by these two parameters, and in particular by the extent of the heart rate reduction (Lechat et al 2001). In CIBIS, retrospective analysis suggested that patients with a LVEF ≤20% benefit more from bisoprolol than other patients (Funck-Brentano et al 2000). However, this was not confirmed in CIBIS-II, where the beneficial effect of bisoprolol was independent of the level of left ventricular dysfunction. Figure 2Survival curves in CIBIS-II patients. BODY.EFFICACY STUDIES: CIBIS TRIALS.CIBIS-III: The third important study with bisoprolol in CHF patients was designed to determine what drug to initiate in a de novo, stable, CHF patient, either ACEI or beta-blocker (Willenheimer et al 2005). Because the beneficial effects of ACEI have been demonstrated first (The CONSENSUS Trial Study Group 1987; The SOLVD Investigators 1991, 1992; Pfeffer et al 1992), all the subsequent studies have studied the impact on mortality of a new drug or device on top of ACEI (Pitt et al 1999; Cleland et al 2005). This question is of importance since the effects of beta-blockers in CHF patients are observed quickly after their introduction, in particular with a reduction of sudden cardiac death, which is the most prevalent cause of death in this population. Moreover, instead of ACEI that blocks one system, beta-blockers effectively inhibit 2 systems, the sympathetic system and the renin-angiotensin system. CIBIS-III was a multicenter, prospective, randomized, open label, blinded end-point evaluation study, with 2 parallel groups (Figure 3). Inclusion criteria were different from the previous CIBIS studies. Eligible patients were patients older than 65 years, in NYHA class II or III, with a LVEF ≤35% and of course receiving neither ACEI nor beta-blocker. All patients were clinically stable for at least 7 days before the inclusion. Figure 3Study design of CIBIS-III. Double titration with monotherapy and combination phases for each arm, bisoprolol-first and enalapril-first. As in CIBIS-II, the titration was forced, depending on patient's tolerance, for both drugs with a target dose of 10 mg/day for bisoprolol and of 20 mg/day for enalapril. After the titration, there was a 6-month monotherapy period, followed by a new titration in order to have the combination of enalapril and bisoprolol for a combined period. The primary end-point was the combination of all-cause mortality or all-cause hospitalization. CIBIS-III was designed as a non-inferiority trial comparing the impact on the primary end-point of the initiation of bisoprolol first compared with enalapril. CIBIS-III enrolled 1010 patients who were followed during a mean period of 1.22 ± 0.42 years. Clinical characteristics of the study population are presented in Table 1. Because of the different inclusion criteria, some characteristics of CIBIS-III patients were different compared with those of patient enrolled in previous CIBIS studies. Patients in CIBIS-III were older, more often female, and less symptomatic (no NYHA class IV patients and half of the population was in NYHA class II). The results did not demonstrate any significant difference between the two strategies (Figure 4). There were 178 patients with the primary end-point in the bisoprolol-first group and 186 in the enalapril-first group (35.2 vs 36.8%). At the end of the monotherapy period, 109 bisoprolol-first patients had a primary end-point compared with 108 enalapril-first patients. There were fewer deaths in the bisoprolol-first group than in the enalapril-first group, but the difference was not statistically significant (65 vs 73, HR: 0.88 [0.63–1.22], p = 0.44). There was a non-significant increase in the number of patients having a hospitalization for worsening CHF in the bisoprolol-first group compared with the enalapril-first group (63 vs 51 patients, respectively, HR = 1.25 [0.87–1.81], p = 0.23). Figure 4Kaplan-Meier curves of the combined primary end-point (death or hospitalization) in CIBIS-III patients. Intention-to-treat analysis. We can conclude, from the results of the CIBIS-III trial, that there was no difference in terms of efficacy and safety between the two strategies of treatment initiation in stable CHF patients. BODY.NON-MORTALITY STUDIES: There are few other studies with bisoprolol in CHF patients, but all these studies are either small or not randomized. A small study analyzed the impact of bisoprolol on LVEF using magnetic resonance imaging (Dubach et al 2002). It was a randomized, double-blind study in 28 patients with a mean age of 57 years, 13 receiving bisoprolol and 15 placebo. Eight patients in each group had an ischemic cardiomyopathy and others had a dilated cardiomyopathy. All the patients were receiving ACEI and 24 were taking diuretics. The mean dose of bisoprolol was 7.19 mg/day. At baseline, at 6 months, and at 1 year after the introduction of bisoprolol, patients performed a cardiopulmonary exercise test and a magnetic resonance imaging of the heart. Bisoprolol produced a significant reduction in heart rate associated with a non-significant increase in peak oxygen consumption. Left ventricular ejection fraction improved at 1 year only in the bisoprolol sub-group, from 25 ± 7% to 36.2 ± 9% (p < 0.05). We performed an observational study in consecutive stable patients with CHF and LVEF <40% (de Groote et al 2004). All the patients received maximal tolerated doses of renin inhibitors and were clinically stable at least 2 months before the introduction of bisoprolol. All the patients performed a cardiopulmonary exercise test, and underwent a radionuclide angiography, before and 3 months after maximal tolerated doses of bisoprolol had been reached. Blood samples were drawn for hormonal determinations. We included 201 patients, with a mean age of 54 ± 12 years; 34% had ischemic cardiomyopathy and the vast majority was in NYHA class I or II (75%). The mean dose of bisoprolol was 8.8 ± 2.4 mg/day. Bisoprolol was associated with an improvement in NHYA functional class, and a significant decrease in heart rate without any effect on blood pressure. There was a small significant improvement in peak oxygen consumption (from 16.1 ± 5 to 16.8 ± 5.5 mL/min/kg, p = 0.015) with a significant decrease in peak expiratory exchange ratio for carbon dioxide production (from 38 ± 7.4 to 34 ± 6.7, p = 0.005). This suggests an improvement in the exercise ventilatory efficacy with bisoprolol. LVEF improved from 31 ± 11 to 41 ± 13% (p < 0.0001). This favorable effect was associated with a reduction in ventricular volumes and with an improvement in the left ventricular filling function. Right ventricular ejection fraction also significantly improved with bisoprolol. Finally, plasma levels of type A and type B natriuretic peptides and norepinephrine were significantly reduced with bisoprolol. Of course, one of the biggest limitations of the study is the lack of a control group. However, at this time it was not ethical to give placebo to CHF patients. In addition, another observational study in 87 CHF patients showed that beta-blockade improved LVEF in the majority of patients. However, significant improvement in LVEF did not enhance functional capacity consistently in CHF patients (Ennezat et al 2005). BODY.DOSES: In CIBIS and CIBIS-II, mean doses were significantly greater in the placebo arm compared with the bisoprolol arm. In CIBIS, respective doses were 4.5 ± 0.1 and 3.8 ± 0.2 mg/day. Half of the patients received 5 mg of bisoprolol in CIBIS, and 43% reached 10 mg in CIBIS-II and 67% at least 5 mg/day. In CIBIS-III, during the monotherapy period, 65% of the patients reached the target dose in the bisoprolol-first group compared with 84% in the enalapril-first group. At the end of the study, in the bisoprolol-first group, 65% of the patients had the target dose of bisoprolol and 67% the target dose of enalapril. In the enalapril-first group, respective percentages were 54% and 77%. A retrospective analysis looking at the doses achieved after the forced titration in CIBIS-II revealed that patients in the lower tertile of doses were older, more often in NYHA class IV, and had a lower blood pressure. However, the beneficial effect of bisoprolol was similar whatever the dose received (Simon et al 2003). BODY.TOLERABILITY OF BISOPROLOL: Curiously, no precise information on non-serious adverse events is available from the three CIBIS studies. Bisoprolol is well tolerated. In CIBIS, percentages of patients with non-serious adverse events were similar in the 2 arms: 26% in the placebo group and 23% in the bisoprolol group. Two cases of sinus bradycardia and 2 cases of atrioventricular blockade were recorded in the bisoprolol group. Significant hypotension was recorded in 3 patients in the placebo group and in 5 in the bisoprolol group. In CIBIS-II, the percentage of premature treatment withdrawal was also similar in the 2 arms (15%), but there were more bradycardia with bisoprolol (14 vs 2, p < 0.004). The main cause of permanent treatment withdrawal was patient's or investigator's personal decision. Age (≥68 years) and heart rate at inclusion were both independent predictors of permanent treatment withdrawal. In patients with a heart rate <72 beats/min at inclusion, the risk of permanent bisopropol withdrawal was 1.97 (1.38–2.80). Of importance, the beneficial mortality effect of bisoprolol was lost in patients having a permanent treatment withdrawal (Funck-Brentano et al 2001). In CIBIS-III, during the monotherapy period, 35 patients (6.9%) had a permanent bisoprolol withdrawal compared with 49 (9.7%) with enalapril. During the combination period, in the bisoprolol-first group, 19 patients (4.2%) had a permanent withdrawal of bisoprolol and 47 patients (10.4%) a permanent withdrawal of enalapril. In the enalapril-first group, corresponding values were 24 (5.5%) and 16 (3.7%). Different post-hoc analyses were performed from the CIBIS-II study population (Erdmann et al 2001). In CIBIS-II, a significant and similar mortality reduction with bisoprolol was observed in the 539 CHF elderly patients (≥71 years) compared with younger patients (HR: 0.68 [0.48–0.97]). Although sudden death was not significantly reduced in the elderly population, rates of pump failure death and CHF hospitalizations were reduced, with a similar permanent treatment withdrawal as compared with younger patients. These results were confirmed by the meta-analysis of both CIBIS trials (Leizorovicz et al 2002). Using the Cockroft Gault equation, 849 patients (32%) had renal impairment with a creatinine clearance <60 mL/min (Erdmann et al 2001). These patients had a similar benefit with bisoprolol compared with patients who had a greater creatinine clearance. However, the rate of permanent treatment withdrawal was significantly higher in patients with renal impairment, reaching almost 25% in patients with a creatinine clairance <60 mL/min and 40% in the 63 patients with a creatinine clearance <30 mL/min. Some other studies have looked at the tolerability of bisoprolol in CHF patients. A small study showed a similar tolerability of the initiation of carvedilol and bisoprolol in 87 patients (Galatius et al 2004). An observational study in elderly CHF patients analyzed the tolerability of bisoprolol. Patients included were older than 70 years, in chronic NYHA class II or III, receiving diuretics and a renin system inhibitor and having a LVEF <40%. As for CIBIS, the inclusion in the study required a period of 6 weeks of clinical stability before the introduction of bisoprolol (Baxter et al 2002). Baxter et al enrolled 51 patients with a mean age of 78 years, with 23 women. After the first dose of 1.25 mg of bisoprolol, the majority of the patients had a hypotension (86%); in 28 of these patients, blood pressure fell below 100 mmHg and in 16 patients the blood pressure fall was greater than 20 mmHg but with a systolic blood pressure >100 mmHg. Of interest, despite a great frequency of hypotension, only 4 patients, all having a blood pressure <100 mmHg, experienced symptoms and complained dizziness. During the titration period, 35 patients tolerated bisoprolol (69%) with a mean dose of 7.6 mg/day. The mean reason for withdrawal was hypotension in 7 patients (including the 4 previous patients) and fatigue (3 other patients). Bradycardia was not a cause for bisoprolol withdrawal. Twenty-one patients tolerated 10 mg/day of bisoprolol and 9 received less than 5 mg/day. The main reasons for not reaching the target dose of 10 mg/day were hypotension in 7 cases, fatigue in 5 cases, and bradycardia in 1 case. In conclusion, this study showed that in elderly CHF patients, hypotension is the major symptom leading to treatment withdrawal or to keep low doses of bisoprolol. However, if bisoprolol was well tolerated, it was possible to reach the target dose of 10 mg/day without problem. Another study looked at the tolerability of bisoprolol after its initiation by the primary care physicians (Schuchert 2005). This prospective study included 328 patients with stable CHF receiving diuretics and renin inhibitors. Mean age was 63 ± 10 years, and 145 patients were in NYHA class III and 1 in class IV. The maximal tolerated dose was 7.2 ± 3.15 mg/day, 61% of the patients receiving at least 7.5 mg of bisoprolol. NYHA class significantly improved, from 2.4 ± 0.5 to 1.8 ± 0.6 (p < 0.0001) at the end of the 24 week study period. At the end, 74% of the patients had an improvement and only 5% a worsening in functional class. Bisoprolol was withdrawn in 57 patients (17%), of whom 40 related to adverse events. No patient had symptomatic bradycardia. This study demonstrated that bisoprolol could safely be introduced by primary care physician who did not have the same level of experience in CHF than physicians involved in the large mortality trials. In summary, bisoprolol is well tolerated, even if in elderly patients treatment withdrawal seems to be more frequent than that observed in the large mortality trials. The main reason for not reaching the target dose of bisoprolol or for bisoprolol withdrawal is hypotension. However, all the previous studies have excluded patients with resting heart rate <60 beats/min, patients with resting systolic blood pressure <100 mmHg and other main contra-indications to beta-blocker such as asthmatic patients. In the future, it will be important to have more information about tolerance of bisoprolol in some subgroups of patients, in particular elderly patients (>75 years), patients with severe renal failure, patients with chronic obstructive pulmonary diseases, and patients in NYHA class IV. Another important question is the management of CHF patients receiving chronic bisoprolol therapy and hospitalized for cardiac decompensation. Currently, the management of these patients depends on their clinical status and the experience of the practitioner. There are three possibilities: no modification, reduction of the doses of beta-blocker (and it is often a half reduction of the dose), or transitory interruption of the beta-blocker. An ongoing French study, B-Convinced, will try to answer to this important question. This study was designed as a non-inferiority trial comparing two strategies after an acute CHF decompensation: to stop or to pursue the beta-blocker. Finally, another unresolved question is to know how long the beneficial effects of beta-blocker therapy in CHF patients will be maintained. BODY.CONCLUSIONS: Bisoprolol fumarate is a potent, highly selective beta-1 adrenergic blocker. Large mortality trials have clearly demonstrated the beneficial effects of bisoprolol on mortality and on morbidity compared with placebo. These favorable effects are associated with a reverse remodelling of the left ventricle and a significant improvement in LVEF. Finally, bisoprolol is well tolerated, with a limited number of side effects leading to its permanent withdrawal.

TITLE: Volumetric analysis of bone substitute material performance within the human sinus cavity of former head and neck cancer patients: A prospective, randomized clinical trial ABSTRACT.BACKGROUND:: In numerous animal and human studies, it could be detected that in bone augmentation procedures, material's physicochemical characteristics can influence the cellular inflammatory pattern and therefore the integration in the host tissue. Histological, histomorphometrical, and clinical analyses of the integration of the biomaterial in the surrounding tissue are well established methodologies; however, they do not make a statement on volume and density changes of the augmented biomaterial. ABSTRACT.AIMS:: The aim of the present study was to assess the volume and density of a xenogeneic (Bio-Oss®, BO) and a synthetic (NanoBone®, NB) bone substitute material in split-mouth sinus augmentations in former tumor patients to complete histological and histomorphometrical assessment. ABSTRACT.METHODS:: Immediately and 6 months after sinus augmentation computed tomography scans were recorded, bone grafts were marked, and the volume was calculated with radiologic RIS-PACS software (General Electric Healthcare, Chalfont St. Giles, Great Britain) to determine the integration and degradation behavior of both biomaterials. ABSTRACT.RESULTS:: Radiographic analysis revealed a volume reduction of the initial augmented bone substitute material (i.e. 100%) to 77.36 (±11.68) % in the BO-group, respectively, 75.82 (±22.28) % in the NB-group six months after augmentation. In both materials, the volume reduction was not significant. Bone density significantly increased in both groups. ABSTRACT.CONCLUSION:: The presented radiological investigation presents a favorable method to obtain clinically relevant information concerning the integration and degradation behavior of bone substitute materials. BODY.INTRODUCTION: In a previously published clinical trial, the performance of two bone substitute materials for sinus augmentation was analyzed. In a group of patients with head and neck cancer and highly atrophic maxillary bone, sinus augmentation was performed with the xenogeneic, bovine- based bone substitute material Bio-Oss® (BO, Geistlich Biomaterials, Wolhusen, Switzerland) and the alloplastic bone substitute material NanoBone® (NB, Artoss, Rostock, Germany) followed by insertion of dental implants 6 months later.[1] The xenogeneic BO and the alloplastic NB have been investigated histologically and histomorphometrically with a focus on inflammatory response in the host tissue, new bone formation, and biomaterial degradation. Therefore, simultaneously with the insertion of dental implants, biopsies of the augmented area were extracted and processed. It was shown that NB granules were embedded in connective tissue, originating from the covering soft tissue and newly formed bone, ingrowing from the residual alveolar crest. Analyzing the fraction of remaining bone substitute material after an observation period of 6 months, the amount of the bovine-based bone substitute material BO was significantly higher than the amount of the synthetic hydroxyapatite (HA) bone substitute material NB. This could be a sign of the degradation of the latter through a foreign body reaction, as multinucleated giant cells and macrophages were present in a high number, as supplied through connective tissue with a high vessel density. According to the different origins, types of processing, and physicochemical structures of both investigated biomaterials, two different processes of biomaterial integration in the human organism were obvious. BO, a well-researched and frequently used xenogeneic bone substitute material, exhibited good integration in the sinus cavity and the formation of a sufficient implantation bed in the above-mentioned split-mouth trial in oral cancer patients. The biomaterial induced almost no signs of a foreign body reaction with only few multinucleated giant or foreign body cells while new bone ingrowth originating from the residual maxillary bone tissue was supported.[1] Patients with atrophic upper alveolar bone who have been successfully treated for oral cancer and who suffer from a reduced life expectance and life quality should be orally rehabilitated to restore articulation and mastication. Therefore, dental implants have been demonstrated to achieve reliable retention for fixed and removable dentures in both healthy and tumor populations.[234] Due to alveolar atrophy or tumor resection, the amount of available bone for the placement of dental implants is reduced in the majority of patients, and bone augmentation becomes necessary to achieve a sufficient implantation bed. To date, autologous bone is still postulated by clinicians and scientists to be the gold standard because of its osteoinductive, osteoconductive, and osteogenic properties.[5] However, the augmentation with autologous bone comes along with several disadvantages, such as limited amount of bone, second surgical site, and risk of donor site morbidity.[6] To avoid the burden of an additional operation, allogeneic bone substitutes from human living or mortal donors, xenogeneic bone substitutes of bovine, porcine, or equine origin, and alloplastic bone substitute materials have been developed and well researched in recent years. In several clinical trials, the high biocompatibility, as well as the support of new bone formation after augmentation in dental and maxillofacial surgery, was demonstrated for these materials.[789101112] Apart from xenogeneic bone substitutes, alloplastic bone substitutes, mainly originating from HA, biphasic calcium phosphate ceramics (BCP), α- and β-tricalcium phosphate ceramics, or bioactive glasses, are widespread alternatives to autologous bone transplantation.[1314] In recent years, alloplastic bone substitute materials have been investigated in several in vivo and clinical trials to analyze the tissue reaction and osseointegration of these materials in humans.[101115] In a preliminary clinical study, the potential of the same nanostructured HA-based biomaterial (NB) was assessed for sinus augmentation in humans. Qualitative histologic analysis of biopsies taken after an integration period of 6 months revealed integration of the biomaterial within the sinus cavity and a high osteoclast activity, resulting in formation of new bone at the margin of the biomaterial.[10] In another clinical trial of sinus augmentation, the de novo bone formation capacity of the nanocrystalline HA bone substitute NB was assessed histologically and histomorphometrically at two time points, 3 and 6 months after implantation, in the human sinus cavity. The bone metabolism within the residual bone and augmented region was emphasized. New bone tissue formation starting from the bone-biomaterial interface could be observed in both study groups while no statistically significant difference in new bone formation could be detected after 3 and 6 months. Therefore, it was concluded that implant insertion in regions augmented with this bone substitute material could be considered already after 3 months.[11] In the present study, the histological, histomorphometrical, and clinical results of the performed split-mouth trial were completed by a three-dimensional radiographic analysis, to determine the volumetric changes of the two different bone substitute materials, which have been augmented in the sinus cavity of patients with cancer anamnesis. Measurements were made at two time points, directly after augmentation and 6 months later at the insertion of dental implants in the augmented region. With RIS-PACS software (General Electric Healthcare, Chalfont St. Giles, Great Britain), the augmented biomaterial was marked in three-dimensional computed tomography (CT) scans in the Digital Imaging and Communications in Medicine (DICOM) format and calculated by rendering in radiographic images. Volume changes were calculated as a percentage of the inserted biomaterial fraction to determine the potential degradation of both xenogeneic and synthetic biomaterial. With this method, previously obtained histologic and histomorphometric results that analyzed the tissue reaction to both materials in the human sinus cavity could be critically reviewed. The aim was to investigate whether the above-mentioned results gained by histomorphometrical analyses can somehow be reproduced by applying the nonsurgical and thus "non-invasive" method of CT. BODY.MATERIALS AND METHODS.STUDY DESIGN/PATIENT POPULATION: The present, randomized, prospective clinical study was approved by the Ethics Committee of the University of Frankfurt am Main and conducted according to the fifth revision of the World Medical Association Declaration of Helsinki 2000. All patients gave informed consent before the sinus augmentation procedure. As previously described, eight partly or completely edentulous patients (five women and three men) with squamous cell carcinoma, affecting different sites of the oral cavity to varying extents, from the Department for Oral, Cranio-maxillofacial and Facial Plastic Surgery, Frankfurt am Main, were enrolled in the study. In all patients, the cancer was completely cured before sinus augmentation was performed in a split-mouth design with each of the bone substitute materials BO and NB inserted randomly on one side. After 6 months of healing, dental implants were inserted in the augmented regions [Table 1]. Simultaneously, bone biopsies were extracted for histological and histomorphometrical analyses.[1] Table 1 Detailed overview of the number and sites of placed implants in the augmented and nonaugmented regions. The content of this figure has already been published by the author in a different form[ 1 ] Time points of CT scan recording were set into regular cancer staging order to avoid unnecessary irradiation of the patients. Immediately after sinus augmentation, CT scans were recorded to control the augmentation results and 6 months later before implant placement to plan the implant position. CT scans were analyzed with the RIS-PACS software from an experienced radiologist who was blinded to the surgical procedure and the distribution of the augmented biomaterials to evaluate volumetric changes of the augmented bone substitute material within the sinus cavity. BODY.MATERIALS AND METHODS.SURGICAL PROCEDURE: According to the previously described methods, sinus augmentation was conducted in eight patients in general anesthesia. Crestal incision and mobilization of a vestibular-based mucoperiosteal flap were performed to obtain access to the processus maxillaris.[1] With a Piezosurgery® device (Mectron, Cologne, Germany), a lateral window was extracted, and the Schneiderian membrane was exposed. Using sinus elevator instruments of different shapes and sizes, the membrane was elevated to enlarge the subantral space. Afterward, both bovine-based bone substitute material BO and alloplastic bone substitute material NB, mixed with blood extracted from the surgical site, were randomly implanted in the sinus cavities of each side. The lateral window was covered with a native collagen membrane (Bio-Gide®, Geistlich, Wolhusen, Suisse) and wound closure was achieved with single sutures. Six months after augmentation, dental implants (CAMLOG® Screw-Line, Camlog Biotechnologies, Basel, Switzerland) were placed simultaneously with the extraction of bone biopsies for the aforementioned histological investigation. A detailed itemization of implant numbers and sites is given in Table 1. BODY.MATERIALS AND METHODS.BONE GRAFTING SUBSTITUTES.NANOBONE: NanoBone® (NB, Artoss, Rostock, Germany), a completely synthetic bone substitute material, is composed of HA crystallites embedded in a matrix of structured silica gel. The granules, with an average size of 60 μm, are manufactured with a sol-gel technique. Thereby, sintering can be avoided. The manufacturing process results in a pore size within the bone substitute material of 100–1000 μm (macropores) or 2–10 μm (micropores), an internal surface of up to 84 m2/g, and a material porosity of 60%–80%.[1617] BODY.MATERIALS AND METHODS.BONE GRAFTING SUBSTITUTES.BIO-OSS: Bio-Oss® (BO, Geistlich Biomaterials, Wolhusen, Switzerland), a xenogeneic deproteinized bone mineral with bovine origin, is processed by sintering in a highly alkaline solution and sterilized by gamma radiation. Organic components are removed by a chemical extraction process to avoid disease transmission. Bone substitute granules have a diameter from 0.25 to 1.0 mm, pore sizes ranging from a few nanometers to 1500 nm, and a material porosity of 70%–75%.[81819] BODY.MATERIALS AND METHODS.RADIOGRAPHIC ANALYSIS.RADIOGRAPHIC IMAGES: Images were recorded for preoperative diagnostics and planning, postoperative augmentation control, and 6 months before implant placement with standardized low-dose CT (Sensation 16 and Volume Zoom, Siemens Healthcare, Erlangen, Germany) and an effective dose of 110 mA. The following settings were used: tube voltage: 120 kV, scan time: 3–10 s, layer thickness: 2 mm (0.75 mm) with sagittal and coronal reconstruction, and table movement/pitch: 0.9. Patients participating in this study were all enrolled in clinical and radiological aftercare due to head and neck cancer anamnesis. CT images were recorded in agreement with the regular radiologic tumor aftercare to avoid unnecessary radiation. BODY.MATERIALS AND METHODS.RADIOGRAPHIC ANALYSIS.ANALYZING SOFTWARE: CT scans were analyzed with the software RIS-PACS AW Suite 2.0 (General Electric Healthcare, Chalfont St. Giles, Great Britain) to determine the augmentation volume immediately after biomaterial insertion and 6 months later. The RIS-PACS system is suitable for visualizing and processing multidimensional images from different equipment (magnetic resonance tomosynthesis, CT, etc.,) and allows analysis and processing images in the DICOM format. First, images, data, and patients' names were anonymized to guarantee objective, blinded analysis. The software shows the recorded CT scan as a number of two-dimensional slices, depending on slice thickness, in three different projections (coronal, axial, and sagittal). Further, a three-dimensional scan could be generated from the different two-dimensional slides. In the present study, from the large number of software tools, "volume viewer" and "volume measurement" were used, to evaluate change of volume and density of the augmented materials directly after the augmentation and 6 months later. Furthermore, the density of the augmented bone substitute material and the zygomatic bone (reference bone) was analyzed with the software tool "density measurement." Analysis of the graft volume and density with the above-mentioned software was performed as follows: Step 1: All layers of the CT in which the augmented area could be identified were chosen for analysis. With the "polygon" tool, the augmented biomaterial was marked at its margins [Figure 1] Step 2: The software calculated the volume of the augmented biomaterial. The transitions between the different scans were interpolated according to the appearance of the augmented biomaterial by the softwareStep 3: The volume fraction of the graft was calculated, describing the volume fraction in cm3/mm3 Step 4: An area in the grafted bone substitute material and in the reference zygomatic bone was marked for the measurement of bone density [Figure 2]. Figure 1Coronal sequence of the analyzed computed tomography images with marked augmentation material in both sinus cavities Figure 2Transversal sequence of the analyzed computed tomography images with the marked reference zygomatic bone for bone density measurements BODY.MATERIALS AND METHODS.STATISTICS: The data from the volumetric and density measurements of both biomaterial groups were compared across the study groups at different time points, immediately after augmentation and 6 months later, with analysis of variance followed by Fisher's least significant difference tests. Post hoc assessments were performed with GraphPad Prism software (Prism 6 V6.01, GraphPad Software Inc., La Jolla, USA). Inter- (*) and intra-individual (•) significant differences were deemed significant when P < 0.05 (*/• P < 0.05) and highly significant when P < 0.01 (**/•• P < 0.01) and 0.001 (***/••• P < 0.001). Finally, the data were presented graphically as mean values ± standard deviations. BODY.RESULTS.VOLUMETRIC CHANGES OF THE GRAFTS.BIO-OSS: The radiologic analysis of the BO graft revealed volumetric reduction in all patients. The average graft volume immediately after augmentation was 2547.75 mm3 (±1287.41 mm3), while 6 months after augmentation, the average graft volume decreased to 1971.00 mm3 (±1046.21 mm3). The average percentage of the BO graft volume after 6 months was 77.36% (±11.68%) of the volume immediately after augmentation [Figures 3 and 4]. The volume reduction of the BO graft was not significant. Figure 3Graphical representation of the average volume of the Bio-Oss® and the NanoBone® grafts immediately and 6 months after augmentation (in mm3) Figure 4Graphical representation of the comparative volume analysis of the Bio-Oss® and the NanoBone® grafts 6 months after augmentation (in %) BODY.RESULTS.VOLUMETRIC CHANGES OF THE GRAFTS.NANOBONE: The radiologic analysis of the NB graft revealed volumetric reduction in all patients. The average graft volume immediately after augmentation was 2180.05 mm3 (±670.02 mm3), while 6 months after augmentation, the average graft volume decreased to 1621.00 mm3 (±702.47 mm3). The average percentage of the NB graft volume after 6 months was 75.82% (±22.28%) of the volume immediately after augmentation [Figures 3 and 4]. The volume reduction of the NB graft was not significant. BODY.RESULTS.CHANGES IN BONE DENSITY OF THE GRAFTS.BIO-OSS: The radiologic analysis of the BO graft revealed increased bone density in all patients. The average density of the BO graft increased significantly from 491.00 Hounsfield units (HU) (±104.94 HU) immediately after augmentation to 859.63 HU (±211.04 HU) 6 months after augmentation (••• P < 0.001). Comparison of the bone density of the region of interest immediately after augmentation and the bone density of the zygomatic bone revealed a significantly higher bone density in the zygomatic bone (average: 703.13 ± 77.79 HU; *P < 0.05) in all cases. Comparing the bone density of the region of interest 6 months after augmentation and the bone density of the zygomatic bone, the bone density of the region of interest was higher than the bone density of the zygomatic bone in 6 of 8 cases [Figure 5]. Figure 5Graphical representation of the comparative density analysis of the Bio-Oss® and the NanoBone® grafts immediately and 6 months after augmentation compared to the density of the zygomatic bone (in Hounsfield units; *P < 0.05; •••P < 0.001) BODY.RESULTS.CHANGES IN BONE DENSITY OF THE GRAFTS.NANOBONE: The radiologic analysis of the NB graft revealed an increase of bone density in all patients. The average density of the NB graft increased significantly from 463.88 HU (±53.90 HU) immediately after augmentation to 771.75 HU (±172.13 HU) units 6 months after augmentation (••• P < 0.001). Comparison of the bone density of the region of interest immediately after augmentation and the bone density of the zygomatic bone revealed a higher bone density in the zygomatic bone (average: 703.13 ± 77.79 HU; *P < 0.05) in all cases. Comparing the bone density of the region of interest 6 months after augmentation and the bone density of the zygomatic bone, the bone density of the region of interest was higher than the bone density of the zygomatic bone in 6 of 8 cases [Figure 5]. BODY.DISCUSSION: In the present study, a three-dimensional radiographic analysis was performed to determine the volumetric changes of the bovine-based bone substitute material BO and the synthetic bone substitute material NB, which were augmented in the sinus cavities of patients with head and neck cancer anamnesis. The aim of the investigation was to compare the changes in volume and density of the augmented regions with the previously published histological and histomorphometrical results of the performed split-mouth trial.[1] Further, it should be clarified that to what extent CT scans are suitable to determine the interaction of biomaterials within the augmentation site in the human organism. The presented increase in bone density and decrease of the augmented volume in both groups represent the reorganization of the augmented bone substitute materials with newly formed bone and lead to the suggestion that the augmented volume reduced by condensation processes or the loss of the liquid component of the augmented volume. However, evaluation of CT scans is not able to provide an objective statement about degradation or dehydration processes. Therefore, histologic and histomorphometric analysis remains the method of choice for detailed analysis of cellular mechanisms. The previously published histological and histomorphometrical analysis of the augmented bone substitute materials revealed good integration of the synthetic NB granules in the peri-implant tissue of the sinus cavity with formation of new bone associated with the bone substitute granules. In addition to the apposition of bone tissue, the HA granules were seeded with TRAP-positive and TRAP-negative multinucleated giant cells, which can be interpreted as an expression of an inflammatory response due to a foreign body reaction to the synthetic bone substitute material.[1] The bovine BO granules were also well integrated in newly formed bone tissue, which seemed to originate from active osteoblasts on the surface of the bone substitute granules. In contrast to the synthetic bone substitute material, very few multinucleated giant cells were visible on the surface of the bovine-based bone substitute granules. The difference in the induction of multinucleated giant cells could be proved by histomorphometric analysis and stated as significant (number of multinucleated giant cells per mm2: NB: 50.40 ± 7.16; BO: 16.37 ± 1.72; P < 0.001). In accordance with the presence of multinucleated giant cells, which are transported in the augmentation bed by a vessel-rich connective tissue, the vascularization and the ratio of connective tissue were significantly higher in the NB group (percentage vascularization: NB: 2.66% ± 0.78%; BO: 0.86% ± 0.07%; P < 0.001; connective tissue: NB: 53.87% ± 5.12%; BO: 34.14% ± 4.45%; P < 0.001). Further, the analysis of the tissue distribution in the implantation bed showed a significantly higher ratio of remaining bone substitute material in BO than that in NB group (NB: 24.28% ± 3.26%; BO: 40.13% ± 3.53%; P < 0.01). Interestingly, although different cellular reactions occurred in the implantation beds, no statistically significant difference in new bone formation could be observed (NB: 21.85% ± 5.96%; BO: 25.73% ± 7.94%). Although the investigated bone substitute materials induced different cellular reactions, the formation of new bone did not differ significantly.[1] Accordingly, the radiologically investigated volumetric and bone density changes in the present study also did not differ significantly between both groups. Graft volumes decreased in both groups over the observation period of 6 months, while the measured bone density increased, most likely due to new bone formation in the augmented regions, also in both groups. It was concluded that the comparable increase in bone density in both groups was in accordance with the new bone formation detected by the histological analysis.[1] A possible explanation for the fact that the augmented volume seemed to decrease and the bone density was significantly higher 6 months after augmentation in both groups, independent of the cellular reactions, is a combination of manual condensation of the augmented bone substitute material by pneumatization of the maxillary sinus and dehydration of the augmented biomaterial after its implantation with a simultaneous ingrowth of newly formed bone tissue in the intergranular space. Further, in a histological and histomorphometrical split-mouth trial, comparing the bone substitute materials NB and BO for sinus augmentation in a healthy patient collective, a significantly higher formation of multinucleated giant cells in the augmentation bed of the synthetic NB compared to the xenogeneic BO was detected while the formation of new bone as well as the ratio of remaining bone substitute showed no statistically significant difference. It could therefore be concluded that multinucleated giant cells act more as foreign body giant cells than as osteoclasts.[20] This assumption is in accordance with the results presented, as the volume reduction and density increase did not show any difference between both groups, although in histological analysis, significantly more multinucleated giant cells were found in the NB group. The performed radiologic analysis presents a suitable supplement to the histological and histomorphometrical investigation of biomaterial integration within the human organism. Clinicians are in daily contact with bone substitute materials and are more interested in the clinical success and durability and reliability of the products they use than in the scientific details, such as cellular reactions. In comparison to the histological and histomorphometrical analysis, the radiologic analysis required less effort, both for the investigator and for the patient, as it is noninvasive and technically supported. By standard techniques, such as CT or digital volume tomography (DVT), which are commonly used in implant procedures, reliable and reproducible statements about density and volume of augmentations can be made, which are easy to understand and clearly interpretable. The ongoing development and dissemination of DVT is another factor, which facilitates the analysis of three-dimensional images at a lower exposure to radiation and makes this methodology viable for patients, clinicians, and scientists. In the planning phase of larger implantation or augmentation procedures and cases of anatomical abnormity, DVT is a standard diagnostic application. Further, it must be mentioned that two-stage sinus augmentation with implant insertion after 6 months causes a longer healing period for the patient compared to simultaneous augmentation and implant insertion. Regarding the ergonomic and patient-friendly trend toward simultaneous augmentation and implantation, it is questionable to what extent biopsies after bone augmentation can still be gained in the future, as in the case of single-stage augmentation and implantation, there is no possibility of obtaining biopsies of bone substitute materials from the augmentation site. Therefore, the analysis of CT or DVT images might become more important as it can also be performed in single-stage augmentation and implantation. The analysis of bone density and the volume of the augmented area can be an indicator of the remodeling and thus the ingrowth of new bone in the augmented bone substitute material. However, it must be mentioned that detailed conclusions about cellular reactions to different biomaterials can only be made by histological and histomorphometrical analysis. Especially, during the early phase of research, histological analysis of biopsies is still important and will not become less relevant. The presented methodology can prove to be a reliable and suitable technique for the analysis of graft volume and density changes and can complete histological analysis or even replace it to a certain degree. Especially for clinicians, this combination of validity and reproducibility in a noninvasive methodology presents major progress in the analysis of the interaction between bone substitute materials and the peri-implant tissue. BODY.CONCLUSION: The presented radiologic analyses were performed to determine the changes in volume and density within the augmented human sinus cavities. Through the analysis of three-dimensional CT images that were recorded immediately after augmentation and 6 months later, changes in the volume and density of the graft over a period of 6 months were determined. In both groups, the volume of the augmented sinus cavities decreased over 6 months while the bone density increased significantly and reached higher values than the referenced zygomatic bone. Considering the results obtained from the histologic and histomorphometric investigation of the cellular response to both biomaterials, it was demonstrated that both biomaterials seemed to undergo volume reduction, while in the center of the augmented sinus cavities, bone remodeling took place, increasing the radiographically measurable bone density. It was shown that the radiologic and volumetric investigation method presented here might be a suitable, noninvasive investigation tool to complete and under certain circumstances avoid histological and histomorphometrical analysis. Moreover, this method of analysis provides suitable information for clinicians to assess the stability and predictability of augmentation procedures in an effective and minimally invasive manner. BODY.CONCLUSION.FINANCIAL SUPPORT AND SPONSORSHIP: This study was supported by a grant from the Camlog Foundation, Basel, Switzerland. BODY.CONCLUSION.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: Randomized controlled trial of oatmeal consumption versus noodle consumption on blood lipids of urban Chinese adults with hypercholesterolemia ABSTRACT.BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death in China and worldwide. Whole grain oats can reduce risk of CVD by reducing total and LDL-cholesterol, major risk factors for CVD. While this association has been established in many populations, data from Asian populations is limited. Thus, this study investigated the impact of oat consumption on cholesterol levels in Chinese adults. Male and female data from this work were previously published separately in mandarin in two Chinese journals. The combined male and female data were reanalyzed and are presented here. ABSTRACT.METHODS: A randomized, controlled, parallel-arm study was conducted at Beijing Hospital, Beijing china. Subjects were adults (men and women) with mild to moderate hypercholesterolemia. The oat group (n=85) consumed 100grams of instant oat cereal versus the control group (n=81) who consumed 100grams of wheat flour-based noodles daily for 6weeks. Laboratory and anthropometric measurements were conducted at baseline and at the end of the 6-week intervention. ABSTRACT.RESULTS: Dietary fiber intake increased significantly in the oat group compared to the control group at the end of the 6-week intervention. Total-, LDL-cholesterol and waist circumference decreased significantly in the oat group compared to the control. HDL-cholesterol decreased significantly in the control group versus the oat group. There were no significant changes in blood pressure, other anthropometric or laboratory measures between the two groups at the end of the intervention. ABSTRACT.CONCLUSIONS: Instant oatmeal consumed daily for 6 weeks significantly increased fiber intake and decreased major risk factors for CVD in Chinese adults with hypercholesterolemia. Increased consumption of whole grains, including oats, should continue to be encouraged. BODY.BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death in China [1] as well as worldwide [2]. Established risk factors for CVD include elevated total cholesterol (TC), elevated low density lipoprotein cholesterol (LDL-C), low high density lipoprotein cholesterol (HDL-C) and hypertension. These risk factors are modifiable by lifestyle factors including diet and exercise [3]. Epidemiological studies suggest that diets high in whole grains are associated with a reduced risk of CVD and mortality [4-7]. In China, recent changes to the traditional diet, including significant reductions in wholegrain intake (104g/day in 1982 to 24g/day in 2002) may be contributing to the increased CVD mortality [8]. Wholegrain oats can significantly lower serum TC and LDL-C and reduce risk for CVD [9]. Authoritative regulatory bodies in several countries, including the United States, Canada, Europe and Malaysia, have reviewed the available evidence and allow a health claim on food labels regarding the relationship between oat consumption and a reduction in blood cholesterol concentration and reduced risk of CVD [10-13]. Data regarding impact of oat consumption on CVD risk factors in Asian populations, including the Chinese population, is limited. Therefore, the present study investigated the relationship between instant oatmeal intake and CVD risk factors in a sample of urban Chinese adults with mild to moderate hypercholesterolemia. BODY.METHODS.STUDY DESIGN: This was a randomized, controlled, parallel-arm study conducted at Beijing Hospital, Beijing China. Screening of potential study subjects was held in August and September 2008. The six week intervention was conducted from October 2008 through December 2008 during which the subjects visited the research site once per week. The study protocol was approved by the ethics committee of the Institute of Nutrition and Food Safety, Chinese center for Disease control and Prevention. Participants provided written informed consent before any study procedures were performed. Due to the nature of the food products serving as control (wheat noodles) and test (oatmeal) product, it was not possible to blind the subjects or the researchers, however, the statistician analyzing the data was blinded to the treatment groups. BODY.METHODS.STUDY PARTICIPANTS: Based on the diagnostic standards in the Chinese Guidelines on Prevention and Treatment of Dyslipidemia in Adults[14] the selection criteria were as follows: (1)males and females 35 to 70 years of age; (2) serum total cholesterol ≥5.18mM, low density lipoprotein cholesterol ≥3.37mM; (3) no serious liver, kidney, digestive tract disease, diabetes or other metabolic diseases; (4) no use of cholesterol lowering medication or foods that have been approved by the China State Food and Drug Administration as functional foods with blood lipid lowering properties in the last 6 months; and (5) did not habitually consume oat products. Screening of study participants was done in August and September 2008. Based on data obtained from a basic health questionnaire, food frequency questionnaire and results of a fasting blood lipid test, 182 subjects (109 females, 73 males) were selected to participate and randomized to either the oat group or the control group. Participants were instructed to consume their normal diet during the study period. Participants who smoked could not have plans to change smoking habits during the treatment period. BODY.METHODS.DIET AND STUDY PRODUCT: A 3-day 24-hour dietary recall was conducted at the start and end of the study to determine food and nutrient intake. The 24 hour dietary recall questionnaire was validated in the 2002 Chinese Nutrition and Health Survey project. SAS 8.2 software was used to calculate nutrient intake based on Chinese Food composition Table. Prior to beginning the intervention, all participants were provided with basic nutrition counseling following the Chinese Food Pagoda guidelines which consisted of advice to consume a variety of foods including cereals, fruits, vegetables, dairy and lean meats and to reduce sodium and limit alcohol intake. Participants were instructed not to change their exercise habits and to maintain their normal dietary habits with the exception of replacing a portion of a staple food product (rice, steamed bread, noodles, etc.) with the intervention product (oats or wheat noodles). Oatmeal (Quaker Instant Oatmeal produced by PepsiCo Foods (China) co., Ltd.) was packaged in 200g (dry wt) packages and 4 packages were provided to participants one week followed by 3 packages the next week. Participants receiving the oatmeal were instructed to prepare 1⁄2 a package (~100g dry wt, providing approximately 3.6g soluble fiber) each day according to the on-pack preparation instructions (with hot water or milk). Consumption of the prepared amount could be split across two or three meals each day for the six week intervention period. Wheat noodles ("Shengchu" brand processed wheat flour based noodles) were purchased in 450g packages at the local grocery store and distributed to participants on a weekly basis. Participants were instructed to prepare and consume 100g (dry wt) each day. All subjects in the control group were shown a 100g portion of dry noodles that had been weighed on a scale to help them visualize the amount that they should prepare and consume each day. Wheat based noodles are a standard staple in the diets of this population and this amount can easily be consumed in one meal. Each participant was asked to record the quantity of the test food they consumed each day. The oat and noodle packs were distributed once each week to facilitate weekly communication with the participants and discuss intake of food and any relevant concerns or issues linked with the daily oat or noodle consumption. BODY.METHODS.ANTHROPOMETRIC AND LABORATORY MEASUREMENTS: Anthropometric measurements were conducted at baseline and at the end of the six week intervention period. Standing height was measured. Participant weight was measured while wearing light weight clothing and without shoes. Waist circumference was measured on a horizontal plane at the level of the iliac crest using a non-stretch anthropometric tape. Two consecutive readings of blood pressure were taken according to methods described in the 2010 Chinese Guidelines for the management of hypertension [15] with the subject in a seated position after 5 minutes of rest. The mean of the two measurements was used for statistical analysis. Fasting blood samples were collected at baseline and at the end of the intervention period. Blood biochemical parameters were measured using a Shimazu 7600 fully automatic biochemical analyzer and included serum total cholesterol (TC), serum triglycerides (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), apolipoprotein A1 (ApoA1), apolipoprotein B (ApoB) and plasma glucose. BODY.METHODS.STATISTICAL METHODS: The statistician was blinded to the treatment groups. All statistical summaries and analyses were performed using SAS V 8.2 for windows (SAS Institute, cary, NC). Categorical data were summarized as frequencies and proportions within each group. When analyzing for group differences among the categories, a 2-sided Fisher's exact test was used. Effect of intervention on dietary composition was analyzed as change from baseline to minimize the bias of any baseline differences. Baseline data and changes from baseline were analyzed using f-test from analysis of covariance (ANCOVA) where the group effect was fixed and the covariates were age, sex, smoking status, alcohol consumption, educational status, and medication use. The effects of intervention on anthropometrics, blood pressure, lipids and glucose were also analyzed for baseline group differences and as change from baseline after six weeks. Summaries of these outcomes were presented as least squares means (LSM) and standard errors (SE) obtained from the ANCOVA model to account for any baseline unbalance in the groups and the effects of the covariates. Statistical significance was set at P<0.05. BODY.RESULTS.STUDY COMPLIANCE: During the study period there were 9 subjects from the control group and 7 subjects from the oat group that dropped out or were eliminated due to non-compliance with the study protocol. Two subjects from each group were eliminated due to incorrect recording of food amounts as determined by unreasonably low calculated daily calorie intake values (< 500 kcal / day),while 2 subjects from the control group and 1 subject from the oat group were eliminated due to use of products that may reduce serum cholesterol. In addition, 5 subjects from control group and 4 subjects from the oat group dropped out due to personal reasons such as losing interest in the study or travel requirements. A total of 85 subjects in the oat group and 81 subjects in the control group were included in the final data analysis. There were no adverse events reported during the intervention period except for some complaints about the taste of the oat product. Baseline characteristics of the two test groups are provided in Table 1. There were no statistically significant differences between groups for any of the demographic characteristics or any of the anthropometric measures. There was a statistical difference between groups in ApoB levels at baseline with the oat group having slightly higher levels than the control group (p=0.044). However, both groups were within the normal reference range of 0.60-1.94g/L. Table 1 Baseline characteristics of subjects Characteristic Oat group (n=85) Control group (n=81) P Between groups Age (y) 1 52.7 (0.69) 53.7 (0.73) 0.341 Male (%) 2 38.8% 39.5% >0.999 Height (cm) 3 165.1 (0.49) 165.3 (0.50) 0.788 Waist circumference (cm) 3 86.7 (0.92) 86.9 (0.94) 0.887 Weight (kg) 3 69.7(0.94) 69.8 (0.97) 0.948 Body mass Index 3 25.5 (0.32) 25.5 (0.33) 0.964 Smoker (%) 1 18.8% 13.6% 0.405 Alcohol Use (%) 1 32.9% 29.6% 0.738 Medication Use (%) 1 50.6% 56.8% 0.441 Systolic BP (mmHg) 3 124.7 (1.74) 129.0 (1.78) 0.085 Diastolic BP 3 80.3 (1.06) 79.7 (1.09) 0.689 Total cholesterol (mmol/L) 3 6.26 (0.074) 6.09 (0.076) 0.129 Triglycerides (mmol/L) 3 2.06 (0.103) 1.89 (0.106) 0.279 High Density Lipoprotein (mmol/L) 3 1.47 (0.029) 1.51 (0.030) 0.337 Low Density Lipoprotein (mmol/L) 3 4.30 (0.075) 4.17 (0.077) 0.237 Plasmaglucose (mmol/L) 3 5.63 (0.097) 5.47 (0.099) 0.277 Apo A1 (g/L) 3 1.59 (0.032) 1.57 (0.033) 0.554 Apo B (g/L) 3 0.96 (0.017) 0.91 (0.018) 0.044 1 LSM (SE) and p-value obtained from ANOVA. 2 Proportion and p-value obtained from 2-sided Fisher’s Exact test. 3 LSM (SE) and p-value obtained from ANCOVA, where covariates were age, sex, smoking status, alcohol consumption, education status, medication use. Covariates were evaluated in the analysis of baseline characteristics to determine their effect on each measure prior to intervention. Age was a significant covariate in systolic blood pressure and blood glucose. Sex was a significant covariate in height, waist circumference, weight, systolic blood pressure, diastolic blood pressure, total cholesterol, triglycerides, HDL-C and ApoA1 levels. Smoking status was a significant covariate in total cholesterol, LDL-C and ApoB levels. Alcohol consumption was a significant covariate in height and diastolic blood pressure. Education status was a significant covariate in waist circumference, BMI and systolic blood pressure. Medication use was not a significant covariate for any characteristics. Due to the mixed and overlapping influences of these covariates, all covariates were kept in the subsequent analyses of dietary compositions and changes in anthropometric and lipid measures after 6 weeks of intervention. BODY.RESULTS.DIETARY INTAKE: Dietary compositions at baseline and after 6 weeks of intervention are presented in Table 2. Baseline dietary composition was consistent between the groups for all components except percent of energy from dietary protein which was significantly higher among the oat group than the control group (p=0.004). After 6 weeks of intervention, dietary fiber significantly increased among the oat group compared to the control group (p<0.001). No other changes in composition were notable between the groups. Table 2 Diet compositions Diet characteristic LSM (SE) 1   Oat group (n=85) Control group (n=81) P Between groups Energy (MJ/d) change from Baseline 0.30 (0.193) 0.32 (0.198) 0.942 Baseline 7.6 (0.20) 7.7 (0.20) 0.687 Week 6 7.9 (0.20) 8.1 (0.20)   Protein (g/d)change from Baseline 4.6 (1.97) 1.7 (2.02) 0.315 Baseline 64.8 (1.83) 61.1 (1.88) 0.168 Week 6 69.3 (1.82) 62.8 (1.86)   Protein (% energy) change from Baseline 0.45 (0.309) -0.14 (0.317) 0.191 Baseline 14.3 (0.24) 13.3 (0.25) 0.004 Week 6 14.8 (0.28) 13.1 (0.29)   Fat (g/d)change from Baseline 2.9 (2.58) 1.2 (2.64) 0.634 Baseline 62.5 (2.77) 61.7 (2.84) 0.841 Week 6 65.5 (2.52) 62.9 (2.59)   Fat (% energy) change from Baseline 0.36 (0.817) -0.58 (0.837) 0.428 Baseline 30.3 (0.86) 29.4 (0.88) 0.472 Week 6 30.6 (0.77) 28.8 (0.79)   Cholesterol (mg/d) change from Baseline -39.7 (21.73) -38.5 (22.27) 0.968 Baseline 312 (19.1) 292 (19.5) 0.460 Week 6 272 (18.1) 253 (18.5)   SFA (% energy) change from Baseline -0.66 (0.261) -0.34 (0.267) 0.389 Baseline 8.7 (0.27) 7.9 (0.27) 0.051 Week 6 8.0 (0.24) 7.6 (0.25)   MUFA (% energy) change from Baseline 0.21 (0.414) -0.52 (0.425) 0.218 Baseline 12.6 (0.41) 12.3 (0.42) 0.602 Week 6 12.9 (0.39) 11.8 (0.40)   PUFA (% energy) change from Baseline 0.38 (0.388) 0.28 (0.398) 0.868 Baseline 7.9 (0.39) 8.2 (0.40) 0.632 Week 6 8.3 (0.34) 8.5 (0.35)   Carbohydrate (g/d) change from Baseline 14.2 (7.12) 16.5 (7.30) 0.825 Baseline 247 (6.4) 259 (6.6) 0.173 Week 6 260 (7.2) 276 (7.4)   Carbohydrate (% energy) change from Baseline 0.41 (0.859) 1.08 (0.881) 0.588 Baseline 54.9 (0.92) 56.6 (0.94) 0.197 Week 6 55.3 (0.83) 57.7 (0.85)   Dietary fiber (g/d) change from Baseline 7.1 (0.60) 1.4 (0.62) <0.001 Baseline 12.2 (0.52) 11.4 (0.53) 0.296 Week 6 19.3 (0.55) 12.9 (0.56)   1 LSM (SE) and p-value obtained from ANCOVA,where covariates were age, sex, smoking status, alcohol consumption, education status, medication use. BODY.RESULTS.ANTHROPOMETRIC MEASURES AND BLOOD PRESSURE: Anthropometric measures and blood pressure after 6 weeks and as change from baseline are presented in Table 3. Waist circumference significantly decreased after 6weeks in the oat group compared to the control group (p=0.002). No other measures changed significantly. Table 3 Changes in anthropometric measures and blood pressure during study period Measures LSM (SE) 1   Oat group (n=85) Control group (n=81) P Between groups Waist circumference (cm) change from Baseline −1.27 (0.473) 0.85 (0.485) 0.002 Baseline 86.7 (0.92) 86.9 (0.94) 0.887 Week 6 85.4 (0.82) 87.7 (0.84)   Weight (kg) change from Baseline 0.46 (0.306) 0.67 (0.313) 0.621 Baseline 69.7(0.94) 69.8 (0.97) .0948 Week 6 69.8 (0.93) 70.4 (0.96)   Body mass Index change from Baseline 0.04 (0.092) 0.25 (0.094) 0.108 Baseline 25.5 (0.32) 25.5 (0.33) 0.964 Week 6 25.6 (0.32) 25.7 (0.32)   Systolic Blood Pressure change from Baseline 1.01 (1.120) 0.85 (1.477) 0.919 Baseline 124.7 (1.74) 129.0 (1.78) 0.085 Week 6 125.7 (1.65) 129.9 (1.69)   Diastolic Blood Pressure change from Baseline −0.13 (0.916) 0.76 (0.938) 0.500 Baseline 80.3 (1.06) 79.7 (1.09) 0.689 Week 6 80.1 (0.97) 80.4 (1.00)   1 LSM (SE) and p-value obtained from ANCOVA, where covariates were age, sex, smoking status, alcohol consumption, education status, medication use. BODY.RESULTS.SERUM LIPIDS AND PLASMA GLUCOSE: Serum lipids and plasma glucose after 6weeks and as change from baseline are presented in Table 4. The oat group showed significant decreases in total cholesterol (p=0.015) and LDL-C (p=0.028) compared to the control group. In addition, HDL-C decreased significantly in the control group compared to the oat group (p=0.017). Effects of intervention were observed for these same lipids when summarized as a percentage change from baseline (Figure 1). There were no significant differences in plasma glucose levels between the two groups. Table 4 Changes in lipids and glucose during study period Measures LSM (SE) 1   Oat group (n=85) Control group (n=81) P Between groups Total cholesterol (mmol/L) change from Baseline −0.41 (0.071) −0.15 (0.073) 0.015 Baseline 6.26 (0.074) 6.09 (0.076) 0.129 Week 6 5.85 (0.085) 5.94 (0.087)   Triglycerides (mmol/L) change from Baseline −0.15 (0.089) −0.04 (0.091) 0.404 Baseline 2.06 (0.103) 1.89 (0.106) 0.279 Week 6 1.91 (0.107) 1.85 (0.110)   HDLC (mmol/L) change from Baseline −0.04 (0.018) −0.10 (0.018) 0.017 Baseline 1.47 (0.029) 1.51 (0.030) 0.337 Week 6 1.43 (0.032) 1.41 (0.033)   LDLC (mmol/L) change from Baseline −0.39 (0.067) −0.17 (0.069) 0.028 Baseline 4.30 (0.075) 4.17 (0.077) 0.237 Week 6 3.91 (0.081) 4.00 (0.083)   Apo A1 (g/L) change from Baseline 0.02 (0.29) −0.02 (0.029) 0.432 Baseline 1.59 (0.032) 1.57 (0.033) 0.554 Week 6 1.61 (0.035) 1.55 (0.036)   Apo B (g/L) change from Baseline −0.06 (0.016) −0.02 (0.017) 0.102 Baseline 0.96 (0.017) 0.91 (0.018) 0.044 Week 6 0.91 (0.017) 0.89 (0.017)   Plasma glucose (mmol/L) change from Baseline −0.30 (0.057) −0.17 (0.059) 0.118 Baseline 5.63 (0.097) 5.47 (0.099) 0.277 Week 6 5.34 (0.088) 5.29 (0.090)   1 LSM (SE) and p-value obtained from ANCOVA, where covariates were age, sex, smoking status, alcohol consumption, education status, medication use. Figure 1 Percentage of change in lipids. BODY.DISCUSSION: The results of this study show that when instant oats are consumed daily in place of another staple food (rice, steamed bread, noodles, etc.) compared to wheat noodles as replacement for another staple food, there is a significant increase in dietary fiber intake and significant decreases in waist circumference, TC and LDL-C in Chinese adults with moderate hypercholesterolemia. During the intervention period there was a 6.2% decrease in TC for the oat group compared to a 2.3% decrease in the control group. There was also an 8.4% decrease in LDL-C for the oat group compared to a 3.5% decrease in the control group. The oat group consumed 100 grams of instant oats per day which provided ~3.6 grams of soluble fiber. Oat products containing β-glucan soluble fiber decrease TC and LDL-C by altering bile acid metabolism and increasing bile acid excretion [16,17]. The reductions in TC and LDL-C observed in this study are similar to previous reports in free living individuals. Van Horn et al. [18] reported a 5.2% reduction in TC in healthy adults consuming 60g/day of either oat bran or oatmeal for 6weeks as part of a low fat diet. Karmally and colleagues [19] provided 3g/day of β-glucan from ready-to-eat (RTE) oat cereal for 6weeks to adults with mild to moderate hypercholesterolemia and observed reductions of 4.5% in TC and 5.3% in LDL-C. In a recent trial a 5.4% reduction in TC along with an 8.7% decrease in LDL-C was reported following consumption of 3g/day of β-glucan from ready-to-eat (RTE) oat cereal for 12 weeks in overweight or obese adults [20]. A 2007 cochrane review reported results of a meta-analysis based on 8 randomized clinical trials that used oats as a wholegrain intervention. A significant effect of oat consumption to lower both TC (P=.0005) and LDL-C concentrations (P=.0008) was observed. The mean percentage reduction in LDL-C from baseline (95% confidence interval) was 4.9% (7.6% to 2.4%) [9]. The prevalence of dyslipidemia in Chinese adults has been reported to be 2.9% for hypercholesterolemia (serum TC ≥5.18 mmol/L) and 11.9% for hypertriglyceridemia (serum TG ≥1.70 mmol/L) based on a nationally representative sample of subjects [21]. However, certain subgroups of the population have a higher reported prevalence of hypercholesterolemia. The prevalence is higher in adults over 60 years of age and has been reported to be 20.2% in males and 38.7% in females in this age group [22]. The prevalence of dyslipidemia has also been reported to be higher for urban (21%) compared to rural (17.7%) Chinese adults [21].The reduction in LDL-C observed in this study is of the magnitude to reduce risk of CHD since every 1% reduction in LDL-C is associated with a decreased risk for CHD of 1% to 3% [23,24]. A dietary pattern characterized by a high intake of vegetables, fruit and soy is one protective lifestyle factor associated with a marked decreased risk of coronary heart disease, cerebrovascular disease, and overall CVD mortality in Chinese men and women [25].The traditional Chinese diet included a high intake of vegetables and coarse grains, which are the main sources of total and insoluble dietary fiber in the Chinese population [8,26]. However significant dietary changes have occurred in recent years which may be contributing to the prevalence of hypercholesterolemia within the population. The national average daily intake of cereals decreased from 510g/day in 1982 to 402g/day in 2002. The amount of coarse grains decreased from 104g/day to 24g/day in the same time period [8]. Average intake of total dietary fiber decreased from 22.6g/day in 1989 to 18.1g/day in 2006 and insoluble dietary fiber intake decreased from 15.1g/day in 1989 to 11.9g/day in 2006 [26]. The 2007 Chinese food based dietary guidelines (FBDGs) issued by the Chinese Nutrition Society include a recommendation to include an appropriate amount of coarse grains. The proposed guidelines recommend a coarse grain intake, including whole grains, of no less than 50 grams per day for adults [8]. In this study the oat group had a significant increase in dietary fiber intake (7.1 grams/day) compared to the control group (1.4grams/day, p<0.001) during the treatment period. Intake of course grains was not calculated in this study. The control group had a significant decrease in HDL-C concentration compared to the oat group during the intervention period (p=0.017). Dietary factors that impact HDL-C concentration include total fat and trans fat intakes along with the ratio of dietary saturated to unsaturated fat. The decrease in HDL-C in this study is puzzling since there were no significant changes in energy or total, saturated, mono- or poly-unsaturated fat intakes during the intervention period. There also was no significant change in ApoA-1 observed during the treatment period in this study. ApoA-1 concentration is also influenced by the ratio of polyunsaturated to saturated fat [27]. Waist circumference decreased 1.27 cm in the oat group compared to a 0.85 cm increase in the control group (p=0.002). Abdominal obesity is strongly associated with metabolic disturbances such as hypertriglyceridemia and insulin resistance [28]. Consumption of whole grains has been associated with smaller waist circumference in population studies [29,30]. Maki et al [20] reported a ~1.5cm reduction in waist circumference in overweight adults consuming 3g/day of β-glucan for 12weeks from RTE oat cereal. The clinical implications of a reduction in waist circumference without simultaneous changes in BMI or weight should be investigated. There was no significant effect of oat consumption on blood pressure at the end of the 6 week treatment period in this study. Previous studies have reported mixed results of the effect of oats, β-glucan or whole grains on blood pressure. Keenan et al [31] reported reduced systolic and diastolic blood pressure in a pilot trial with oats. Whole-grain diets reduced blood pressure in mildly hypercholelsterolemic men and women [32]. Other investigators reported no effect on blood pressure after consumption of foods containing oat β-glucan [33], oats [34] and RTE oat cereal [20]. One confounding factor in this study is that slightly more than 50% of the total subjects were using medications including antihypertensive medication during the intervention. One limitation of this study is exercise/activity levels were not reported however the subjects were instructed not to make any changes to their habitual diet or other lifestyle factors during the intervention. Another limitation is that antihypertensive medications were allowed during the trial and therefore the impact of the oat intervention on blood pressure was confounded. As the soluble fiber content of the diet before and after intervention could not be calculated, it is not possible to determine if there were changes to soluble dietary fiber intake over the course of the study. BODY.CONCLUSIONS: Consumption of 100 grams of instant oatmeal per day significantly reduced TC, LDL-C and waist circumference in moderately hypercholesterolemic Chinese men and women. Replacement of a staple food with oatmeal significantly increased dietary fiber intake. Recent Chinese dietary guidelines include a recommendation to consume an appropriate amount of coarse grains including whole grains. The results of this study demonstrate that consumption of a wholegrain oat cereal has beneficial effects on risk factors for CVD. BODY.ABBREVIATIONS: CVD: Cardiovascular Disease; TC, Total cholesterol; LDL-C: Low Density Lipoprotein cholesterol; HDL-C: High Density Lipoprotein cholesterol; TG: Triglycerides; ApoA1: Apolipoprotein A1; ApoB: Apolipoprotein B; ANCOVA: Analysis of covariance; LSM: Least Square mean; SE: Standard Error; RTE: Ready To Eat; FBDGs: Food Based Dietary Guidelines. BODY.COMPETING INTERESTS: Authors AK and JC are employees of PepsiCo, Inc. and Pepsi Co china Foods, respectively. Authors JZ, LL, PS, QM, LM and CW are employees of Institute of Nutrition and Food Safety, Chinese center for Disease control and Prevention, and declare no conflicts of interests. BODY.AUTHORS’ CONTRIBUTIONS: The study was designed by JZ. LL, QM and CW carried out the study and collected the data (under the supervision of JZ). PS was responsible for the dietary survey and nutrient calculations. LM and AK were responsible for data analyses. JZ and AK were responsible for interpretation of results and manuscript writing. JC, AK and JZ reviewed manuscript drafts and the final manuscript. All authors read and approved the final manuscript.

TITLE: Effectiveness of Comprehensive Health Education Combining Lifestyle Education and Hot Spa Bathing for Male White-Collar Employees: A Randomized Controlled Trial with 1-Year Follow-Up ABSTRACT.BACKGROUND: Physical activity is known to prevent obesity and metabolic syndrome in middle-aged and elderly people; however, the effectiveness of a comprehensive health education program for male white-collar employees is uncertain. ABSTRACT.METHODS: Forty-three men volunteered to participate in this study and were randomly assigned into 2 groups. The intervention group participated in a 2-hour program comprising comprehensive health education and hot spa bathing, offered once every 2 weeks, in addition to individualized programs once a week, for 24 weeks. The control group received only general health guidance. We compared their lifestyle characteristics and physical and mental health criteria at baseline, immediately after the intervention, and 1 year after the end of the intervention. ABSTRACT.RESULTS: Rates of adherence to individualized programs were 60.0 ± 27.2% and 30.5 ± 29.6% at the end of the intervention and at 1 year after the end of the intervention, respectively. Significant (P < 0.05) interaction of criteria was observed for cluster of differentiation 4+ (CD4+) cells and the ratio of cluster of differentiation 4+ to 8+ (CD4/8) cells, which were used to represent the participants' immunological function. We divided the intervention group into 2 subgroups on the basis of their attendance. Among the resulting 3 groups, significant interaction of criteria was observed for CD4+ and CD4/8 cells. In addition, the high attendance group had the highest CD4+ count and CD4/8 ratio. ABSTRACT.CONCLUSIONS: Participants who attended classes and/or performed the supplementary individualized programs tended to maintain their immunological function and to experience a decrease in body fat percentage. However, few effects were noted in participants with poor adherence, even in the intervention group. BODY.INTRODUCTION: The prevalences of obesity and metabolic syndrome are increasing in many industrialized countries.1 In Japan, the prevalence of metabolic syndrome, as determined using Japanese criteria, was 18.4% and 5.8% for men and women, respectively.2 Among the indicators of metabolic syndrome, high blood pressure was most frequently observed, followed by dyslipidemia; high fasting plasma glucose was least frequent among both sexes. The health benefits of physical activity for middle-aged and elderly people are well documented, and an increase in physical activity is effective in preventing coronary heart disease, stroke, diabetes, obesity, and hypertension, and for improving quality of life and mental health.3–10 An increasing number of people enjoy bathing in hot spas, and more hot spa facilities are being built throughout Japan.11 Spa bathing (balneotherapy, or spa therapy) is a popular alternative medical treatment, and is a very popular treatment for arthritis in many European countries, as well as in Israel and Japan.12–14 Hot spas exert a thermal action, hydraulic pressure, a chemical action, and a general conditioning action,15 all of which are known to affect humans favorably. Several studies suggest that the warmth and buoyancy of spa water block nociception by acting on thermal receptors and mechanoreceptors and by enhancing blood flow, which is thought to help in dissipating algogenic chemicals and in possibly facilitating muscle relaxation.16,17 The hydrostatic effect may relieve pain by reducing peripheral edema18 and by dampening sympathetic nervous activity.19 Balneotherapy may also have potential for augmenting immunological function (suppressor T cells, natural killer cell activity, B cells, cluster of differentiation 4+ cells, etc.) and may relieve stress.20 We hypothesized that in Japan, where hot spa bathing is a part of daily life and custom, hot spa bathing (including bathing at home), when combined with a health education program focused on enhancing conventional physical and health activities, should improve health. However, no randomized controlled trials (RCTs) have been performed to determine the effects of such a comprehensive health education program for male white-collar employees. In the present study, we instructed male white-collar employees on exercise, diet, and daily life activities once every 2 weeks for 24 weeks. They were also encouraged to take hot spa baths so that we could evaluate the health effects of spa bathing. Serum lipid levels and immune function were the main outcome measures and were monitored for 1 year. BODY.METHODS.PARTICIPANTS AND RANDOMIZATION: Figure 1 shows the study flow diagram. This study was announced between August and September 2006 on a website used exclusively by municipal office personnel in Unnan, Japan. The eligibility criteria were male sex, age from 30 to 57 years, and no contraindications for exercise or spa bathing. Workers were informed of the study by means of the website in the municipal office, intrasectional circular, and direct letters and e-mails to all eligible people. Of 311 male white-collar employees between the ages of 30 and 57 years, 43 volunteered to participate in this study. After explanatory meetings were held on September 25–26, all 43 volunteers (14% participation rate) agreed to be included in this study, regardless of whether they were assigned to the control group or the intervention group (no blinding for participants). Twenty-two and 21 volunteers were randomly assigned by lottery to the intervention and control groups, respectively. We first randomized the list of volunteer names using a common bingo lottery device, after assigning unique numbers to the volunteers in the list in the order of their application. We then assigned 22 signed sticks (intervention group) and 21 unsigned sticks (control group), which were drawn from sealed boxes, to the numbers for the volunteers. The lottery operation was performed by a third party, ie, a person other than authors, and the person in charge of intervention and evaluation. Concealment was confirmed after completion of the assignments. Figure 1.Study flow diagram BODY.METHODS.RESEARCH DESIGN: In this RCT we compared comprehensive health education to limited health education. No dropouts were reported during the intervention period; therefore, all analyses were intention-to-treat (ITT). In addition, the intervention group was divided into high (n = 10) and low (n = 12) adherence groups, and subgroup analysis was then performed between the 3 groups, ie, the high and low adherence groups and the control group. The trial procedure, analysis, and description were reported according to the CONSORT statement21 and the CLEAR NPT checklist (a checklist to evaluate reports of nonpharmacological trials).22 BODY.METHODS.INTERVENTION AND SETTING: A 2-hour program encompassing comprehensive health education with hot spa bathing was offered once every 2 weeks for 24 weeks (Table 1). Guidance on lifestyle, physical exercise, and diet consisted of lectures (comprehensive health education) and various forms of physical exercise, as shown in Table 1. Each session required approximately 60 minutes. The lectures and exercise sessions were delivered by physicians, dieticians, academics, public health nurses, and exercise instructors with more than 20, 5, 10, 15, and 5 years, respectively, of clinical or pedagogical experience. Table 1. Protocol for lifestyle education, exercise, and bathing a Session Date b Instructor Main program (contents) 1 Oct. 30–31 public health nurse A lecture on project significance and setting goals 2 Nov.13–14 orthopedist A lecture on preventing backache and stiff shoulders 3 Nov.27–28 exercise instructor Light exercise: stretching 4 Dec.11–12 internist A lecture on preventing lifestyle-related diseases 5 Dec.25–26 exercise instructor Light exercise: stretching and walking 6 Jan.15–16 psychiatrist A lecture on releasing mental stress 7 Jan.29–30 exercise instructor Light exercise: recreation (1) 8 Feb.13–14 exercise instructor Light exercise: recreation (2) 9 Feb.26–27 academic expert A lecture on modifying behavior for health 10 Mar.12–13 dietician A lecture on appropriate eating for office workers 11 Mar.27–28 exercise instructor Light exercise: recreation (3) 12 Apr.9–10 public health nurse A lecture on self-management of health a All lectures and exercises were 60 min, and were followed by spa bathing in earthy gypsum salt springs for 30–45 min. b To increase participation, the lectures and exercise sessions were held on more than 1 day. The participants took half-baths (up to the chest) in an open-air bath (earthy gypsum salt spring; bath temperature, 40 °C). The duration of the bath was approximately 20 minutes (2 baths of 10 minutes each). The total time for the bathing process was approximately 60 minutes, which included 40 minutes for changing clothes, washing the body, and resting (drinking beverages). The public multipurpose hall, located at a distance of 200 meters from the municipal hall, and the spa facility, which was immediately adjacent to the multipurpose hall, were used as settings for the intervention. All participants were able to visit the spa facility, which was within a 10-minute drive from their home or workplace. In addition to the intervention listed in Table 1, the volunteers also participated in an individualized program once a week. This program focused on targets set by the participants themselves, and included physical activities in addition to spa bathing (for example, a 30-minute walk before returning home, in addition to their daily activities). Based on studies showing that written reports circulated after the completion of an intervention resulted in higher rates of persistent effects,23,24 we sent copies of a newsletter to the participants once every 3 months (4 times) during the 1-year observation period. These digests (1 sheet of A4-sized paper) contained a review of recommended methods for eating, sleeping, exercising, and bathing. Participants in the control group received general health guidance on a single occasion, in addition to instructions on stretching and dietary guidance. They were also encouraged to continue with their usual daily activities. BODY.METHODS.INSTRUCTIONS ON DAILY LIVING: Guidance on daily living during the intervention emphasized increased physical activity, such as walking and using stairs instead of an escalator or elevator. Regarding dietary guidance, a leaflet was distributed that indicated the suggested level of energy intake for the program. Daily bathing at home or a spa was recommended at a suitable water temperature (40°–41 °C) in a half-bath (water level at the chest while seating). In addition, participants were asked to perform their individualized programs as frequently as possible, although these instructions were delivered in a nonsupervisory manner, so as to give priority to the participant's free will, work conditions, and family situation. BODY.METHODS.OUTCOME AND PROCESS MEASUREMENTS: The baseline assessment was performed in October 2006, the interim assessment was performed immediately after completion of the intervention in May 2007, and the final assessment was done after a 1-year observation period, in May 2008. Serum HDL cholesterol (HDL-C) was the main outcome measure, and hemoglobin A1c (HbA1c) level and the ratio of cluster of differentiation 4+ and 8+ (CD4/8) were defined as secondary outcomes. Other outcome measures included body measurements, physical strength, blood profile, and mental state. The body measurements were height, weight, body mass index (BMI), waist circumference, hip circumference, and body fat percentage, as measured by the impedance method (TBF-102, Tanita Corp., Japan). The blood profile comprised serum glucose, total cholesterol (T-C), triglyceride (TG), LDL cholesterol (LDL-C), free fatty acid (FFA), GOT, GPT, γ-GTP, cholinesterase (ChE), natural killer cell activity (NK), T cell percentage, B cell percentage, and uric acid (UA). These were examined between 9 AM and 11 AM, after a minimum 12-hour fast. Measures of strength were grip strength, abdominal strength, back strength (Sensor EG-230, Sakai Corp., Japan), and anteflexion. Aerobic capacity was measured on a bicycle ergometer (Ergociser EC-1600, Cateye Corp., Japan) by using a ramp test with a continuous increase in load, starting from an initial load automatically programmed for sex, age, and weight. Physical Work Capacity (PWCHRmax) was calculated as 100% of HRmax, estimated from sex and age. The Profile of Mood States (POMS)25 was used to assess mental state. Participants were asked, while in a quiet and private room, to describe frankly their mood states during the past week. Process measurements were obtained by using a questionnaire. The lifestyle items were taken from the Japan Arteriosclerosis Longitudinal Study Physical Activity Questionnaire (JALSPAQ)26 and included questions on moderate exercise, along with questions on behavior patterns. The number of active modifications of lifestyle was assessed by questionnaire items on the following behaviors: regular breakfast, number of sleep hours, snacking frequency, drinking status, amount of alcohol consumed, smoking status, daily stress, awareness of physical activities, practice of physical activities, spa-bathing times, and bathing times. All measurements of the body and physical strength were performed by skilled evaluators with more than 5 years of experience. All the measurements of abdominal and back strength were performed by a single evaluator, after sufficient training, because it was considered likely that the evaluator's skill would influence the evaluation. The evaluator and the interventionist were not the same person; neither was blinded to the participants' group assignment. Collection and examination of blood were outsourced to the Unnan Hospital; both the phlebotomist and the examiner were blinded to the participants' group assignment. Classroom attendance rates were regularly confirmed by reviewing an attendance book, and staff confirmed the attendance of participants at each session. Attendance rates were calculated by dividing the number of attendees by the number of sessions. The participants were interviewed about their adherence (percentage of compliance during the previous period) to the individualized program at the end of the intervention and at the final evaluation. The rates of adherence represent the percentage of program compliance during the intervention period and at 1 year after the end of the intervention. Participants were informed of the study plan, both verbally and in writing, before their written consent was obtained. The information clearly indicated that they were allowed to quit the study at any time, and included an explanation of all the possible disadvantages of participating in the study. To avoid the appearance of unfairness, participants in the control group received a single session of general health guidance, which included instruction on stretching and dietary guidance. In addition, they received several leaflets on improving fitness, as well as coupons for the spa facility and the gym. The methodology of this study was approved by the Ethics Board of Tokyo University of Agriculture. The present study was registered as ID 000000607 by the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR) in Japan. BODY.METHODS.STATISTICAL ANALYSIS: Because HDL-C was the primary outcome measure and CD4/8 and HbA1c were secondary outcomes of interest, power calculations were performed for the primary outcome. We used data from the National Nutrition Survey in Japan 200427 on people aged 30 to 59 years to determine the sample size. The standard deviation of the mean was approximately 15 mg/dL in males, and the significant difference level between groups in the current study was 15 mg/dL. Statistical power was set so that the probability of type 1 error (α) was 5% and the probability of type 2 error (β) was 20%. The required sample size was calculated to be 16 or more participants in each arm. A 2-sample t test (Welch test) was used to compare continuous variables between groups. Fisher's exact probability test, the Mann-Whitney test, and the Kruskal-Wallis test were used for analysis of discrete variables. Repeated measures of variance (ANOVA) was used to investigate differences in changes between groups (2 groups × 3 times on ITT analysis; 3 groups × 3 times on subgroup analysis). Variables not filling Mauchly's test of sphericity were analyzed after Greenhouse-Geisser correction. Differences within and among groups were judged to be significant when the significance level was 5% or lower. SPSS 14.0J for Windows was used for statistical analysis. BODY.RESULTS: Table 2 shows the salient medical history of the participants. No significant differences were found between the 2 groups in age, internal diseases, or orthopedic diseases. The attendance rate for the intervention course was 56.2 ± 24.2% (mean ± SD). Rates of adherence to the individualized programs were 60.0 ± 27.2% and 30.5 ± 29.6% during the intervention period and at 1 year after the intervention, respectively. Table 2. Clinical characteristics of participants   Intervention Group Control Group n 22 21 Age (yrs) a 41.1 ± 7.5 46.3 ± 7.0 Medical history (Internal medicine)     ​ Diabetes 1 (4.5%) 0 (0%) ​ Dyslipidemia 1 (4.5%) 0 (0%) ​ Hyperuricemia 1 (4.5%) 0 (0%) ​ Hyperparathyroidism 0 (0%) 1 (4.8%) ​ Aortic stenosis 0 (0%) 1 (4.8%) Medical history (Orthopedics)     ​ Knee Osteoarthrosis 0 (0%) 1 (4.8%) ​ Lumbar spine Osteoarthrosis 0 (0%) 2 (9.5%) ​ Osteoporosis 1 (4.5%) 0 (0%) The numbers represent the number of patients (percentage), unless otherwise indicated. a Mean ± SD Table 3 shows results of the ITT analysis between groups at baseline, at the interim evaluation, and at the final evaluation, 1 year after the end of the intervention. No significant differences in HDL-C were observed between groups. Significant (P < 0.05) interaction was observed for the secondary outcome measure CD4/8, which was used to represent participants' immunological function. Related to this result, significant (P < 0.05) interaction was found between groups for CD4+ and CD8+. No significant difference in HbA1c was found between groups. There was a tendency for serum glucose and vigor to differ between groups, (P = 0.057 and P = 0.069, respectively). No significant differences were found for the other variables. Table 3. Time-series comparison of physical status, blood profile, and mental state (mean ± SD) in the intervention and control groups Outcome Measurements Intervention group ( n = 22) Control group ( n = 21)   P -value baseline* 6 months later follow-up baseline* 6 months later follow-up   Body measurements                 ​ Weight, kg 70.7 ± 9.7 70.4 ± 10.0 70.2 ± 8.6 64.2 ± 5.8 64.1 ± 6.3 63.6 ± 5.6   0.875 ​ Body mass index, kg/m 2 24.2 ± 2.8 24.1 ± 3.1 24.0 ± 2.6 22.8 ± 2.5 22.7 ± 2.5 22.5 ± 2.3   0.772 ​ Waist circumference, cm 85.2 ± 7.7 84.8 ± 7.0 85.3 ± 7.0 80.4 ± 5.4 80.7 ± 6.4 80.4 ± 6.1 # 0.619 ​ Hip circumference, cm 93.6 ± 4.9 93.4 ± 4.7 93.5 ± 4.5 89.4 ± 4.9 90.1 ± 3.7 89.9 ± 3.2 # 0.530 ​ Body fat percentage, % 20.2 ± 5.2 18.7 ± 4.7 17.8 ± 3.8 17.8 ± 3.8 16.5 ± 3.8 15.5 ± 3.1 # 0.972 Physical strength                 ​ Right-hand grip, kg 47.6 ± 7.2 49.8 ± 6.3 48.6 ± 6.5 45.5 ± 6.2 46.5 ± 6.0 46.4 ± 7.5   0.537 ​ Left-hand grip, kg 45.6 ± 6.7 45.8 ± 7.3 44.7 ± 6.4 44.0 ± 7.1 43.8 ± 7.7 44.4 ± 7.6   0.338 ​ Anteflexion, cm 38.3 ± 7.6 41.6 ± 8.9 43.8 ± 8.3 37.9 ± 9.8 38.4 ± 7.5 41.1 ± 9.3   0.212 ​ Maximal physical working capacity, W 193.6 ± 61.9 189.2 ± 50.5 189.3 ± 48.3 180.8 ± 38.5 186.8 ± 28.0 187.2 ± 24.0   0.274 ​ Abdominal strength, kgf 25.7 ± 7.4 28.7 ± 5.1 33.3 ± 5.4 21.4 ± 6.7 24.9 ± 5.1 28.4 ± 5.9   0.942 ​ Back strength, kgf 26.8 ± 6.2 34.1 ± 3.8 37.9 ± 5.1 24.5 ± 6.6 31.2 ± 5.7 35.1 ± 5.0   0.855 Blood profile                 ​ Serum glucose, mg/dL 99.8 ± 26.5 99.0 ± 20.7 96.7 ± 22.5 95.5 ± 9.0 99.7 ± 12.1 99.8 ± 12.3   0.057 ​ Hemoglobin A 1c , % 5.2 ± 0.8 5.1 ± 0.6 4.9 ± 0.7 5.1 ± 0.4 5.0 ± 0.4 4.9 ± 0.4   0.434 ​ Lactic acid, mg/dL 9.1 ± 3.5 8.1 ± 3.0 8.5 ± 2.4 8.9 ± 4.3 9.9 ± 4.3 9.3 ± 3.6   0.350 ​ Total cholesterol, mg/dL 197.1 ± 27.0 212.6 ± 28.7 211.8 ± 37.8 190.2 ± 14.8 189.2 ± 48.8 201.8 ± 30.3   0.205 ​ Triglyceride, mg/dL 125.0 ± 114.5 141.0 ± 128.0 188.4 ± 290.0 105.0 ± 52.7 118.7 ± 58.6 142.2 ± 92.0 # 0.579 ​ High-density lipoprotein cholesterol, mg/dL 59.5 ± 17.0 63.7 ± 16.0 60.0 ± 15.7 59.0 ± 15.1 61.8 ± 14.5 59.1 ± 14.5   0.792 ​ Low-density lipoprotein cholesterol, mg/dL 112.7 ± 32.3 120.7 ± 28.3 120.7 ± 26.7 110.1 ± 17.9 112.7 ± 24.5 115.7 ± 28.9 # 0.667 ​ Free fatty acid, mEq/L 0.45 ± 0.22 0.34 ± 0.16 0.28 ± 0.11 0.44 ± 0.15 0.30 ± 0.10 0.30 ± 0.11   0.806 ​ Glutamic oxaloacetic transaminase, IU/L 22.0 ± 5.5 20.5 ± 4.9 26.4 ± 9.9 21.0 ± 4.8 19.2 ± 3.6 22.1 ± 3.7 # 0.125 ​ Glutamic pyruvic transaminase, IU/L 27.5 ± 16.7 25.4 ± 13.3 29.5 ± 13.8 20.6 ± 8.8 22.3 ± 7.9 25.0 ± 6.6 # 0.396 ​ γ-glutamyl transpeptidase, IU/L 52.5 ± 43.3 46.1 ± 29.1 59.0 ± 54.0 43.8 ± 55.2 40.1 ± 47.5 41.0 ± 43.3 # 0.130 ​ Cholinesterase, IU/L 346.8 ± 57.8 351.0 ± 55.3 349.5 ± 54.5 357.4 ± 55.6 363.3 ± 58.4 354.4 ± 59.6   0.589 ​ NK cytotoxicity, % 33.6 ± 17.9 36.5 ± 15.2 29.7 ± 11.3 35.2 ± 14.9 41.1 ± 13.9 36.1 ± 12.7   0.395 ​ T cell, % 87.7 ± 5.6 88.5 ± 4.4 87.3 ± 4.9 87.3 ± 4.0 87.6 ± 4.0 86.2 ± 4.1 # 0.776 ​ B cell, % 5.7 ± 5.4 3.1 ± 1.7 5.6 ± 3.1 4.3 ± 3.3 2.6 ± 1.2 5.3 ± 3.0 # 0.558 ​ Cluster of differentiation 4+, % 39.7 ± 8.3 37.8 ± 6.1 37.9 ± 6.8 43.7 ± 8.7 39 ± 9.9 37.7 ± 7.3   0.027 ​ Cluster of differentiation 8+, % 35.9 ± 8.7 36.9 ± 8.1 36.2 ± 7.5 33.0 ± 7.2 34.6 ± 6.4 35.8 ± 7.2   0.027 ​ Cluster of differentiation 4/8 1.21 ± 0.49 1.09 ± 0.37 1.12 ± 0.41 1.44 ± 0.67 1.20 ± 0.50 1.12 ± 0.45 # 0.042 ​ Uric acid, mg/dL 6.3 ± 0.9 6.1 ± 1.3 6.1 ± 1.2 5.9 ± 1.3 5.8 ± 1.1 5.5 ± 1.2   0.350 Mental state                 ​ -Tension 48.6 ± 9.0 50.7 ± 9.0 49.9 ± 7.9 45.8 ± 5.2 46.3 ± 8.1 47.1 ± 7.7   0.728 ​ -Depression 50.6 ± 7.7 50.1 ± 7.4 50.5 ± 7.6 46.9 ± 6.7 48.2 ± 8.7 47.8 ± 7.3   0.666 ​ -Anger 50.0 ± 8.0 50.5 ± 6.6 50.0 ± 7.1 44.4 ± 6.5 46.6 ± 7.9 44.7 ± 5.3   0.724 ​ -Vigor 43.2 ± 6.7 47.4 ± 9.1 45.5 ± 7.5 43.7 ± 11.3 43.0 ± 11.7 43.3 ± 10.6   0.069 ​ -Fatigue 51.0 ± 10.1 52.0 ± 8.6 50.6 ± 8.2 48.8 ± 8.9 50.6 ± 11.0 48.6 ± 10.4   0.900 ​ -Confusion 52.7 ± 10.0 50.7 ± 8.2 52.1 ± 9.2 51.0 ± 6.9 51.0 ± 7.0 52.0 ± 9.1   0.457 #: Variables not fulfilling Mauchly's test of sphericity were analyzed after Greenhouse-Geisser correction. *: There were no significant differences between the 2 groups at baseline (2-sample t test). Table 4 shows changes in the lifestyle process measures of the intervention and control groups from baseline through to the final examination. Time spent on moderate exercise increased over the study period (P = 0.053) in the control group. No significant differences were found in other lifestyle process measures. Regarding adherence during the 1-year observation period in the intervention group, the cut-off value that divided the group into 2 equal parts was 25%. Subgroup analysis was performed using 3 groups: a high adherence group (more than 25%), a low adherence group (less than 25%), and the control group (Table 5). Significant (P < 0.05) effects were observed in CD4+ and CD4/8, which were maintained in the high adherence group, but tended to decrease in the low adherence and control groups. A greater decrease in body fat percentage was observed in the high adherence group, but was not significant (P < 0.07). Antiflexion also tended to improve in the high adherence group. Table 4. Time-series comparison of lifestyle characteristics of the intervention and control groups Process Measurements Intervention group ( n = 22) Control group ( n = 21) P -value baseline# 6 months later follow-up baseline# 6 month later follow-up Regular breakfast yes 19 (86%) 18 (82%) 18 (82%) 17 (81%) 18 (86%) 19 (91%) 0.162 0.083   no 3 (14%) 4 (18%) 4 (18%) 4 (19%) 3 (14%) 2 (10%)     Number of sleep hours* hours per day 6.9 ± 0.9 6.9 ± 0.9 7.0 ± 0.9 6.9 ± 0.9 6.7 ± 0.8 6.8 ± 0.7 0.825 Snacking frequency Seldom 10 (46%) 8 (36%) 9 (41%) 8 (38%) 6 (29%) 10 (48%) 0.917 0.592   1–2 times a month 0 (0%) 2 (9%) 2 (9%) 1 (5%) 2 (10%) 1 (5%)       1–2 times a week 3 (14%) 3 (14%) 6 (27%) 4 (19%) 3 (14%) 5 (24%)       3–4 times a week 6 (27%) 3 (14%) 2 (9%) 2 (10%) 4 (19%) 4 (19%)       Almost every day 3 (14%) 6 (27%) 3 (14%) 6 (29%) 6 (29%) 1 (5%)     Drinking status Drinker 18 (82%) 20 (91%) 16 (73%) 16 (76%) 16 (76%) 16 (76%) 0.984 0.249   Former drinker 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)       Infrequent drinker 4 (18%) 2 (9%) 6 (27%) 5 (24%) 5 (24%) 5 (24%)     Amount of alcohol consumed* Units # 1.6 ± 0.8 1.2 ± 0.8 1.2 ± 1.0 1.0 ± 0.8 1.4 ± 1.1 1.1 ± 1.0 0.301   (1 unit = 500 mL/day of beer, or equivalent) #             Smoking status Smoker 8 (36%) 7 (32%) 6 (27%) 8 (38%) 7 (33%) 8 (38%) 0.323 0.083   Former smoker 6 (27%) 6 (27%) 6 (27%) 3 (14%) 5 (24%) 3 (14%)       Nonsmoker 8 (36%) 9 (41%) 10 (46%) 10 (48%) 9 (43%) 10 (48%)     Daily stress Very high 5 (23%) 5 (23%) 2 (9%) 2 (10%) 3 (14%) 1 (5%) 0.416 0.866   High 9 (41%) 8 (36%) 11 (53%) 7 (33%) 7 (33%) 4 (19%)       Normal 6 (27%) 6 (27%) 6 (27%) 9 (43%) 8 (38%) 15 (71%)       Low 2 (9%) 3 (14%) 3 (14%) 3 (14%) 3 (14%) 1 (5%)     Middle strength exercise* min per month 404.5 ± 423.1 334.5 ± 377.5 215.1 ± 293.0 162.1 ± 218.9 223.6 ± 298.2 319.5 ± 456.4 0.053 Awareness of the need for Always aware 1 (5%) 3 (14%) 0 (0%) 0 (0%) 2 (10%) 1 (5%) 0.607 0.467 physical activity Usually aware 10 (46%) 8 (36%) 13 (59%) 11 (52%) 7 (33%) 11 (52%)       Sometimes aware 6 (27%) 8 (36%) 7 (32%) 5 (24%) 10 (48%) 7 (33%)       Seldom aware 5 (23%) 3 (14%) 2 (9%) 5 (24%) 2 (10%) 2 (10%)     Practice of Precontemplation 3 (14%) 1 (5%) 2 (9%) 5 (24%) 3 (14%) 6 (29%) 0.434 0.334 physical activities Contemplation 6 (27%) 4 (18%) 8 (36%) 4 (19%) 5 (24%) 7 (33%)       Preparation 5 (23%) 11 (50%) 6 (27%) 5 (24%) 7 (33%) 4 (19%)       Action 4 (18%) 0 (0%) 0 (0%) 2 (10%) 3 (14%) 1 (5%)       Maintenance 4 (18%) 6 (27%) 6 (27%) 5 (24%) 3 (14%) 3 (14%)     Frequency of spa bathing* times per month 1.5 ± 1.6 2.0 ± 2.4 2.0 ± 2.3 1.0 ± 1.9 1.3 ± 1.9 1.3 ± 2.6 0.910 Frequency of bathing at home* times per month 22.9 ± 8.5 24.2 ± 6.6 23.0 ± 8.1 23.9 ± 7.5 23.5 ± 6.6 24.1 ± 6.3 0.552 *: Continuous variables are shown as mean ± standard deviation and were tested after Greenhouse-Geisser correction. Categorical variables are shown as frequency (percentage). The Mann-Whitney test was adapted to test the difference in 6-month change between groups (left P -value); it was also adapted to the change from baseline to follow-up (right P -value). #: There were no significant differences between the 2 groups at baseline (2-sample t test for continuous variables and Fisher's exact probability test or Mann-Whitney test) Table 5. Subgroup analysis of physical status, blood profile, and mental state (mean ± SD) Outcome Measurements High adherence group ( n = 10) Low adherence group ( n = 12) Control group ( n = 21) P -value baseline 6 months later follow-up baseline 6 months later follow-up baseline 6 months later follow-up Body measurements                     ​ Weight, kg 72.9 ± 10.7 73.0 ± 11.7 71.7 ± 9.0 68.8 ± 8.9 68.2 ± 8.2 68.9 ± 8.4 64.2 ± 5.8 64.1 ± 6.3 63.6 ± 5.6 0.188 ​ Body mass index, kg/m 2 24.8 ± 3.3 24.8 ± 3.7 24.4 ± 2.7 23.7 ± 2.4 23.5 ± 2.6 23.7 ± 2.6 22.8 ± 2.5 22.7 ± 2.5 22.5 ± 2.3 0.232 ​ Waist circumference, cm 86.0 ± 8.2 85.9 ± 7.4 85.5 ± 7.5 84.5 ± 7.5 83.9 ± 6.8 85.1 ± 6.9 80.4 ± 5.4 80.7 ± 6.4 80.4 ± 6.1 0.664 ​ Hip circumference, cm 94.8 ± 4.9 94.9 ± 5.1 94.7 ± 4.4 92.6 ± 4.9 92.3 ± 4.1 92.5 ± 4.6 89.4 ± 4.9 90.1 ± 3.7 89.9 ± 3.2 0.902 ​ Body fat percentage, % 22.4 ± 6.6 19.8 ± 5.3 17.8 ± 2.6 18.3 ± 2.7 17.8 ± 4.2 17.7 ± 4.8 17.8 ± 3.8 16.5 ± 3.8 15.5 ± 3.1 0.066 Physical strength                     ​ Right-hand grip, kg 50.5 ± 5.2 52.1 ± 6.4 50.9 ± 6.2 45.2 ± 7.9 47.9 ± 5.8 46.7 ± 6.4 45.5 ± 6.2 46.5 ± 6.0 46.4 ± 7.5 0.783 ​ Left-hand grip, kg 46.6 ± 7.1 48.2 ± 6.1 47.4 ± 6.1 44.8 ± 6.6 43.8 ± 7.9 42.5 ± 5.9 44.0 ± 7.1 43.8 ± 7.7 44.4 ± 7.6 0.199 ​ Anteflexion, cm 39.7 ± 8.1 45.4 ± 8.4 46.6 ± 6.7 37.2 ± 7.2 38.4 ± 8.3 41.3 ± 9.1 37.9 ± 9.8 38.4 ± 7.5 41.1 ± 9.3 0.067 ​ Maximal physical working capacity, W 205.3 ± 67.6 201.0 ± 54.5 199.7 ± 49.4 183.9 ± 57.8 179.4 ± 47.0 179.9 ± 47.6 180.8 ± 38.5 186.8 ± 28.0 187.2 ± 24.0 0.611 ​ Abdominal strength, kgf 26.9 ± 6.8 29.4 ± 5.2 35.1 ± 4.5 24.6 ± 7.9 28.0 ± 5.1 31.6 ± 5.9 21.4 ± 6.7 24.9 ± 5.1 28.4 ± 5.9 0.875 ​ Back strength, kgf 29.6 ± 5.3 33.7 ± 3.8 38.7 ± 3.9 24.5 ± 6.1 34.4 ± 3.9 37.2 ± 6.0 24.5 ± 6.6 31.2 ± 5.7 35.1 ± 5.0 0.285 Blood profile                     ​ Serum glucose, mg/dL 97.3 ± 5.8 95.4 ± 6.7 93.3 ± 7.5 101.9 ± 36.1 102.1 ± 27.5 99.5 ± 30.1 95.5 ± 9.0 99.7 ± 12.1 99.8 ± 12.3 0.207 ​ Hemoglobin A 1c , % 5.1 ± 0.4 5.1 ± 0.3 4.9 ± 0.4 5.2 ± 1.0 5.2 ± 0.8 5.0 ± 0.9 5.1 ± 0.4 5.0 ± 0.4 4.9 ± 0.4 0.703 ​ Lactic acid, mg/dL 9.0 ± 3.5 8.4 ± 2.9 8.7 ± 2.1 9.1 ± 3.7 7.9 ± 3.2 8.3 ± 2.7 8.9 ± 4.3 9.9 ± 4.3 9.3 ± 3.6 0.640 ​ Total cholesterol, mg/dL 212.6 ± 30.0 227.5 ± 26.1 218.3 ± 35.9 184.3 ± 16.1 200.3 ± 25.4 206.4 ± 40.0 190.2 ± 14.8 189.2 ± 48.8 201.8 ± 30.3 0.294 ​ Triglyceride, mg/dL 109.6 ± 56.1 127.5 ± 53.4 141.8 ± 97.7 137.8 ± 148.5 152.3 ± 169.2 227.3 ± 386.2 105.0 ± 52.7 118.7 ± 58.6 142.2 ± 92.0 0.629 ​ High-density lipoprotein cholesterol, mg/dL 60.2 ± 16.3 63.2 ± 14.9 58.4 ± 14.5 59.0 ± 18.3 64.1 ± 17.5 61.3 ± 17.0 59.0 ± 15.1 61.8 ± 14.5 59.1 ± 14.5 0.520 ​ Low-density lipoprotein cholesterol, mg/dL 130.5 ± 29.0 138.9 ± 23.9 130.8 ± 29.2 97.9 ± 27.7 105.6 ± 22.6 112.3 ± 22.2 110.1 ± 17.9 112.7 ± 24.5 115.7 ± 28.9 0.419 ​ Free fatty acid, mEq/L 0.4 ± 0.1 0.3 ± 0.2 0.3 ± 0.1 0.5 ± 0.3 0.4 ± 0.2 0.3 ± 0.1 0.4 ± 0.1 0.3 ± 0.1 0.3 ± 0.1 0.784 ​ Glutamic oxaloacetic transaminase, IU/L 23.9 ± 6.1 23.2 ± 4.9 29.7 ± 11.7 20.3 ± 4.6 18.3 ± 3.8 23.7 ± 7.5 21.0 ± 4.8 19.2 ± 3.6 22.1 ± 3.7 0.256 ​ Glutamic pyruvic transaminase, IU/L 33.7 ± 18.8 31.5 ± 15.5 32.8 ± 13.5 22.3 ± 13.3 20.3 ± 8.8 26.8 ± 14.0 20.6 ± 8.8 22.3 ± 7.9 25.0 ± 6.6 0.403 ​ γ-glutamyl transpeptidase, IU/L 64.4 ± 58.4 49.1 ± 32.7 65.2 ± 68.1 42.7 ± 23.4 43.7 ± 27.0 53.8 ± 41.4 43.8 ± 55.2 40.1 ± 47.5 41.0 ± 43.3 0.094 ​ Cholinesterase, IU/L 326.5 ± 39.0 342.1 ± 37.5 335.9 ± 28.9 363.8 ± 66.7 358.3 ± 67.5 360.8 ± 68.4 357.4 ± 55.6 363.3 ± 58.4 354.4 ± 59.6 0.273 ​ NK cytotoxicity, % 31.6 ± 19.1 35.3 ± 18.3 27.1 ± 12.1 35.3 ± 17.6 37.6 ± 12.8 31.8 ± 10.6 35.2 ± 14.9 41.1 ± 13.9 36.1 ± 12.7 0.723 ​ T cell, % 86.4 ± 6.5 88.6 ± 3.8 87.2 ± 4.0 88.8 ± 4.8 88.5 ± 5.0 87.3 ± 5.8 87.3 ± 4.0 87.6 ± 4.0 86.2 ± 4.1 0.425 ​ B cell, % 6.7 ± 6.6 3.1 ± 1.9 5.9 ± 3.1 4.8 ± 4.3 3.1 ± 1.7 5.3 ± 3.2 4.3 ± 3.2 2.6 ± 1.2 5.3 ± 3.0 0.679 ​ Cluster of differentiation 4+, % 40.7 ± 7.0 38.2 ± 5.5 40.2 ± 8.1 38.8 ± 9.4 37.6 ± 6.8 36.1 ± 5.0 43.7 ± 8.9 39.0 ± 9.9 37.7 ± 7.3 0.047 ​ Cluster of differentiation 8+, % 36.7 ± 6.8 38.2 ± 6.8 36.4 ± 7.2 35.3 ± 10.3 35.9 ± 9.2 36.1 ± 8.1 33.0 ± 7.2 34.6 ± 6.4 35.8 ± 7.2 0.051 ​ Cluster of differentiation 4/8 1.2 ± 0.4 1.0 ± 0.3 1.2 ± 0.5 1.2 ± 0.6 1.1 ± 0.4 1.1 ± 0.3 1.4 ± 0.7 1.2 ± 0.5 1.1 ± 0.5 0.031 ​ Uric acid, mg/dL 6.7 ± 0.9 6.5 ± 1.0 6.6 ± 1.2 5.9 ± 0.9 5.9 ± 1.0 5.6 ± 1.0 5.9 ± 1.3 5.8 ± 1.1 5.5 ± 1.2 0.473 Mental state                     ​ -Tension 52.3 ± 9.6 54.2 ± 6.9 53.5 ± 5.8 45.5 ± 7.6 47.8 ± 9.7 46.8 ± 8.4 45.8 ± 5.2 46.3 ± 8.1 47.1 ± 7.7 0.959 ​ -Depression 55.6 ± 7.2 51.5 ± 5.9 53.6 ± 7.9 46.4 ± 5.3 48.9 ± 8.5 48.0 ± 6.7 46.9 ± 6.7 48.2 ± 8.7 47.8 ± 7.3 0.177 ​ -Anger 52.6 ± 5.9 51.2 ± 6.0 52.2 ± 7.6 47.8 ± 9.1 48.8 ± 8.0 48.3 ± 6.4 44.4 ± 6.5 46.0 ± 8.3 44.7 ± 5.3 0.875 ​ -Vigor 42.1 ± 5.6 46.9 ± 7.2 45.3 ± 5.0 44.2 ± 7.6 48.4 ± 10.4 45.6 ± 9.3 43.7 ± 11.3 43.0 ± 11.7 43.3 ± 10.6 0.229 ​ -Fatigue 54.3 ± 11.8 54.7 ± 6.6 52.0 ± 8.1 48.3 ± 9.4 49.0 ± 9.2 49.4 ± 8.5 48.8 ± 8.9 50.6 ± 11.0 48.6 ± 10.4 0.893 ​ -Confusion 58.4 ± 11.1 54.8 ± 13.1 55.4 ± 10.5 48.0 ± 6.0 49.8 ± 7.7 49.4 ± 7.3 51.0 ± 6.9 53.0 ± 8.9 52.0 ± 9.1 0.267 #: Variables not fulfilling Mauchly's test of sphericity were analyzed after Greenhouse-Geisser correction. Table 6 shows the results of subgroup analysis of the process measures. A significant difference with the intervention group (P < 0.05) was observed only with smoking status. Table 6. Subgroup analysis of lifestyle characteristics Process Measurements High adherence group ( n = 10) Low adherence group ( n = 12) Control group ( n = 21) P -value baseline 6 months later follow-up baseline 6 months later follow-up baseline 6 month later follow-up Regular breakfast yes 9 (90%) 8 (80%) 8 (80%) 10 (83%) 10 (83%) 10 (83%) 17 (81%) 18 (86%) 19 (91%) 0.212 0.153   no 1 (10%) 2 (20%) 2 (20%) 2 (17%) 2 (17%) 2 (17%) 4 (19%) 3 (14%) 2 (10%)   Number of sleep hours* hours per day 7.3 ± 0.8 7.0 ± 0.8 7.1 ± 0.9 6.63 ± 0.9 6.89 ± 1.0 6.88 ± 0.9 6.86 ± 0.9 6.74 ± 0.8 6.81 ± 0.7 0.285 Snacking frequency Seldom 5 (50%) 3 (30%) 3 (30%) 5 (42%) 5 (42%) 6 (50%) 8 (38%) 6 (29%) 10 (48%) 0.891 0.722   1–2 times a month 0 (0%) 1 (10%) 1 (10%) 0 (0%) 1 (8%) 1 (8%) 1 (5%) 2 (10%) 1 (5%)     1–2 times a week 0 (0%) 1 (10%) 2 (20%) 3 (25%) 2 (17%) 4 (33%) 4 (19%) 3 (14%) 5 (24%)     3–4 times a week 3 (30%) 2 (20%) 2 (20%) 3 (25%) 1 (8%) 0 (0%) 2 (10%) 4 (19%) 4 (19%)     Almost every day 2 (20%) 3 (30%) 2 (20%) 1 (8%) 3 (25%) 1 (8%) 6 (29%) 6 (29%) 1 (5%)   Drinking status Drinker 10 (100%) 10 (100%) 7 (70%) 8 (67%) 10 (83%) 9 (75%) 16 (76%) 16 (76%) 16 (76%) 1.000 0.131   Former drinker 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (%) 0 (%) 0 (%)     Infrequent drinker 0 (0%) 0 (0%) 3 (30%) 4 (33%) 2 (17%) 3 (25%) 5 (24%) 5 (24%) 5 (24%)   Amount of alcohol consumed* Units # 1.59 ± 0.8 1.35 ± 0.6 1.36 ± 1.0 1.18 ± 0.8 1.06 ± 0.9 1.13 ± 1.0 1.03 ± 0.8 1.41 ± 1.1 1.03 ± 1.0 0.137   (1 unit = 500 mL/day of beer, or equivalent) # Smoking status Smoker 3 (30%) 3 (30%) 3 (30%) 5 (42%) 4 (33%) 3 (25%) 8 (38%) 7 (33%) 8 (38%) 0.612 0.017   Former smoker 3 (30%) 3 (30%) 3 (30%) 3 (25%) 3 (25%) 3 (25%) 3 (14%) 5 (24%) 3 (14%)     Never-smoker 4 (40%) 4 (40%) 4 (40%) 4 (33%) 5 (42%) 6 (50%) 10 (48%) 9 (43%) 10 (48%)   Daily stress Very high 3 (30%) 4 (40%) 1 (10%) 2 (17%) 1 (8%) 1 (8%) 2 (10%) 3 (14%) 1 (5%) 0.684 0.596   High 6 (60%) 3 (30%) 6 (60%) 3 (25%) 5 (42%) 5 (42%) 7 (33%) 7 (33%) 4 (19%)     Normal 1 (10%) 3 (30%) 2 (20%) 5 (42%) 3 (25%) 4 (33%) 9 (43%) 8 (38%) 15 (71%)     Low 0 (0%) 0 (0%) 1 (10%) 2 (17%) 3 (25%) 2 (17%) 3 (14%) 3 (14%) 1 (5%)   Middle strength exercise* min per month 391 ± 342.3 370 ± 420.2 212 ± 187.4 415 ± 495.6 323 ± 355.7 218 ± 367.7 162 ± 218.9 224 ± 298.2 320 ± 456.4 0.177 Awareness of the need for Always aware 0 (0%) 1 (10%) 0 (0%) 0 (0%) 3 (25%) 0 (0%) 1 (5%) 1 (5%) 1 (5%) 0.568 0.190 physical activity Usually aware 5 (50%) 3 (30%) 4 (40%) 8 (67%) 4 (33%) 9 (75%) 8 (38%) 8 (38%) 11 (52%)     Sometimes aware 2 (20%) 4 (40%) 5 (50%) 3 (25%) 4 (33%) 2 (17%) 6 (29%) 10 (48%) 7 (33%)     Seldom aware 3 (30%) 2 (20%) 1 (10%) 1 (8%) 1 (8%) 1 (8%) 6 (29%) 2 (10%) 2 (10%)   Practice of Precontemplation 0 (0%) 1 (10%) 1 (10%) 3 (25%) 0 (0%) 1 (8%) 5 (24%) 3 (14%) 6 (29%) 0.736 0.236 physical activities Contemplation 4 (40%) 0 (0%) 2 (20%) 2 (17%) 4 (33%) 6 (50%) 4 (19%) 5 (24%) 7 (33%)     Preparation 2 (20%) 5 (50%) 2 (20%) 3 (25%) 6 (50%) 4 (33%) 5 (24%) 7 (33%) 4 (19%)     Action 2 (20%) 0 (0%) 0 (0%) 2 (17%) 0 (0%) 0 (0%) 2 (10%) 3 (14%) 1 (5%)     Maintenance 2 (20%) 4 (40%) 5 (50%) 2 (17%) 2 (17%) 1 (8%) 5 (24%) 3 (14%) 3 (14%)   Frequency of spa bathing* times per month 1 ± 1.2 2.3 ± 2.5 2.5 ± 3.0 1.83 ± 1.8 1.75 ± 2.4 1.5 ± 1.6 0.95 ± 1.9 1.29 ± 1.9 1.24 ± 2.6 0.191 Frequency of bathing at home* times per month 23.2 ± 9.2 26.4 ± 2.5 24.3 ± 6.3 22.6 ± 8.4 22.5 ± 8.5 21.8 ± 9.5 23.9 ± 7.5 23.5 ± 6.6 24.1 ± 6.3 0.698 *: Continuous variables are shown as mean ± standard deviation and were tested by the paired t -test. Categorical variables are shown as frequency (percentage). The Kruskal-Wallis test was adapted to test the difference in 6-month change between groups (left P -value), it was also adapted to the change from baseline to follow-up (right P -value). Rates of intervention attendance for the high and low attendance groups were 57.0 ± 22.3% and 8.3 ± 9.4%, respectively, and were significantly different (P < 0.001). Participants who performed their individualized programs attended the classroom more frequently. There were no adverse events during the intervention or examination. There were also no cases of hospitalization or severe morbidity during the study period. BODY.DISCUSSION: In this study, the intervention was based on the working conditions of male white-collar employees, and was designed to be feasible for industrial health. The study was completed successfully: no dropouts were observed and a complete ITT analysis was performed. This allowed us to precisely evaluate the duration of the effects from the end of the intervention to 1 year after the intervention. No significant effects were observed in the primary outcome measures, HDL-C. Also, a minor inclination toward beneficial activities was observed in the process measurements used to explain causal relationships. The ineffectiveness of the intervention might have been due to low attendance and poor adherence (56.2 ± 24.2% and 30.5 ± 29.6% in the intervention and observation periods, respectively). In addition, we did not attempt to forcefully persuade participants to engage in physical activity or to change their behavior, out of consideration for their job-related fatigue and the importance of time for their family life. A recent study reported that variability in adherence to an unsupervised exercise regimen in obese women was poor28; an unsupervised individualized program was also employed in the present study. A systematic review1 showed that booster interventions using the telephone, e-mail, or the Internet strengthened the long-term duration of the effects, and an RCT29 showed that brief, monthly personal contact provided a modest benefit in sustaining weight loss. These findings suggest that distributing black-and-white leaflets only 4 times after the intervention was insufficient to augment the effects of the present intervention. In addition, only a small number of participants had specific diseases that necessitated immediate changes in their behavior, and the intervention was performed for the purpose of general health improvement. Therefore, the results might be partially due to insufficient participant receptiveness to the intervention. Regarding the criteria used for immunological evaluation, CD4+ represented the role of helper T cells in relation to cytokine generation and macrophage activation. We found that high values of CD4+ were maintained in the intervention group, especially in the high adherence group, and that values tended to decrease in the control group. CD4/8, a secondary outcome measure, also showed some interaction. Participants who performed satisfactorily in the individualized programs had better class attendance. This suggests that those who had diligently learned techniques for improving their health from the specialist trainers—and who had good adherence to their self-constructed weekly additional physical activities and hot spa bathing—maintained and improved their immunological function for a period as long as 18 months. Improvements in health-related behavior, stress relief, and relaxation due to spa bathing20 may influence immunological function via automatic nervous system activity,19 but the mechanism underlying such an effect is unclear. In addition, participants in the high adherence group experienced a greater decrease in body fat percentage than did the other groups. All the participants in this study were automobile commuters, as they resided in areas where the mass transportation system was less developed than in the major urban areas of Japan. This limited the physical activity in their daily lives. A nationwide population-based study in Australia supported recommendations to cycle to work or use public transport, rather than cars, as a strategy to maintain healthy weight in men.30 Moreover, a recent study reported that, among middle-aged white-collar office workers, full-time work was associated with lower rates of achieving recommended physical activity levels.9 The study also discussed techniques for increasing physical activity levels during automobile commuting and other times. Recently, the concept of nonexercise activity thermogenesis (NEAT)31,32 has received attention as a way to increase energy consumption. This concept involves increasing the duration of physical activity by means of non-exercise activities, such as posture maintenance, walking for commuting or shopping, or load carrying during work. On the JALSPAQ, no significant difference was observed between the intervention and control groups with respect to moderate exercise. Considering that all the participants were automobile commuters living a sedentary lifestyle, an intervention that encouraged moderate physical activity might have been insufficient. Studies have shown that adherence to healthy lifestyle behaviors is associated with a lower risk of acute coronary syndrome among obese individuals,6 and that current health and health-related behaviors were stronger predictors than social factors of early mortality among older women.7 These findings highlight the importance of comprehensive health education on factors such as diet, smoking cessation, and sleep hygiene, in addition to the importance of physical activity. In the present study, hot spa bathing was employed as a unique intervention, and we recommended that participants bathe rather than shower at home. Despite the many studies on balneotherapy, evidence for its effectiveness is weak, due to difficulties in obtaining definite results and methodological problems with the RCTs that have been performed.33–35 However, hot spas are being increasingly utilized in many countries,12–14 with almost no adverse reactions or health hazards reported,35 and this has enhanced the utilization of hot spa bathing in health promotion campaigns. It was reported that, even after a 1-year observation period, improvements in HbA1c, aerobic capacity, back pain, vigor, fatigue, and depression were maintained in middle-aged women who had received comprehensive health education that included instruction on lifestyle and exercise, in combination with hot spa bathing once per week for 6 months.36 A cohort study reported that repeated comprehensive health education classes that included instruction for lifestyle, exercise, diet, and spa bathing, although available only biweekly, may have been effective preventive care for middle-aged and elderly people.37 Both the effectiveness and the cost-benefit of an intervention—especially as it relates to curbing medical expenses—are important concerns. To cite one example, a cohort study showed that medical expenses for subjects walking more than 1 hour per day were lower than for those walking less.38 The present report also emphasized the importance of maintaining a favorable lifestyle, from the perspective of medical economics. Unfortunately, the office in charge of health insurance for the participants was unable to provide information on individual medical expenses incurred during the study period. Comprehensive intervention by means of health education once every 2 weeks for 24 weeks, as performed in this study, is feasible in many white-collar labor environments, although poor results should be expected. However, substantial effects might be observed with a group approach that used definite selection criteria for the participants (eg, improvements in obesity or lipid metabolism) and specified a target based upon such criteria. In addition, regular communication with participants might maintain their motivation. This study had some potential limitations. First, although comprehensive health education was performed by specialists in a number of fields, improvements in diet were limited. Second, a type 2 error may have occurred in the subgroup analysis due to the small number (10 to 12) of subjects in each arm of the intervention groups. Third, because the participants were government employees of a single municipality, participants in both groups might have met one another and learned their group assignments. As a result, participants in the control group could have been unintentionally informed of the activities in the intervention group. The tendency observed in the control group—improvements in healthy behaviors (increased physical activity) and in physical measurements and some blood profile parameters—might not have been due exclusively to the Hawthorne effect, but could have also been influenced by such hearsay. This possibility may necessitate the use of a different study design, such as cluster RCT. Fourth, although it was a considerable achievement that there were no dropouts during this study, there were participants whose rates of attendance and adherence were very poor. We were unable to identify the reasons for these poor outcomes; thus, evidence-based measures could not be established in this study. Finally, we could not perform subgroup analysis between participants within and outside of reference values for each primary outcome measurement. BODY.CONCLUSION: Participants who attended classes and/or participated in additional individualized programs showed a tendency to maintain their immunological functions and to experience a decrease in body fat percentage. Lesser effects were observed in participants with poor adherence, even in the intervention group. However, we cannot quantify the effectiveness of each intervention (eg, spa bathing, exercise, healthy behavior, etc.) due to difficulties in interpretation that result from the inclusion of a comprehensive education program for the participants.

TITLE: The OnControl bone marrow biopsy technique is superior to the standard manual technique for hematologists-in-training: a prospective, randomized comparison ABSTRACT: The purpose of this study was to compare a novel bone marrow device with the standard marrow needle in a prospective, randomized study in a teaching hospital employing hematologists-in-training. The new device, the OnControl Bone Marrow (OBM) Biopsy System, utilizes a battery-powered drill to insert the needle. Fifty-four bone marrows (27 standard and 27 OBM) were performed by 11 fellows under the observation and supervision of 3 attending hematologists and 1 research technologist. The primary endpoint of the study, the mean length of the marrow biopsy specimens, a surrogate for marrow quality, was determined by a pathologist in a blinded manner. The mean length of the marrow biopsy specimens was significantly longer (56%) for the OBM group (15.3 mm) than for the standard bone marrow (SBM) group (9.8 mm), P<0.003. An objectively determined secondary endpoint; mean procedure time, skin-to-skin; also favored the OBM group (175 s) versus the SBM group (292 s), P<0.007. Several subjective secondary endpoints also favored the OBM group. Only minor adverse events were encountered in the OBM and SBM study groups. It was concluded that bone marrow procedures (BMPs) performed by hematologists-in-training were significantly faster and superior in quality when performed with the OBM compared to the SBM. These data suggest that the OBM may be considered a new standard of care for adult hematology patients. OBM also appears to be a superior method for training hematology fellows. BODY.INTRODUCTION: The human bone marrow is often evaluated in patients with various hematological disorders, including anemia, thrombocytopenia, pancytopenia, leukemia and other hematological malignancies.1 Multiple bone marrow procedures (BMP) are often required in patients with hematological malignancies to guide their treatment. The instrument customarily employed, the Jamshidi needle, is designed to yield both an aspirate and a marrow biopsy. The BMP has changed very little in the last 40 years and involves a manual rotary insertion of the Jamshidi needle into the marrow cavity of the posterior aspect of the iliac bone. Although local anesthesia for the skin, subcutaneous tissues and periosteum is universally administered, the BMP is regarded by patients and physicians alike as a painful and uncomfortable procedure.2–4 In addition, suboptimal specimens including dilute aspirates and small core biopsies are often obtained, limiting the diagnostic potential of the procedure. The conventional manual rotary technique causes excessive periosteal stimulation contributing to the pain. In addition, especially in heavy patients, the biopsy length is often suboptimal due to limitation of depth that can be reached in the bone by manual pressure. A new FDA-approved device for performing bone marrows, the OnControl Bone Marrow Biopsy System (OBM) was recently introduced by the Vidacare Corporation (Shavano Park, TX, USA). The OBM utilizes a battery-powered drill to insert the marrow needle into the iliac bone of adult hematology patients. Initial clinical studies utilizing the OBM system indicated that it was faster and easier to use for bone marrow aspirations than the traditional method.5 A few prospective studies comparing the OBM with the standard bone marrow procedure (SBM) have been carried out to date.6–8 While the duration of the procedure has been consistently shorter, and the core samples larger for patients undergoing OBM in these reports, no studies have been carried out in teaching hospitals to determine whether or not the OBM system will be more readily mastered by hematologists-in-training. Instruction of hematology fellows in the BMP technique has never been standardized, varies greatly between different fellowship programs and seldom receives high priority. As a result, fellows completing training are sometimes not well versed in the performance of the BMP and tend to avoid it in their post-fellowship careers. We conducted a prospective, randomized study to compare the OBM procedure with the SBM procedure in adults. The length of marrow biopsy specimens (a surrogate for marrow quality), aspirate quality and spicule content and procedure time were assessed objectively by the attending hematologist and pathologist. The patient, fellow and attending also completed questionnaires grading the pain, procedure difficulty, specimen quality and patient acceptability. BODY.MATERIALS AND METHODS: The study protocol was approved by the Biomedical Research Alliance of New York Institutional Review Board. Two kits were used, the OBM and SBM. The OBM system consists of a battery-powered driver and a biopsy needle set. The driver resembles a small hand-held drill, and powers a single lumen needle into the medullary cavity of the adult iliac bone. The needle set consists of two parts: an outer cannula, 11 gauge by 4 or 6 inches (102 or 152 mm) long; and a bevel-tip inner stylet- used to penetrate the cortex. The SBM device used in the study was typically a Jamshidi bone marrow biopsy needle (11 gauge by 4 or 6 inches), which has a two-piece T-handle design, a trocar-tapered stylet point and a triple-crown cannula tip. All fellows satisfactorily completed at least one SBM and OBM procedure under the supervision of an attending hematologist, before they were deemed certified to start enrolling to the study. Each fellow was initially randomized to perform either the SBM or the OBM. After the initial randomization the fellow then alternated between OBM and SBM procedures in a sequential fashion. Randomization was performed for the fellows rather than the patients to minimize differences between individual fellows as to previous experience and variable aptitude. After giving informed consent, adult patients requiring bone marrow sampling procedures underwent either a SBM or an OBM. Bone marrow aspiration and core biopsy were obtained utilizing a one needle/one puncture approach. The fellows were observed and supervised throughout the procedure by an attending hematologist or research technologist. The planned sample size was 102 patients, 51 by each technique. The primary endpoint of the study was the mean length in millimeters of the bone marrow specimens yielded by the two techniques. The measured length of the marrow biopsy specimen is a generally accepted surrogate for the quality of the marrow biopsy. It was chosen as the primary endpoint because that measurement was made in the pathology department by a person who had no knowledge of whether the specimen was OBM or SBM and who was not involved in the study in any way. Secondary endpoints included the skin to skin procedure time in seconds as well as other endpoints derived from the questionnaires. The questionnaires were completed by the patients, fellows and the attending hematologist/research technologist immediately after the procedure. All questions were answered on a 0 to 10 numerical scale. The patient questionnaire included questions regarding the level of pain experienced, the patient-perceived ease/difficulty of the procedure and the degree of patient willingness to have a repeat BMP if medically recommended. The questionnaires completed by the fellows included questions on the patient's level of pain, the ease/difficulty of the procedure and the perceived quality of the bone marrow aspirate and biopsy obtained. The questionnaires completed by the attending hematologist or by the research hematology technologist (who observed the procedure) included questions regarding the patient's apparent level of pain, the ease/difficulty of the procedure and the perceived quality of the bone marrow aspirate and biopsy obtained. Complications or adverse events were recorded during the procedure and at patient follow-up evaluation. Outpatients were called 1 or 2 days after the BMP and their responses recorded. Inpatients were visited 1 or 2 days after the BMP. Statistical testing was conducted using SAS Version 9 for Windows (SAS Institute, Cary, NC). Continuous parameters were summarized and compared between groups using a 2 sample t-test. Categorical parameters were summarized as proportions and compared using Fisher's Exact test. Because most fellows used OBM and SBM multiple times, linear mixed effects models were fit to the data to evaluate differences between the OnControl and Standard methods and adjust for the potential correlation in repeated measurements from the same fellow. A priori significance level was set at 0.05. An interim analysis was planned after the first 51 patients were accrued to determine if significant endpoints had been reached to permit early termination of the study. BODY.RESULTS: Two hospitals in the Bronx, NY participated in the study, Jacobi Medical Center and Montefiore Medical Center. A total of 54 BMPs (27 SBM and 27 OBM) were performed by 11 hematology fellows under the observation of 3 attending hematologists and 1 research technologist. The mean age of the 54 patients was 58.9 (±15.1) years and 61.1% were male. The mean height and weight were 168.1 (±11.0) cm and 77.9 (±19.3) kg, respectively. For these demographic parameters, there was statistical homogeneity between the two groups (Table 1). Of the 54 patients in the study (Table 2), 11 (20.4%) had myeloma, the most frequently-occurring diagnosis. There was no significant difference in the frequency of diagnoses between the two groups (P=0.563). Table 1Patient demographics.DemographicSBMOBMPNumber of male/female16/1116/110.609Mean age (years±standard deviation)60.7±16.357.2±14.00.399Mean height (cm±standard deviation)167.4±10.3168.0±12.10.842Mean weight (kg±standard deviation)76.3±17.078.8±22.20.634Body Mass Index27.2±5.627.7±6.10.755Race/Ethnicity: numbers of:0.730 Black1410 Hispanic912 Asian23 White22 Table 2Patient diagnoses.DiagnosisSBMOBMMyeloma74Pancytopenia35Lymphoma, non-Hodgkin's33Anemia25Myeloproliferative disorder32Monoclonal gammopathy of undetermined significance22Myelodysplastic syndrome13Thrombocytopenia21Hodgkins lymphoma11Acute myeloid leukemia10IgM paraprotein10Metastatic carcinoma10Eosinophilia01 The primary study endpoint, the mean marrow biopsy length was significantly longer in the OBM group (15.3 mm) than in the SBM group (9.8 mm), P<0.003 (Table 3). The mean procedure time, a secondary endpoint, was significantly shorter in the OBM group (175 s) compared to the SBM group (292 s), P<0.007. Table 3Study results: means±standard deviation.VariableOBMSBMPObjective Device efficacy Biopsy specimen length (mm)15.3±7.39.8±6.70.003* (primary endpoint) Procedure time (seconds)174.6±105.1292.1±210.00.007*Subjective Pain Scores 0–10 Perceived by patient4.7±2.85.9±2.80.11 Reported by fellow3.2±2.24.9±2.70.002* Reported by attending2.9±1.74.5±2.40.008* Willingness by patient to repeat BMP1.0±2.22.9±3.50.03* Procedure ease/difficulty (0–10) Reported by patient1.1±1.72.2±3.30.11 Reported by fellow2.6±2.65.0±2.80.002* Reported by attending3.0±2.65.1±3.40.006* Perception of specimen adequacy (0–10) Aspirate- fellow5.0±3.35.4±2.70.59 Aspirate- attending4.9±3.65.6±3.10.47 Core biopsy-fellow6.4±1.95.1±3.10.07 Core biopsy-attending6.7±2.04.8±3.40.01* Number (proportion) of dry taps7.0 (25.0%)4.0 (15.4%)0.505*Indicates statistical significance Other secondary endpoints which significantly favored the OBM group included the mean pain scores recorded by the fellows (P<0.002) and by the attendings (P<0.008). In regard to the mean pain scores reported by the patients in the 2 study groups, a lower score was tabulated in the OBM group (4.7) than in the SBM group (5.9), but the difference was not significant (P=0.11). However, patients indicated a greater willingness to have a repeat OBM (1.0) than a repeat SBM (2.9), P<0.03. Both the fellows (2.6 vs. 5.0, P<0.002) and attendings (3.0 vs 5.1, P<0.006) perceived the OBM to be easier to perform than the SBM. The attendings, but not the fellows, reported superior biopsy specimens in the OBM vs. the SBM group. The superiority of the OBM specimens was verified by objective blinded measurements in the pathology department. However, the fellows and the attendings scored the quality of the marrow aspirates as about equal. There were a greater number (proportion) of aspirates scored 0 (dry tap) in the OBM group (7/25%) than in the SBM group (4/15.4%), but the difference was not significant (P=0.505). There were no serious adverse events in either the OBM or the SBM study groups. One patient withdrew consent after an OBM was begun. The needle had penetrated the cortex of the iliac bone, but the needle could not be detached from the driver in order to proceed with the aspiration and biopsy. That needle was withdrawn from the ilium. The BMP was subsequently performed without incident using a standard marrow needle. The event was classified as a device-related complication. Later, the manufacturer modified the OBM needle attachment. After that, no further incidents of that type were reported. A minor adverse event occurred in a 56 year old man hospitalized for gastrointestinal bleeding, cirrhosis with portal hypertension, thrombocytopenia and coagulation abnormalities. He was being treated with red cell transfusions and fresh frozen plasma. Five days after an OBM, there was a spontaneous local drainage of a soft tissue hematoma from the posterior iliac marrow biopsy site. This responded to local and systemic therapy; the patient improved and was subsequently discharged. Another minor adverse event occurred in a 50 year-old female undergoing an outpatient SBM, which was successfully completed. Immediately after the procedure, the patient complained of numbness and weakness in the right lower extremity and was unable to stand. The patient was transferred via wheel chair to the emergency department, where an aortic sonogram and an echocardiogram were performed and reported normal. Two hours after the SBM, the patient was examined by a neurologist who found that the patient had completely recovered. The consultant felt unable to distinguish between an excess of local anesthetic adjacent to the right sciatic nerve versus an anxiety reaction to a difficult procedure. The patient was discharged from the emergency department and followed in clinic. She had no further right lower extremity weakness or numbness or other neurological symptoms. There were no other adverse events encountered in either study group. BODY.DISCUSSION: Since the introduction of the Jamshidi needle for BMPs 40 years ago, few technical advances have been made in the field. Commercial introduction of the OBM, a battery powered drill with attached needle for bone marrow aspirations and biopsies, followed FDA approval of the device in 2007. Preliminary studies indicated that the OBM was safe and yielded adequate aspirates in a short period of time.5 A prospective randomized study by Berenson et al.,6 comparing OBM and SBM, indicated that the OBM yielded bone marrow biopsy specimens of significantly greater volume, in a shorter period of time, with less residual pain in adult patients. A prospective, randomized study of OBM versus SBM by Swords et al.,7 using experienced operators, indicated that significantly longer biopsy cores were obtained with the OBM method. The mean biopsy lengths obtained in that study (13.1 mm OBM and 8.2 mm SBM) were very similar to those obtained in the present study (15.3 mm OBM and 9.8 mm SBM), as shown in Table 3. Thus, the primary endpoint in the present study confirmed the observation that the length of the marrow biopsy is significantly longer with the OBM method than with the SBM method. The length of the marrow biopsy is widely regarded as a surrogate for biopsy quality.7 Since the only reason for subjecting patients to this painful procedure is to obtain diagnostic information, the device which yields the most must be regarded as superior. The fact that the primary endpoint of the study, the length of the marrow core, was objectively determined in the pathology laboratory (which had no involvement in the study and no information as to how individual specimens were obtained), underscores the objectivity and validity of the conclusion. An element of operator bias appeared unlikely, since neophyte hematologists are maximally motivated to obtain optimal marrow specimens. A pre-planned interim analysis detected significant differences, and, as a result, the study was terminated early. Other OBM studies have been conducted with experienced operators in clinical practice settings.5–7 The present study is the first to be conducted in teaching hospitals utilizing inexperienced operators, i.e. hematologists-in-training. The fact that the results were similar whether the operators were experienced or not confirms the ease with which the OBM technique is mastered. Indeed, the majority of the participating fellows expressed a preference for the OBM method which suggests that OBM is a better training tool for teaching programs. The key secondary endpoint of the study was the duration of the procedure, skin-to-skin. The mean procedure time was significantly shorter with the OBM (174.6 s) than with the SBM (292.1 s). In other words, even with inexperienced operators, OBM changed BMP from a 5 minute procedure to a 3 minute procedure, on an average. Other comparative studies, employing experienced operators, have reported even faster OBM procedure times, and all have demonstrated significantly shorter times compared to a SBM control group.6–8 According to Kuball et al, BMP time is particularly relevant to patient pain.2 Patients are generally willing to undergo the BMP and a reasonable level of pain, providing that the procedure time is relatively short. Pain scores, as reported by the patients, the fellows and the attending hematologists/ research technologist were also secondary endpoints of the study. The patient-reported pain scores showed a trend favoring OBM, but the difference was not significant (P=0.11). A similar result was reported by Berenson et al who opined that the overall patient-reported pain score is largely dominated by the sharp pain of marrow aspiration. The latter would not be expected to differ between various needles. Both the fellows' and the attendings' perceptions of patient pain were significantly less with OBM compared to SBM, a result subject to observer bias. These data suggest there is a need for clinicians to reconsider current pain treatment options. These could include identifying patients at risk for significant pain during bone marrow procedures, discussing analgesia and even sedation options with patients including associated risks, and possibly re-dosing during the procedure, particularly if multiple punctures are required. Interestingly, the OBM patients expressed a significantly greater willingness to have a repeat BMP than the SBM patients (P<0.03). That result may be criticized as possibly subject to physician influence. However, another possible explanation for the higher level of patient willingness to have a repeat procedure may be the difference in degree of procedure difficulty between the two procedure types. Kuball noted that technical difficulties showed a trend toward a more painful procedure.2 As shown in Table 3, the Fellows did have more difficulty with the SBM than with the OBM procedure. Other secondary endpoints included the scores assessing ease/difficulty of the procedure by patients, fellows and attendings. On a scale of 0–10 for procedure difficulty, patients recorded a mean value of 1.1 for OBM and 2.2 for SBM. However, the difference was not statistically significant, owing to the wide standard deviation in both groups. On the other hand, the fellows and attendings rated the OBM procedure as significantly less difficult than the SBM, a result subject to observer bias. The questionnaire scores regarding quality of marrow aspirates and biopsies from the fellows and the attendings/research technologist did not show notable differences between the OBM and the SBM groups and were also subject to observer bias. Refusal by patients to undergo BMPs, especially in diseases like myeloma, leukemia and lymphoma, lead to delays in the diagnosis and treatment which may have fatal consequences. Any device, such as OBM, which promotes greater patient acceptance of a painful, but necessary, procedure may be anticipated to improve quality of care and to enhance favorable clinical outcomes. Cost effectiveness is an ever present criterion in the field of medical devices. The data presented in this and other OBM studies leave little question as to the superior effectiveness of OBM when compared to SBM. In a study involving 767 patients, Bishop et al reported that only 42% of bone marrow biopsy specimens were long enough for definitive diagnosis.9 The following points are relevant to the cost portion of the equation. First, the battery powered driver has a negligible cost since it can be used for about 500 procedures. Second, the OBM sterile disposable trays retail for about $40 more than many SBM trays. But the lower cost of SBM trays is offset by the inferior quality of the marrow specimens, delays in diagnosis and treatment, the necessity of repeating some BMPs, to say nothing of the longer procedure time and the inferior patient acceptance with the SBM. The long term costs and consequences of training future hematologists with inferior devices are not readily calculable. BODY.DISCUSSION.LIMITATIONS: There were several limitations in this study. As with other device studies, this study was limited by the absence of blinding of patients , operators or observers. The noise and the vibration of the OBM driver limited ability to blind the study for the patients ; the operator or observer could not be blinded for obvious reasons. Although the primary endpoint (biopsy length) and a secondary endpoint (procedure time) were objectively determined, other secondary endpoints derived from the various questionnaires could not be free of observer bias or physician influence. There was also potential operator bias in the primary endpoint since bone penetration by the operator might be influenced by the fellow's personal preference for the SBM vs. OBM. Another limitation was including data from multiple marrows for different fellows, as opposed to one marrow of each type per fellow, which would have taken years to complete. There was also variability in the number of marrows performed by each fellow, however this was partially offset by each fellow alternating between the SBM and OBM. Some senior fellows also had greater prior experience with the SBM before they were certified for the study. However greater experience with the SBM would have shifted the results in favor of the SBM rather than OBM. Finally, a detailed, blinded comparison of the pathologic quality of OBM versus SBM specimens was not carried out, owing to the omission of a specific consent phrase in the patient consent form. Nonetheless, blinded observations of the OBM and the SBM biopsy specimens by the hematopathologists did not reveal any notable differences in the amount of marginal necrosis (as might be caused by heat denaturation), hemorrhage, aspiration artifact, or crush artifact. Both OBM and SBM biopsy specimens displayed variable aspiration artifact, which was expected since the study design mandated a one puncture/one needle approach. BODY.CONCLUSIONS: The results of this first prospective, randomized trial in two teaching hospitals comparing BMPs in OBM and SBM patients, as performed by novice hematologists, indicate that significantly longer and better quality marrow biopsy cores may be obtained in a much shorter period of time, and with less patient pain when using the OBM device. Adverse events were inconsequential. The slightly greater expense of OBM appears justifiable in balance. Wider acceptance of OBM as a preferred BMP device in teaching hospitals may lead to greater acceptance of BMP as a necessary procedure by patients, to improvements in the diagnosis and treatment of hematology/ oncology patients and better training for fellows.

TITLE: Thoracic epidural analgesia reduces myocardial injury in ischemic patients undergoing major abdominal cancer surgery ABSTRACT.BACKGROUND AND OBJECTIVES: Major abdominal cancer surgeries are associated with significant perioperative mortality and morbidity due to myocardial ischemia and infarction. This study examined the effect of perioperative patient controlled epidural analgesia (PCEA) on occurrence of ischemic cardiac injury in ischemic patients undergoing major abdominal cancer surgery. ABSTRACT.PATIENTS AND METHODS: One hundred and twenty patients (American Society of Anesthesiologists grade II and III) of either sex were scheduled for elective upper gastrointestinal cancer surgeries. Patients were allocated randomly into two groups (60 patients each) to receive, besides general anesthesia: continuous intra and postoperative intravenous (IV) infusion with fentanyl for 72 h postoperatively (patient controlled intravenous analgesia [PCIA] group) or continuous intra and postoperative epidural infusion with bupivacaine 0.125% and fentanyl (PCEA group) for 72 h postoperatively. Perioperative hemodynamics were recorded. Postoperative pain was assessed over 72 h using visual analog scale (VAS). All patients were screened for occurrence of myocardial injury (MI) by electrocardiography, echocardiography, and cardiac troponin I serum level. Other postoperative complications as arrhythmia, deep venous thrombosis (DVT), pulmonary embolism, pneumonia, and death were recorded. ABSTRACT.RESULTS: There was a significant reduction in overall adverse cardiac events (myocardial injury, arrhythmias, angina, heart failure and nonfatal cardiac arrest) in PCEA group in comparison to PCIA group. Also, there was a significant reduction in dynamic VAS pain score in group PCEA in comparison to PCIA at all measured time points. Regarding perioperative hemodynamics, there was a significant reduction in intra-operative mean arterial pressure (MAP); and heart rate in PCEA group in comparison to PCIA group at most of measured time points while there was not a significant reduction in postoperative MAP and heart rate in the second and third postoperative days. The incidence of other postoperative complications such as DVT, pneumonia and in hospital mortality were decreased in PCEA group. ABSTRACT.CONCLUSION: Perioperative thoracic epidural analgesia in patients suffering from coronary artery disease subjected to major abdominal cancer surgery reduced significantly postoperative major adverse cardiac events with better pain control in comparison with perioperative IV analgesia. BODY.INTRODUCTION: Life expectancy has extended worldwide and a growing number of patients with multiple comorbidities including ischemic heart diseases and cancer have undergone surgeries. Consequently, postoperative cardiovascular complications are expected to increase,1 and perioperative acute myocardial infarction (AMI) has become a major health concern.2,3 It is well known that surgery induces a stress response, its extent is directly dependent on the magnitude of tissue destruction, and may be modified by the type of perioperative analgesia used. This stress response can lead to an increase in heart rate (HR) and blood pressure, which can precipitate episodes of myocardial ischemia.4 Perioperative myocardial infarction (PMI) is one of the most important predictors of short- and long-term morbidity and mortality associated with noncardiac surgery.5–8 Prevention of PMI is thus a prerequisite for the improvement in overall postoperative outcome. Thoracic epidural anesthesia (TEA) has been established as a cornerstone in perioperative care after thoracic and major abdominal surgery providing most effective analgesia.9,10 Beyond its analgesic properties, TEA's effects on postoperative neurohumoral stress response, cardiovascular pathophysiology, and intestinal dysfunction have been in the focus of both clinical and experimental investigations for years.11–15 The aim of the study was to test whether epidural analgesia added to a general anesthetic, compared with systemic, opioid-based standard care analgesia in ischemic patients undergoing major abdominal cancer surgery, provided any reduction in adverse cardiac events. BODY.PATIENTS AND METHODS: This study was approved by the local ethics committee of the South Egypt Cancer Institute, Assiut University, Assiut, Egypt. After obtaining written informed consent from each patient, 120 adult patients, complaining of coronary artery disease (CAD), classified as American Society of Anesthesiologists grade II and III and New York Heart Association class II and III, scheduled for elective major abdominal cancer surgery were consecutively enrolled. Patients with coagulopathy, active neurological disease, cutaneous disorders at the epidural insertion site, and allergy to the studied medications were excluded from the study. Every patient was evaluated by a cardiologist and anesthesiologist for medical history, physical examination, electrocardiography (ECG), and echocardiography. Anti-ischemic and antihypertensive drugs were continued during the perioperative period, including the morning of surgery; however, angiotensin-converting enzyme inhibitors, diuretics, and calcium channel blockers were suspended the day before surgery. The day before surgery, all patients were taught how to evaluate their own pain intensity using the visual analog scale (VAS), scored from 0 to 10 (where 0= no pain and 10= worst pain imaginable) and how to use the patient controlled analgesia (PCA) device (Abbott Laboratories, North Chicago, IL, USA). Each patient was given oral ranitidine tablet, 50 mg and lorazepam tablet, 3 mg on the night of surgery. Patients were randomly assigned into two groups, 60 patients each, by using opaque sealed envelopes containing a computer generated randomization schedule; the opaque envelopes were sequentially numbered and were opened before application of anesthetic plan. In the patient controlled intravenous analgesia (PCIA) group (N=60), patients received intraoperative analgesia with intravenous fentanyl bolus dose, 0.5 μg/kg, followed by continuous infusion of 1 μg/kg/h till the end of surgery. Postoperative analgesia consisted of intravenous fentanyl PCA, 10 μg/mL, background infusion 2 mL/h, bolus dose 3 mL and lockout interval 15 min. In the patient controlled epidural analgesia (PCEA) group (N=60), where patients received PCEA in conjunction with GA, intraoperative analgesia was started before skin incision by epidural bolus dose of 0.1 mL/kg of 0.125% bupivacaine/fentanyl 10 μg/mL, followed by continuous infusion of 0.1 mL/kg/h of 0.125% bupivacaine/fentanyl 5 μg/mL until the end of surgery. Postoperative analgesia was provided through PCEA for 72 h postoperatively (background infusion of 0.1 mL/kg/h of 0.125% bupivacaine/fentanyl 3 μg/mL, bolus dose of 3 mL, lockout interval was set at 20 min). BODY.PATIENTS AND METHODS.THE TECHNIQUE OF THORACIC EPIDURAL: Before induction of GA and under strict aseptic precautions, thoracic epidural catheter was inserted using a 16 gauge, Tuohy epidural needle by a paramedian approach. T8–T9 interspace was chosen for the injection. Skin at insertion site was anesthetized by 3 mL of lidocaine 1%, the epidural space was identified by the loss of resistance technique, the catheter was introduced ~2–4 cm into the epidural space, and epidural test dose of 3 mL of lidocaine 2% with 1:200,000 adrenaline was injected to confirm its position. The epidural was loaded with 0.1 mL/kg of 0.125% bupivacaine/fentanyl 10 μg/mL to obtain T4 sensory level; if the injected dose was not enough to achieve T4, another dose of 0.05 mL/kg was injected. BODY.PATIENTS AND METHODS.GA: After preoxygenation for 3 min, anesthesia was induced with IV propofol (1.5 mg/kg) and fentanyl 2 μg/kg. Tracheal intubation was performed after adequate neuromuscular blockade with cisatracurium 0.15 mg/kg. Anesthesia was maintained by isoflurane 1–1.5 minimum alveolar concentration (MAC); cisatracurium 0.03 mg/kg was administered when indicated. Fentanyl 0.5 μg/kg was given to maintain HR and blood pressure within 20% of the basal value. Patients were mechanically ventilated to maintain end tidal CO2 between 35 and 40 mmHg. The inspired oxygen fraction (FIO2) was 0.5 using oxygen-and-air mixtures. At the end of surgery, neuromuscular block was antagonized in all patients with neostigmine 0.05 mg/kg and atropine 0.02 mg/kg and finally the patients were extubated in the operating room. Hypotension was determined as systolic blood pressure <85 mmHg and was managed with IV ephedrine 0.1 mg/kg. Bradycardia was determined as HR slower than 50 beats/min and was taken care of by atropine 0.01 mg/kg. All patients were admitted to the surgical intensive care unit (ICU), and were followed up for 2 weeks by the following observations: 12-lead ECG was recorded daily and if there was any suspicion of ischemic attacks.Vital signs were recorded every 1 h in the ICU.Echocardiography was requested if there ECG findings (on continuous monitoring) suggested ischemic episodes or if the patient's complaint was consistent with angina. (All ECGs and echocardiography were analyzed by a consultant cardiologist who was blinded to the patients' condition.)VAS was recorded every 4 h for 3 days postoperatively.Venous blood samples for troponin I measurement were withdrawn routinely every day and at any time if there were ECG findings suggestive of ischemia. The primary endpoint was the overall occurrence of adverse cardiac events that include new ECG findings suggestive of ischemia such as new ST segment changes, new pathologic Q wave, or new T wave inversion;new echocardiographic findings suggestive of ischemia (new regional wall motion abnormalities);new critical arrhythmia, such as atrial flutter and fibrillation, second or third degree heart block, and any other arrhythmia affecting the hemodynamics;postoperative myocardial infarction diagnosed clinically, and by ECG and echocardiography, and in conjunction with cardiac troponin I, level >0.23 ng/mL was considered the cut-point for diagnosis of myocardial injury (MI);nonfatal cardiac arrest;heart failure, diagnosed clinically (new in-hospital signs or symptoms of dyspnea, orthopnea, paroxysmal nocturnal dyspnea, increased jugular venous pressure, pulmonary rales on physical examination) and by measuring B-type natriuretic peptide (BNP) level (the decision cut-point of BNP level for the diagnosis of heart failure was identical to that of 100 pg/mL).16 The secondary endpoints were intensity of pain measured by VAS (resting and dynamic);occurrence of other systems adverse events and complications;all in-hospital 30 days mortality. BODY.PATIENTS AND METHODS.TECHNIQUE OF MEASUREMENT: Blood samples for troponins I and plasma BNP levels were collected in non-pyrogenic, sterile falcon tubes. Troponin I and BNP were measured by a newly developed high-sensitive Elecsys analyzer (fully automated enzyme linked immunosorbent assay [ELISA] EVOLIS; Bio-Rad Laboratories Inc., Hercules, CA, USA). BNP kit uses competitive ELISA as the method while the cardiac-specific troponin I (cTnI) ELISA test is based on the principle of a solid phase ELISA. BODY.PATIENTS AND METHODS.STATISTICAL ANALYSIS: Statistical analysis was carried out on a personal computer using Statistical Package for the Social Sciences (SPSS) version 20 software. The sample size included all eligible patients admitted to the institute from August 2014 to August 2016 who were consecutively enrolled. Normality of continuous data distribution was tested using the Kolmogorov–Smirnov test. Normally distributed continuous data were described as mean and standard deviation. Independent samples Student's t-test was used for comparison between two independent groups (PCIA and PCEA). Skewed data were presented as median (interquartile range) and differences between the two groups were compared nonparametrically using Mann–Whitney U test. P<0.05 was considered statistically significant. BODY.RESULTS: The flow of the patients through the study is illustrated in Figure 1. The demographic data and the characteristics of the patients were similar between groups (Table 1). There was a significant decrease in overall adverse cardiac events (myocardial injury, ventricular and atrial arrhythmia, angina, heart failure and nonfatal cardiac arrest) in PCEA group in comparison to PCIA group (Table 2). The level of troponin I was significantly higher in group PCEA in comparison to group PCIA at all measured time points (P<0.038) (Figure 3). The number of patients with increased troponin level was higher in PCIA group (Figure 4). Regarding post-operative pain, the VAS pain score at rest was similar between groups (Table 3), while the VAS pain score during movement was significantly decreased in PCEA group in comparison to PCIA group at all measured time points (P<0.04) (Figure 2). Regarding perioperative hemodynamics, there was a significant reduction in intra-operative MAP and heart rate in PCEA group in comparison to PCIA group at most of measured time points while there was no significant reduction in postoperative MAP and heart rate in the second and third post-operative days (Table 4). The incidence of other postoperative complications such as DVT, pneumonia and in hospital mortality were decreased in PCEA group (Table 2). BODY.DISCUSSION: The present study showed that perioperative thoracic epidural analgesia in patients suffering from CAD subjected to major abdominal cancer surgery reduced significantly postoperative major adverse cardiac events in comparison with perioperative IV analgesia (Table 2). Moreover, the intensity of pain during movement was significantly decreased in PCEA group in comparison to PCIA group. The choice of 72 h as the period for PCA (either IV or epidural) because of the large proportion of clinically unrecognized AMI is related to the fact that most AMI occur during the early postoperative period.17 In agreement with the present study, previous study showed that cardiac morbidity was lower among patients undergoing major vascular surgery after the administration of GA combined with postoperative epidural analgesia compared to the administration of GA alone and postoperative systemic opioid analgesia.18 MASTER Trial showed a significant reduction in PMI with thoracic epidural catheters in comparison with control groups among 11 randomized studies involving 1,173 patients. Their inclusion criteria demanded that epidural analgesia should continue for at least 24 h after surgery, but their article does not state how they accounted for mortality among those patients randomized to the epidural group who may have died within the first 24 h.19 The Cochrane study in 2016 concluded from a review of 15 clinical trials that epidural analgesia provides better pain management than systemic opioids. It significantly reduces the number of people who suffer heart damage, time to return of unassisted respiration, gastrointestinal bleeding, and ICU length of stay. No difference was found in death rates at 30 days.20 A study was conducted by Mohamed et al in the same institute to observe 60 ischemic patients, assigned into two groups, who underwent elective major abdominal cancer surgery; 30 patients receiving GA (G1) and the others receiving combined general and epidural anesthesia (G2). They concluded that lumbar epidural anesthesia combined with general anesthesia in high-risk patients with ischemic heart disease undergoing major abdominal cancer surgery provided better pain relief, and ischemic cardiac events were similar in both groups.22 According to Moltner, dysrhythmias are common complications in the immediate postoperative period, even more common after upper abdominal and thoracic surgeries.22 Scott et al presented the first randomized evaluation of the impact of perioperative TEA on outcome in a large series of 400 patients with normal ventricular function undergoing coronary artery bypass grafting, wherein epidural catheters were placed immediately before surgery. There was a reduction in the incidence of supraventricular arrhythmias.23 In agreement with the present analysis, a study conducted by Giroban et al registered dysrhythmias in the postoperative period of 20% of 185 patients undergoing thoracoabdominal surgeries.24 The occurrence of arrhythmias can be explained by many factors such as preexisting cardiac pathology, intraoperative events, and arrhythmia triggers. Autonomic imbalance after operation has been implicated as a possible trigger, and is thought to be characterized by increased sympathetic tone and lower vagal tone.25 In this study, PCEA resulted in a better optimization of HR and mean arterial pressure during the intra and postoperative period in comparison with the PCIA group. This result showed the advantage of TEA over IV PCA by means of decreased HR and improved coronary blood flow. Consistent with these results, Kessler et al compared HR between patients who received GA together with TEA (group1) and those who received only GA (group2) during coronary artery bypass surgery performed on a beating heart and reported that the HR in group 1 was lower than preoperative values, during sternotomy and anastomosis compared to group 2. In that study, IV esmolol was administered in the group that received GA because of a high HR.26 Berendes et al and Fillinger et al, however, reported contradictory results as they did not observe a difference in hemodynamic findings between the control group and the TEA treatment group when they studied TEA in patients undergoing coronary artery bypass grafting.27,28 All the above studies resulted in low morbidity and mortality in patients receiving TEA and this reflected on hospital ICU stay. In contrast, the present study showed no significant difference with regard to ICU and hospital stay between the two groups. This is consistent with the observations of Kessler et al who found no differences in ICU and hospital stay between the TEA and GA groups.29 In contrast, Priestly et al found no difference in troponin levels between GA alone and GA plus high TEA groups.30 TEA modifies the electrical activity of the heart in addition to ventricular function and wall motion. Improvements in regional blood flow and reduction of major determinants of cardiac oxygen consumption lead to less severe ischemic injury.31 Large coronary epicardial arteries and coronary arterioles are densely innervated by sympathetic adrenergic nerve fibers. Cardiac sympathetic stimulation results in vasoconstriction of both normal and diseased coronary arteries in animals and in humans.32,33 In a canine model of experimentally induced cardiac ischemia, cardiac sympathectomy by TEA has been shown to increase regional cardiac blood flow, and redistribute coronary blood flow in favor of the endocardium in both normal and diseased areas.31 Davis et al observed favorable alteration in myocardial oxygen supply demand ratio.34 In patients with severe CAD, TEA relieved angina and improved myocardial oxygen supply by lowering systolic blood pressure and HR as well as pulmonary capillary wedge pressure with no significant improvement in coronary perfusion pressure.35,36 Cardioselective epidural blocks can increase the luminal diameter of stenosed segments of epicardial coronary arteries without affecting the diameter of non-stenosed segments.37 BODY.DISCUSSION.STUDY LIMITATIONS: Small sample size that may hinder providing well-drawn results with smaller statistical error and better conclusions with shorter duration follow-up period. BODY.CONCLUSION: Perioperative thoracic epidural analgesia in patients suffering from CAD subjected to major abdominal cancer surgery reduced significantly postoperative major adverse cardiac events with better pain control in comparison with perioperative IV analgesia.

TITLE: A randomized controlled trial of an ambulatory approach versus the hospital-based approach in managing suspected obstructive sleep apnea syndrome ABSTRACT: Comparisons of home-based versus hospital-based approach in managing patients with suspected obstructive sleep apnoea syndrome(OSAS). A prospective, controlled CPAP parallel study of new referrals with suspected OSAS randomized into group A) home-based or B) hospital-based approach. Following detection of AHI ≥ 15/hr by Embletta sleep study (group A) or polysomnography (group B), patients received CPAP for 3 months after an overnight autoCPAP titration at home or in hospital respectively. Modified intention-to-treat analysis of those with AHI ≥ 15/hr on CPAP (n = 86 vs 86) showed no difference in Epworth sleepiness score, the primary endpoint, but greater improvement in Sleep-Apnoea-Quality-of-Life-Index[difference 0.3,(95%CI 0.02, 0.6), p = 0.033] at 3 months in group A. The mean costs for the patients in group A and group B were HK$8479(989) and HK$22,248(2407) respectively. The mean difference between groups was HK$-13,769(USD 1770 equivalent) per patient with 95% CI. (−14324, −13213), p < 0.001. The waiting time of patients with AHI ≥ 15/hr who were started on CPAP treatment from the first clinic consultation to the diagnostic sleep test, autoCPAP titration, and CPAP treatment was 189.6, 148.8 and 145.0 days shorter in group A than group B respectively. Home-based approach is non-inferior to hospital-based approach in managing patients with suspected OSAS, with shorter waiting time, and substantial cost savings. BODY: Untreated obstructive sleep apnoea syndrome (OSAS) causes daytime sleepiness, cognitive function impairment and is associated with hypertension, atrial fibrillation, heart failure, sudden death, and stroke1. Attended polysomnography (PSG) is the standard investigation for patients with suspected OSAS, but the waiting time is often lengthy2. In recent years, several portable monitoring devices have been validated as useful alternatives of PSG, with the potential advantages of reducing the waiting time and healthcare cost34. The algorithms of managing patients with suspected OSAS were discussed by the American Academy of Sleep Medicine (AASM), and other professional societies56. The AASM task force has recommended that at a minimum, portable monitoring device must record airflow, respiratory effort, and blood oxygenation7. Several research groups have examined different models of care involving initial home-based sleep test in diagnosing OSAS, identifying patients who may benefit from continuous positive airway pressure (CPAP), and reducing the need for hospital-based PSG and CPAP titration with encouraging results891011121314. However very few studies have addressed healthcare costs, with conflicting results1314, and there are unresolved issues as to what disease management models based on economic outcome and what patient population would be appropriate for ambulatory management1516. This comprehensive study aimed to assess the role of an ambulatory approach with home diagnostic sleep test followed by home autoCPAP titration versus the hospital-based approach in managing patients with suspected OSAS with reference to 1) Improvement of subjective sleepiness; 2) Quality of life; 3) Cognitive function; 4)CPAP usage; and 5) Healthcare costs. We hypothesized that the ambulatory approach would be non-inferior to the conventional approach in managing patients with OSAS in terms of clinical outcome but the former approach would lead to substantial cost savings. BODY.METHODS.SUBJECTS AND METHODS: We conducted a prospective, randomized, controlled CPAP parallel study on new referrals to the Respiratory Clinic, Prince of Wales Hospital, Shatin, with suspected OSAS from 25 September 2013 to 31 August 2014. OSAS was defined by apnea-hypopnea index (AHI) ≥ 5/hr of sleep plus excessive daytime sleepiness or two of the following symptoms: choking or gasping during sleep, recurrent awakenings from sleep, unrefreshed sleep, daytime fatigue, and impaired concentration17. BODY.METHODS.INCLUSION CRITERIA: All patients, age 18–80 years, with suspected OSAS underwent assessment at the clinic with the Epworth sleepiness score (ESS)18 and symptoms evaluation. Patients who had ESS score >9 or at least two OSAS symptoms as described above were invited to join the study. Exclusion criteria included patients with (a) unstable cardiovascular diseases (e.g. recent unstable angina, myocardial infarction, stroke within the previous 6 months or severe left ventricular failure), (b) neuromuscular disease affecting respiratory muscles, (c) moderate to severe respiratory disease or documented hypoxemia or awake SaO2 <92% or (d) psychiatric disease that limited the ability to give informed consent. They were randomized by a random table into either group (A) home-based management approach or group (B) hospital-based management approach by a third party not involved in the trial. In both groups, the patients first went through the evaluation phase and if they had symptoms of OSAS with AHI ≥ 15/hr on home Embletta sleep test (Group A) or hospital-based PSG (Group B), they would then be enrolled to the CPAP outcome study (i.e. the second phase). BODY.METHODS.INCLUSION CRITERIA.GROUP A: Patients in group A underwent a level 3 home sleep study with the Embletta device (Medcare, Iceland), which had been validated against hospital-based PSG in the Chinese population in Hong Kong4. The EmblettaTM PDS is a pocket-sized, digital, multi-channel recording device that measures airflow through a nasal cannula connected to a pressure transducer, providing an AHI based on recording time. It detects respiratory and abdominal efforts through the effort sensor and can differentiate between obstructive and central events. Built-in position sensors can differentiate supine from non-supine events. Patients were instructed by nurses how to operate the Embletta device for the sleep recording and estimate their time of sleep4. Respiratory events are scored when desaturations of ≥4% occurs in the absence of moving artefacts and irrespective of co-existing changes in snoring or heart rate. The EmblettaTM PDS default settings for apneas and hypopneas were used in this study. An apnea was defined as a decrease in airflow by 80% of baseline for ≥10 seconds. A hypopnea was defined as a decrease in airflow by 50% of baseline for ≥10 seconds. The EmblettaTM PDS AHI used for analysis was automatically analyzed by the EmblettaTM PDS software4. Following detection of OSAS with AHI ≥ 15/hr on home Embletta sleep study, each patient was interviewed by the physician on duty and invited to participate in the overnight home autoCPAP titration study. We offered symptomatic patients with AHI ≥ 15/hr early CPAP treatment as this group of patients were most likely to be adherent to CPAP19, and had the greatest risk of adverse health outcomes related to OSAS2021. Those who had failed the Embletta sleep study were arranged to have a second home Embletta sleep study. Patients who were symptomatic of OSAS with a negative home Embletta sleep study were arranged to have a hospital-based PSG. Patients with AHI ≥ 15/hr were offered a basic CPAP education package by nurses and a 30-minute CPAP acclimatization trial with the AutoSet device (ResMed, NSW, Australia) in the afternoon at the clinic before an unattended overnight autoCPAP titration study at home (22). BODY.METHODS.INCLUSION CRITERIA.GROUP B: Overnight PSG(Alice LE, Respironics, USA) was performed as at the hospital for every subject in group B recording electroencephalogram, electro-oculogram, submental electromyogram, bilateral anterior tibial electromyogram, electrocardiogram, chest and abdominal wall movement by inductance plethysmography, airflow measured by a nasal pressure transducer [PTAF2, Pro-Tech, Woodinville, WA, USA] and supplemented by oronasal airflow thermistor, plus finger pulse oximetry. (22) Apnea was defined as cessation of airflow for >10 seconds with drop in the peak thermal sensor excursion by ≥90% of baseline whereas hypopnea as a reduction of nasal pressure airflow of ≥30% of baseline for >10 seconds plus an oxygen desaturation of ≥4% (23). Patients with AHI ≥ 15/hr on PSG underwent attended autoCPAP titration in the hospital setting after receiving a basic CPAP education package by nurses and a 30-minute CPAP acclimatization trial with the AutoSet device in the afternoon at the clinic (22). The CPAP level for both groups A and B was set at the median 95th centile pressure needed during autoCPAP titration at home (group A) and hospital setting (group B) to abolish snoring, obstructive respiratory events and airflow limitation. The patients in both groups were followed up at the CPAP clinic at 1 month and at 3 months by nurses to manage any problem with the CPAP device or mask fit and monitor the CPAP compliance. Subjects in both groups were prescribed nasal CPAP units with time clocks to assess objective CPAP usage (run time). In both arms of care, extra nursing consultations and phone advice were possible and were recorded. Those with mild OSAS (AHI 5–15/hr) in both group A and group B were advised by physicians on treatment alternatives such as dental device, lifestyle modifications22 if appropriate or a trial of CPAP if agreeable but they were not included in the CPAP outcome study. BODY.METHODS.OUTCOME ASSESSMENT: Prior to commencement of home or conventional CPAP treatment, all patients underwent baseline assessment including ESS18, sleep-apnoea-specific-quality-of-life index(SAQLI)23, and cognitive function tests24, and these were reassessed at 3 months after CPAP treatment. The ESS is a questionnaire specific to symptoms of daytime sleepiness and the patients are asked to score the likelihood of falling asleep in eight different situations with different levels of stimulation, adding up to a total score of 0 to 24 (18). The SAQLI has 35 questions organized into four domains: daily functioning, social interactions, emotional functioning and symptoms with a fifth domain, treatment-related symptoms, to record the possible negative impacts of treatment (25). Cognitive function tests including trail-making, digit-symbol, digit-span and Stroop colour testing were performed to provide objective evidence for improvement in daytime function on CPAP treatment, as in our previous study. (22) The trail-making test estimates the minimum time required to connect a structured number sequence and the lower the score, the better the performance. The digit symbol and span tests involve the immediate memory and recall of number sequences while the stroop colour test evaluates the correct matching of colour and their corresponding characters. For the Stroop color, digit-symbol, and digit-span tests, a higher score indicated superior performance (22). The primary endpoints included the difference in changes in ESS, SAQLI, cognitive function tests, objective CPAP usage after 3 months of CPAP treatment between the 2 groups. Secondary endpoints included difference in healthcare costs. BODY.METHODS.SAMPLE SIZE ESTIMATION: The study was powered to demonstrate non-inferiority of the ambulatory approach versus the conventional approach with respect to change in ESS, the primary outcome measure. A sample size of 86 patients in each group achieved 90% power to detect non-inferiority using a one-sided, independent samples t-test. The margin of equivalence was set at 2, i.e. a difference of this size or less on the ESS scale was not considered to be clinically important. The one-sided significance level (alpha) of the test was set at 0.025. It was assumed that the common standard deviation for change in ESS in moderate-severe OSAS was 425. As our respiratory clinic generally attracted 75% of patients with at least moderate OSAS, in order to allow for 10% drop-out over the course of the study, we proceeded to recruit at least 130 patients in each group. BODY.METHODS.STATISTICAL ANALYSIS: Data were analysed on an intention-to-treat (ITT) basis comparing the two groups in terms of demographics, and waiting time for diagnostic tests, CPAP titration and treatment by including all patients randomized. Data were analysed on a modified ITT basis by including patients with AHI ≥ 15/hr starting CPAP therapy in both groups. For comparisons of baseline characteristics, health economics, CPAP compliance and magnitude on change in outcomes (treatment minus baseline) between the 2 groups, independent samples t-test was used for continuous variables and chi-squared test for categorical variables. To compare the measurements before and after CPAP treatment, paired samples t-test was used. Data analysis was performed with IBM SPSS (IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp). As a sensitivity analysis, treatment per-protocol (TPP) analysis was conducted for those with AHI ≥ 15/hr and had completed the 3-month CPAP outcome study. BODY.METHODS.COST-EFFECTIVENESS ANALYSIS: Costs were divided into 2 parts, namely within-study costs and implementation costs in practice1115. Cost consequences analysis, i.e. a comparison of costs with different outcomes, was conducted26. Within study costs included the resource use and cost over the study period from randomization (eg, staff cost, CPAP device and its education package, attended training for the patients at clinic or hospital, overnight testing with PSG versus home monitoring, any Embletta recording failure and need for repeating the test, productivity loss due to sick leave of patients, and etc). The implementation cost in practice not only included the within-study cost but also other factors comparable to the reality (eg, the prevalence of patients with different severity, the waiting time for having PSG or unattended home CPAP titration, etc) (15). All costs associated with the continuum of care (CPAP equipment and disposables, dental appliance or surgical intervention, physician visits, etc) were taken into account (15). More details of the cost-effectiveness analyses are available in the Supplementary Information. This study protocol was approved by the Ethics Committees of the Chinese University of Hong Kong (CREC-2011.215-T) and registered at ClinicalTrials.gov(Identifier: NCT01828216) on 8 April 2013. The methods were carried out in accordance with the relevant guidelines and regulations. Written informed consent was obtained from all subjects enrolled in this study. BODY.RESULTS [MEAN(SD) UNLESS STATED OTHERWISE].BASELINE CHARACTERISTICS: In the first phase, altogether 316 subjects were recruited and randomized into group A (n = 157) and group B (n = 159) as shown in Fig. 1. Eighty six subjects were found to have AHI ≥ 15/hr in both Group A and B. Sixty two subjects in Group A and 69 subjects in Group B completed the 3-month follow-up. Table 1 showed the demographic data between Group A and B. The two groups were similar for most of the variables including the oxygen desaturation index (ODI) except for lower AHI and ESS in Group A (Table 1). The waiting time of patients with AHI ≥ 15/hr who were started on CPAP treatment from the first clinic consultation to the diagnostic sleep test, autoCPAP titration, and CPAP treatment was 59.3 (35.2), 117.3 (41.0) and 154.7 (69.5) days for group A versus 248.9 (186.0), 266.1 (192.5) and 299.7 (198.7) days for group B respectively. All the waiting time for sleep tests and treatment in group A was significantly shorter than group B (p < 0.001) (Table 2). Using a modified ITT analysis of those with AHI ≥ 15/hr (n = 86 vs 86), group A had greater improvement in SAQLI at 3 months [difference 0.3, (95% CI 0.02, 0.6), p = 0.033]. There were no significant differences in ESS and most of the cognitive function tests except for greater improvement in stroop colour testing with words in group A [difference 2.6, (95% CI 0.1, 5.1), p = 0.038] (Table 3). The results using TPP approach were similar to those analyzed by the modified ITT approach (Tables 4 and 5). Since home sleep test could underestimate the AHI, additional analysis was performed by comparing moderate OSA (AHI 15–30/hr by home Embletta) in group A with severe OSA (AHI > 30/hr by PSG) in group B by modified ITT (n = 43 vs n = 48) and TPP (n = 33 vs n = 39) respectively. Apart from some differences in trail making with words and stroop colour, differences in other endpoints were not significant (supplemental file Table S2a–d). BODY.RESULTS [MEAN(SD) UNLESS STATED OTHERWISE].BASELINE CHARACTERISTICS.HEALTHCARE COSTS: For those who had dropped out after commencement of CPAP, a last observation carried forward approach was used to impute their wage loss and transportation expense. The approach should be acceptable in this scenario as it is expected their salary and the route to go to hospital would be the same. Following modified ITT analysis, the mean (SD) costs for the patients in group A (n = 86) and group B (n = 86) were HK$8479 (989) and HK$22,248 (2407) respectively. The mean difference between groups was HK$-13,769 per patient with 95% CI. (−14324, −13213), p < 0.001. Status of those with AHI < 15/hr (n = 98) who were excluded from the 3-month CPAP outcome study is shown in the Supplementary Table S1. Patients who were symptomatic of OSAS with a negative home Embletta sleep study (n = 10) were arranged to have a hospital-based PSG, which revealed that 2 patients had AHI ≥ 15/hr. Those who had failed the first Embletta sleep study (n = 7) were arranged to have a second home Embletta sleep study. A "failed" home study means that a case could not complete the home sleep test successfully due to either technical problems (poor signals) or poor sleep (TST < 4 hrs), despite having repeated the test. Of these 7 patients who had undergone PSG, 3 were found to have AHI ≥ 15/hr. Thus altogether 10 + 7 = 17 patients in group A finally required PSG, with a waiting time of 384.4 (312.4) days for PSG. BODY.DISCUSSION: This randomized controlled trial (RCT) compared a home-based approach using a validated level 3 sleep diagnostic device4 for diagnosis followed by one night of autoCPAP titration against the hospital-based sleep laboratory approach in managing clinic patients with suspected OSAS. The waiting time of patients with AHI ≥ 15/hr who were started on CPAP treatment from the first clinic consultation to the diagnostic sleep test, autoCPAP titration, and CPAP treatment was 189.6, 148.8 and 145.0 days shorter in group A than in group B respectively. The much longer waiting time for PSG and autoCPAP titration in the hospital setting was due to the limited number of hospital beds designated for sleep medicine service whereas the home-based approach offered much more flexibility. Using a modified ITT analysis of those with AHI ≥ 15/hr who had commenced home CPAP treatment, Group A had slightly greater improvement in SAQLI and stroop colour testing with words at 3 months whereas there was no difference in other cognitive function tests between the 2 groups. As importantly, there was significant cost saving of HK$13,769 (USD1770 equivalent) per patient in favour of group A. However, whether there is significant cost saving using home management approach would depend on the local healthcare system and costs. A RCT in Spain has shown that patients with a high probability of OSAS could be diagnosed and treated in a home setting, with a high level of CPAP compliance and lower cost (€590 vs €894 vs €644) than using either a hospital-based approach or home respiratory polygraphy/hospital follow-up.(13) In contrast, another RCT in the USA has shown cost saving of US$264(95% CI $39, $496, P = 0.02) in favor of home-based management from the patient's perspective but US$40 (95% CI -$213, $142, P = 0.66) in favor of the laboratory-based arm under the base case from the provider perspective14. Our study findings add more strength to the growing literature that the ambulatory approach is an alternative strategy in managing clinic patients with suspected OSAS. In a RCT conducted by Skomro et al.8 comparing an ambulatory approach (home-based level 3 testing followed by one week of autoCPAP titration and then fixed-pressure CPAP based on 95th centile pressure) vs in-laboratory PSG and CPAP titration, the laboratory approach did not lead to superior four-week outcomes in sleepiness scores, sleep quality, quality of life, blood pressure, and CPAP adherence. Another RCT by Berry et al.9, comparing a level 3 portable monitoring(Watch PAT-100) and autoCPAP titration(2–3 nights) vs PSG for the diagnosis and treatment of OSAS, has shown that the former approach resulted in CPAP adherence and clinical outcomes similar to the one using PSG. In a RCT that compared standard PSG against ambulatory CPAP titration (autoCPAP titration for 1 week followed by CPAP set at 95th centile pressure) in high-risk patients identified by a diagnostic algorithm involving symptom score and a level 3 portable sleep diagnostic device, Mulgrew et al.10 have shown no advantage of PSG over the ambulatory approach in terms of diagnosis, CPAP titration(AHI on CPAP), ESS, and SAQLI over 3 months whereas adherence to CPAP therapy was better in the ambulatory group. A nurse-led model of care by Antic et al.11 using 4 nights of home auto-adjusting device after overnight home oximetry to set therapeutic CPAP has demonstrated non-inferior results(change in ESS, CPAP adherence at 3 months) to physician-directed care, which involved two laboratory PSG to diagnose and treat patients with moderate to severe OSAS. Kuna et al.12, who randomized patients with suspected OSAS to either home testing with the Embletta device followed by at least 3 nights of autoCPAP titration or laboratory-based management, found that functional outcome and treatment adherence in patients evaluated through the home testing approach was not clinically inferior to that in patients receiving standard in-laboratory PSG. Pressure for alternative approaches to the in-laboratory/hospital management of patients with OSAS will continue to increase given the cost of PSG, the limited number of hospital or laboratory-based facilities and the growing demand for more rapid access to testing16. Since 2007, the AASM has approved the use of home portable monitoring and recommended that unobserved registers with type 2–3 monitors be used as an alternative to PSG diagnosis7, and unattended autoCPAP titration to determine a fixed CPAP treatment pressure in patients with a high probability of moderate-to-severe OSAS without significant medical comorbidities27. It is important to shorten the waiting time for sleep investigation and treatment especially for those with a high clinical probability of OSAS, as many studies have shown that untreated OSA is associated with increased risks of hypertension28, platelet activation29, diastolic dysfunction30, mortality20313233, sudden death2134, and stroke213536. CPAP has been shown to reduce systemic blood pressure3738, daytime sleepiness39, risk of driving accidents40, and platelet activation29 although it remains to be seen by proper RCTs whether it may reduce mortality in patients with OSAS41. One limitation of this study was that the Embletta diagnostic device had underestimated the AHI because it only recorded the number of obstructive events/hour of recording instead of actual sleep time although the ODI in both groups was comparable. Moreover, only 62 and 69 out of 86 patients completed 3 months of CPAP treatment in group A and group B respectively. Without adequate contact with health professionals, more patients in group A were likely not inclined to follow-up care. In conclusion, the ambulatory approach for diagnosis and treatment was non-inferior to the conventional hospital-based approach in managing clinic patients in Hong Kong with suspected OSAS in terms of CPAP usage and improvement of some clinical endpoints, with the advantages of much shorter waiting time, and substantial cost savings. BODY.ADDITIONAL INFORMATION: How to cite this article: Hui, D. S. et al. A randomized controlled trial of an ambulatory approach versus the hospital-based approach in managing suspected obstructive sleep apnea syndrome. Sci. Rep. 7, 45901; doi: 10.1038/srep45901 (2017). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. BODY.SUPPLEMENTARY MATERIAL: Supplementary File

TITLE: Magnetic Resonance Findings of Acute Severe Lower Back Pain ABSTRACT.OBJECTIVE: To determine abnormal MRI findings in adults hospitalized with acute severe axial LBP. ABSTRACT.METHOD: Sixty patients with back pain were divided into 3 groups consisting of 1) 23 adults with acute axial severe LBP who could not sit up or stand up for several days, but had not experienced previous back-related diseases or trauma (group A), 2) 19 adults who had been involved in a minor traffic accident, and had mild symptoms but not limited mobility (group B), and 3) 18 adults with LBP with radicular pain (group C)., Various MRI findings were assessed among the above 3 groups and compared as follows: disc herniation (protrusion, extrusion), lumbar disc degeneration (LDD), annular tear, high intensity zone (HIZ), and endplate changes. ABSTRACT.RESULTS: The MRI findings of A group were as follows: disc herniation (87%), LDD (100%), annular tear (100%), HIZ (61%), and end plate changes (4.4%). The findings of disc herniation, annular tear, HIZ, and LDD were more prevalent in A group than in B group (p<0.01). HIZ findings were more prevalent in A group than in group B or group C (p<0.05). ABSTRACT.CONCLUSION: Patients with acute severe axial LBP were more likely to have disc herniation, LDD, annular tear, HIZ. Among LBP groups, there was a significant association of HIZ on MRI with acute severe axial LBP. BODY.INTRODUCTION: Acute lower back pain is one of the most common reasons for hospital visits. Approximately 33% of adults experience lower back pain in their life,1 and the lifetime prevalence of lower back pain is reported to be 70-85%.2 Despite this high prevalence rate, the cause of lower back pain is difficult to accurately diagnose, and its treatment is costly and results in considerable work day loss. In most cases, the cause of lower back pain, if without any red-flag signs, is a self-controllable disease, and is known to be a benign disease. Red-flag signs include serious neurological defects or organic diseases as tumors, infections, paralytic syndrome, or fractures that require immediate assessment and treatment.3 Thus, the first diagnostic approach to determine the cause of acute lower back pain is to investigate whether the patient has a neurological defect or an organic disease by investigating his/her medical history and through a physical assessment. This assessment may be helpful in determining the type of lower back pain.4,5 Diagnostic imaging may have a critical impact on the proper diagnosis of lower back pain and on the treatment decision by providing accurate anatomical information from a therapeutic viewpoint. Magnetic resonance imaging (MRI) enables the visualization of abnormal vertebral findings that could not have previously been revealed.6,7 For example, MRI is generally recommended for patients who need surgical intervention to verify the existence of a tumor or infection and the degree of disc herniation.1,8 MRI can also reveal various abnormal findings associated with the lumbar disc and the bone, and can be used as a diagnostic tool for identifying various causes of pain.7 Abnormal MRI findings include reduced disc height, reduced signal intensity, change in the disc contour, annular tear, a high-intensity zone (HIZ), and endplate change.1,7,9-12 Despite these findings, the clinical importance of MRI in the diagnosis of the cause of lower back pain remains controversial, as the incidence of lower back pain is high even in patients who do not have abnormal MRI findings.13 Abnormal MRI findings can be observed in -33% of patients younger than 40 years, and in almost all patients aged 60-80 years, but its exact cause remains uncertain.14-16 Moreover, radiological findings in patients with acute severe lower back pain have not yet been well-studied. This study was conducted to focus on abnormal MRI findings and their clinical relevance in patients who complain of lower back pain that is so severe as to require hospitalization. BODY.MATERIALS AND METHODS.SUBJECTS: Twenty-three patients who were hospitalized with acute lower back pain in the Department of Rehabilitation of the Kunkuk University Medical Center in Seoul, Korea during the period of August 2005 to July 2010, were included in group A. An inclusion criterion for group A was having visited the hospital for lower back pain for the first time in their life, and having pain limited to their lower back, without a particular history associated with the lower back and without red-flag signs (infection, fracture, or neurological defects). Severe lower back pain was defined as pain so severe that the patient had difficulty sitting on a bed at the time of his/her presentation to the hospital. Patients aged ≥60 years were excluded from this study, as most of these patients showed abnormal findings.14-16 Of the patients who had no particular history of low back pain, 19 patients who complained of mild lower back pain caused by a simple vehicle accident were assigned to control group B, and 18 patients who complained of severe radiating pain without trauma history and did not let them lead a normal life were assigned to control group C. Although the control group should have consisted of asymptomatic patients, it was difficult to find such patients. In recent studies, MRI did not reveal meaningful findings for patients who had mild lower back pain caused by a sports activity or a simple vehicle accident.17,18 Thus, assuming that of the patients without a particular history related to their lower back, those who complained of mild lower back pain after a simple vehicle accident would not show a change in their MRI findings, those patients were assigned to control group B. In addition, most of the patients with severe radiating pain and who had a history of lower back pain that lasted for a mean of 5.6±9.4 months were assigned to control group C for comparison of chronic lower back pain. Of the patients with acute severe lower back pain (group A), 13 were male and 10, female. Of the patients with mild lower back pain caused by a simple vehicle accident (control group B), 8 were male and 11 were female. Of the patients with severe radiating lower back pain (Control group C), 11 were male and 7 were female. The patients' mean ages were 34.4±8.6 years (group A), 39.4±9.3 years (control group B), and 36.3±12.0 years (control group C) (Table 1), with no significant differences among the groups (p<0.05). BODY.MATERIALS AND METHODS.METHODS: The MRI findings for the 3 groups were compared. The MRI instrument used in this study was a Signa EXCITE HD 1.5T (General Electronics Co., Milwaukee, USA). T1 (TR/TE, 550/12), and T2 (TR/TE 4000/120) weighted images were obtained in the sagittal plane, and T1 (TR/TE, 550/12) and T2 (TR/TE, 4,000/120) weighted images were obtained in the axial plane. The MRI findings showed disc herniation, lumbar disc degeneration (LDD), annular tear, high signal intensity (HIZ), and end plate change (Modic change). Disc herniation, including disc protrusion and disc extrusion, was defined as an abnormal finding, because disc bulging is known not to have a meaningful association with lower back pain or other abnormal findings for the disc.12,15 In addition, annular tear was identified on the T2 weighted image based on the change in the signal of the posterior annulus fibrosus, and the high intensity zone (HIZ) was defined as the local area with a high intensity signal in the posterior annulus fibrosus in the sagittal T2 weighted image, according to Aprill and Bogduk's definition (Fig. 1).19 The scale described by Pfirrmann20 was used for lumbar disc degeneration (LDD), and a grade ≥3 (a grade at which the overall disc signal is weaker, the boundary between the nucleolus pulposus and the annulus fibrosis is obscure, and the disc height starts decreasing on the MRI scan) was defined as degeneration (Fig. 2).21 The Modic change, which Modic et al.22,23 defined was used for abnormal findings in the end plate change, and was grouped based on the presence or absence of abnormal findings instead of being classified into types, as the frequency of each type was low (Fig. 3, 4). Statistical analysis was performed using SPSS 17.0, and the frequencies of abnormal findings between the groups were compared using the chi-square test with a significance level of p<0.05. BODY.RESULTS: The frequencies of the MRI findings were as follows: In patients with acute lower back pain (group A), disc herniation (87%), LDD (100%), annular tear (100%), HIZ (61%), and Modic change (4.4%); in patients with mild lower back pain caused by a simple vehicle accident (control group B), disc herniation (31.6%), LDD (47.4%), annulus tear (26.3%), HIZ (21.1%), and Modic change (10.5%); and in patients with severe radiating lower back pain (control group C), disc herniation (100%), LDD (100%), annulus tear (94.4%), HIZ (16.7%), and Modic changes (27.7%) (Table 1). In all groups, (80%) of the disc changes were in L4-5 and L5-S1 (Table 2). There were significant differences in the disc herniation, HDD, annulus tear, and HIZ between the patients with acute severe lower back pain (group A) and the control group B (p<0.05) (Table 3). The proportion of HIZ incidence was significantly higher in group A than in control group B, and in the patients with severe radiating pain (control group C) (p<0.05) (Table 3). It was difficult to give clinical meaning to the Modic change, as its frequency was low. BODY.DISCUSSION: In this study, MRI findings in patients with acute severe pain limited to their lower back were analyzed. The anatomical structures that may cause lower back pain include the bone, muscle, fascia, ligaments, disc (exterior annulus fibrosus and posterior disc regions), and nerve roots, and exclude the nucleus pulposus and the ligament flavum. With the introduction of MRI, it is now possible to reveal abnormal vertebral findings which could not be previously shown.6,7 Abnormal findings from an MRI scan are common in asymptomatic patients and many physicians consider them part of degenerative changes due to aging. In addition, the incidence of annulus tear and disc herniation has been reported to increase with age.13-16,24 A recent study reported that disc degeneration in the lumbar region reached the intermediate level in a patient in his early 20s.21 Thus, LDD and disc herniation without an acute traumatic history can be interpreted as degenerative changes due to aging.13-16,21,24 Some studies have reported that in a number of cases, the presence of HIZ indicated that a disc change might have been causing the pain,19,25-29 and other studies have reported that no association could be found between these indications.29-31 In 2006, Peng et al.32 investigated histological changes in specimens obtained from patients with lower back pain to show pathological changes in HIZ. As a result, Peng et al.32 observed granulation tissue wherein capillary vessels were affluent, and that vascular proliferation and invasion of inflammatory cells were present. Considering these reliable indicators of damage of the exterior annular fibrosus in patients with lower back pain, Peng et al.32 reported a meaningful association between the findings and lower back pain. In this study, the incidence of HIZ was significantly higher in patients with acute severe lower back pain than in patients in the control groups (Table 3), which suggests that HIZ in patients with acute severe lower back pain could be attributable to inflammatory pathological changes. There was no significant difference in LDD, which is considered part of degeneration, disc herniation, and annulus fibrosus between patients with severe lower back pain (group A) and patients with radiating lower back pain (control group C). The incidence of LDD, disc herniation, and annulus fibrosus was higher in group A and control group C than in patients with lower back pain caused by a simple vehicle accident (control group B) (Table 3). This indicates that when there was no significant difference in age between the groups, the frequency of all the degenerative changes was higher in patients with severe lower back pain without trauma (group A and control group C) than in control group B. The differences in occupation, lifestyle, and patient history aggravated the chronic degenerative change; and these fundamental degenerative changes made many patients prone to lower back pain or aggravated their lower back pain.33 Though the patients might have experienced lower back pain only once before, interior micro changes caused by environmental or intrinsic factors might have accelerated degenerative changes, and thus, these patients became prone to severe lower back pain.33 Though bad posture and management were found to be related to lower back pain in daily life, investigations of patients and their guardians' histories indicate that environmental factors are considerably related to degenerative changes. In addition, most of the patients who experienced severe lower back pain reported that they felt tearing pain in their lower back and a sensation of something being cut off in the pain site after they sprained their back while suddenly bending their lower back or moving. The MRI findings in these patients showed that HIZ was significantly more frequent in group A than in control group B and the patients with acute lower back pain (control group C) (Table 2). In addition, a recent study showed that HIZ alone has no relationship with discogenic LBP, but has meaning when it accompanies disc protrusion.34 However, this study showed, that HIZ was present in 86% (12/14) of the patients with disc protrusion in group A (n=19) but without disc extrusion, and disc protrusion, was present in 80% (4/5) of the patients with HIZ, which reveals no meaningful association, as the low number of patients with severe lower back pain (group A) does not show a meaningful association. It is difficult to make a conclusion based on a single study regarding whether or not HIZ can reflect acute inflammatory change in the annulus fibrosus and be a cause of acute lower back pain, but this study is believed to provide clues to the causes of acute lower back pain. BODY.CONCLUSION: MRI is the most ideal diagnostic tool for explaining basic anatomical abnormalities. The analysis of abnormal MRI findings in this study showed that disc herniation, LDD, annulus tear, and HIZ were significantly higher in patients with severe lower back pain (group A) than in control group B, and when compared to groups B and C. HIZ was the only parameter that demonstrated a significant difference.

TITLE: Role of noninvasive ventilation in weaning from mechanical ventilation in patients of chronic obstructive pulmonary disease: An Indian experience ABSTRACT.BACKGROUND:: Endotracheal intubation and mechanical ventilation (MV) are often needed in patients of chronic obstructive pulmonary disease (COPD) with acute hypercapnic respiratory failure. The rate of weaning failure is high and prolonged MV increases intubation associated complications. ABSTRACT.OBJECTIVE:: To evaluate the role of Noninvasive ventilation (NIV) in weaning patients of chronic obstructive pulmonary disease (COPD) from MV, after T piece trial failure. ABSTRACT.DESIGN:: A prospective, randomized, controlled study was conducted in a tertiary care centre. 30 patients of acute exacerbation of COPD with acute on chronic hypercapnic respiratory failure, who were mechanically ventilated, were included in the study A T-piece weaning trial was attempted once the patients achieved satisfactory clinical and biochemical parameters. After T-piece failure, defined as pH < 7.35, PaCO2 >50 mmHg, PaO2 <50 mmHg, HR >100/min, RR >35, patients were randomized to receive either NIV or PSV. ABSTRACT.RESULTS:: Demography, severity of disease and clinical profiles were similar in both groups. No significant difference between the two groups in duration of MV (6.20 ± 5.20 days vs. 7.47 ± 6.38 days, P > 0.05), duration of weaning (35.17 ± 16.98 and 47.05 ± 20.98 hours, P > 0.05) or duration of ICU stay (8.47 ± 4.79 and 10.80 ± 5.28 days, P > 0.05) in Gp I and Gp II, respectively. Five patients developed VAP in the PSV group, where as only one patient had pneumonia in the NIV group. Lesser number of deaths in the NIV group at discharge from ICU (3 vs. 5 patients, respectively) and at 30 days (5 vs. 9 patients, respectively), it did not achieve statistical significance (P > 0.05). ABSTRACT.CONCLUSION:: NIV is as useful as PSV in weaning and can be better in weaning failure especially in COPD for earlier weaning, decrease ICU stay, complications and mortality. BODY.INTRODUCTION: Removal of patients from mechanical ventilation (MV) has been termed liberation, discontinuation, withdrawal and most commonly weaning. The process of permanent removal of the artificial airway is extubation.[1] A balance must be achieved between the risk associated with early discontinuation and delay in extubation. Premature withdrawal causes loss of airway protection, cardiovascular stress, suboptimal gas exchange, muscle overload and fatigue. Delayed withdrawal exposes to complications associated with ventilation like infections, barotrauma, stretch injury, sedation, airway trauma and costs.[2] Weaning strategies clearly affect the outcome and weaning should be aggressive but monitored carefully to minimise the risks. Two large trials have been performed comparing T-piece, assist control ventilation (ACV), intermittent mandatory ventilation (IMV) and pressure support ventilation (PSV) as approaches for weaning.[34] PSV was superior in one trial and T-piece in the other. Consensus regarding the best mode of weaning from these studies is difficult to derive. Patients of COPD requiring MV frequently suffer from persistent weaning failure, so early extubation with NIV decreases the duration of MV, length of intensive care unit (ICU) stay, incidence of nosocomial pneumonia and improves survival when compared to conventional weaning.[5] Noninvasive ventilation (NIV) is one of the new strategies in the weaning of patients of chronic obstructive pulmonary disease (COPD) needing MV. There are few studies which have systematically evaluated the role of NIV in weaning of patients of COPD on MV. Studies by Nava and Hilbert supported the role of NIV in weaning and postextubation respiratory failure.[67] Girault and Ferrer also found NIV as a suitable method for systematic extubation and weaning technique in patients with acute on chronic respiratory failure and persistent weaning failure.[89] The objective of this study is to evaluate the effectiveness of NIV as a weaning method in patients of COPD on MV, in comparison to PSV. BODY.MATERIALS AND METHODS: A two group, parallel, prospective randomized controlled trial was carried out in a tertiary care centre. Patients who were admitted in the intensive care ward, with acute exacerbation of COPD and needing intubation were eligible for the study. Acute hypercapnic respiratory failure in COPD was defined as severe dyspnoea in the absence of objectively documented causes such as pneumonia, and with arterial blood gas analysis (ABGA) findings of: pH <7.33 (breathing at room air)PaO2 <50 mmHg.PaCO2 >50 mm Hg To make a decision for intubation as objectively as possible, the following guidelines were followed.[10] Major: Respiratory arrestLoss of consciousnessPsychomotor agitation requiring sedationHemodynamic instability with systolic BP <70 mmHg or >180mm HgHeart rate (HR) <50 beats/min with loss of alertness Minor: Respiratory rate (RR) >35/minpH <7.30PaO2 <50 mm HgPresence of weak cough reflex with accumulation of secretionsWorsening of the neurological state (encephalopathy score) The presence of one major criterion was considered as an indication for immediate intubation. The presence of two minor criteria after the failure of one hour of medical treatment and NIV was an indication for intubation. Patients who had concomitant neurological disease (other than hypercapnic encephalopathy), cardiac arrest, cardiogenic pulmonary oedema, cardiogenic shock, acute myocardial infarction, gastrointestinal perforation/obstruction, metabolic coma, coagulopathy and postoperative respiratory failure were excluded from the study. Intubation was done through the orotracheal route. All patients were initially ventilated with control / assist control mode, in a stepwise manner (considering their level of consciousness, sedation level and improvement in ABG. Muscle relaxants and sedation were used as required. Standard ventilator settings for COPD i.e., respiratory rate of 12/min., tidal volume 8–10 mL/kg, FiO2 to obtain a saturation of 90% with a PEEP of 5 cm H2O and an I: E ratio of 1:2.5-3.0 was initiated. T-piece weaning trial was given to the patients when they were judged to have reached satisfactory neurological status, clinical and biochemical parameters with a SaO2 of 88% or more for a FiO2 of 40% after a minimum of 24 hours of ventilation. The two hours T-piece trial failure was considered when the patient had any of the following: PaO2 <50 mm for a FiO2 of 40%pH < 7.35RR > 35/min.HR >145 bpmSystolic BP >180 mmHg or <70 mmHgSignificant arrhythmiaAgitation, anxiety or diaphoresis Patients with T-piece trial failure were randomized into two groups to receive either NIV (group I) or continued weaning with invasive pressure support ventilation (group II). The Kendall and Babington table was used to randomize patients. Immediately after T-piece failure, patients were put back on CMV/ACV mode until previous PaCO2 and pH values were reached, with a respiratory rate of 30 per minute or less and then considered for the respective modality of weaning intervention. After randomization, Group l patients to be weaned with NIV were then extubated and switched on to noninvasive pressure support ventilation with a full face mask using a BiPAP ventilatory assist system (RESMED, Sullivan-VPAP ST-II). Patients received NIV continuously except during meals and for expectoration. A particular level of IPAP and EPAP support that achieved t satisfactory blood gases and a RR <25/min were used. Once that was achieved, the pressure support was decreased by 2 cm of H2O every 4 hours with a good tolerance, and with close monitoring for any change in oxygen saturation and respiratory rate. As soon as we could reduce the inspiratory positive airway pressure (IPAP) and expiratory positive airway pressure (EPAP) levels to 8 and 4 cm of H2O, respectively, with a satisfactory ABG of PH ≥7.35, SaO2 ≥90%, FiO2 ≤40% and RR<30, patients were allowed to breathe spontaneously on a venturi mask. Group II, patients received pressure support ventilation with a particular level of pressure support that achieved satisfactory blood gases and a RR <25/min. Once that was achieved, the pressure support was decreased by 2 cm of H2O every 4 hours with a good tolerance and with close monitoring for any change in oxygen saturation and respiratory rate. As soon as the pressure support and PEEP reached 10 and 5 cm of H2O, respectively, with a satisfactory ABGA of PH ≥7.35, SaO2 ≥90%, FiO2 ≤40% and RR<30, patients were extubated and allowed to breathe spontaneously receiving oxygen therapy via a venturi mask. Success of weaning was assessed after 2 hours of spontaneous breathing on a venturi mask by SaO2, FiO2, pH, RR, hemodynamic status, dyspnoea and a good neurological status. Successful weaning was defined when the patient maintained SaO2 ≥90%, FiO2 ≤40%, pH ≥7.35, respiratory rate <30/min with no dyspnoea and intact cognition. Absence of even one of these criteria was considered as weaning failure. Weaning was also considered a failure if the patient could not be taken off the ventilator after 30 days, or needed reintubation within 72 hours of disconnection from the ventilator, or if death related to MV occurred. Nosocomial infection, pneumothorax, ischemic event or fatal arrhythmias during the weaning process were considered causes of death associated with MV. Arterial blood gas analysis (ABGA) was done at presentation and at 1, 4, 8 and 12 hours following the start of MV and also during the weaning process. Neurological score and APACHE II score were calculated at presentation. Ventilator associated pneumonia (VAP) was defined as the presence of new and persistent (>48 hours) lung infiltrates on chest radiography combined with fever, total leukocyte count (TLC) >10,000/μl after 48 hours on ventilator. Pulmonary function tests were performed with a portable spirometer as soon as the patients' clinical condition allowed testing before discharge from the intensive care unit. The predicted values for the local population were calculated by the previously published regression formula.[11] The outcomes of treatment between the two groups were compared with the following parameters: Duration of MV, i.e. from the day of intubation to the day of extubation from the artificial airway (in group I before randomization and in group II before randomization plus weaning duration after randomization).Duration of ICU stay (from the day of admission to the day of discharge from ICU)Duration of weaning (after randomization).Incidence of nosocomial pneumoniaMortality at discharge from ICU and at 30 days discharge Institutional review board approval was taken. Statistical analysis was done by using SPSS version 10.0 Results are given as mean ± SD. Chi-square test was applied for discrete variables and Mann-whitney U test for continuous variables where ever applicable. We also used the Cochrane Q test and Fisher exact test. Fisher exact test was used because sample size and values were small. Cochran's and Mantel-Hanazel test was used for test of independence between dichotomous variable and dichotomous response. BODY.RESULTS: A total of 140 patients of acute exacerbation of COPD with acute on chronic type II respiratory failure were admitted in the intensive care unit (ICU) over a period of 18 months. Ten patients were excluded from the study, due to pneumonia (n =5), ischemic heart disease (n = 2), cardio-respiratory arrest (n = 2) and postoperative respiratory failure (n = 1). 100 patients were initially treated with NIV, of which 15 patients needed MV after one hour trial on NIV. 30 patients were taken on MV directly. A total of 45 patients needed MV and were eligible for the study. 5 patients died immediately after intubation. 10 patients were extubated directly after successful T-piece trial. The remaining 30 patients were randomized into 2 groups of 15 each [Figure 1]. Figure 1Methodology of the study The two groups were similar in terms of age, sex, smoking history, previous treatment, precipitating factors, severity of the disease, clinical and biochemical parameters during admission and at the time of randomization [Tables 1 and 2]. All patients were on irregular treatment with theophyllines, beta agonists and anticholinergics. Table 1 Demographic profile Group I Group II Age (in years) 57.73 ± 11.23 61.13 ± 8.18 * Sex Male 12 (80%) 9 (60%) * Female 3 (20%) 6 (40%) * Smoking (Pack Years) 46.43 ± 17.03 35.45 ± 11.93 * Glasgow Coma Scale 8.40 ± 1.12 8.67 ± 1.45 * Heart Rate (per min) 133.47 ± 13.97 125.0 ± 19.90 * Respiratory Rate (per min) 33.80 ± 16.75 32.27 ± 15.92 * Mean Arterial Pressure (mm Hg) 72.93 ± 18.40 65.80 ± 18.44 * Total leukocyte count (per μl) 12,466.67 ± 4870.36 11,273.33 ± 3732.19 * PH 7.13 ± 0.06 7.13 ± 0.07 * PaCO 2 95.98 ± 21.28 102.54 ± 28.36 * * P > 0.05 Table 2 Severity of disease Group I Group II FEV1 % 29.77 ± 6.98 29.33 ± 5.61 * APACHE II 30.73 ± 5.35 29.47 ± 5.00 * * P > 0.05 The mean IPAP and EPAP (inspiratory/expiratory positive airway pressure) initially used were 15.07 ± 1.27 and 6.21 ± 0.43 cm of H2O, respectively, in group I. The mean initial PSV used was 18.21 ± 1.1cm of H2O above PEEP (5 cm of H2O) in group II. There was no difference in the time spent on mechanical ventilation between the two groups i.e. 6.20 ± 5.20 day's vs. 7.47 ± 6.38 days in Gp I and Gp II, respectively (P > 0.05, Table 3). No difference in duration of weaning was observed between NIV and PSV groups (35.17 ± 16.98 and 47.05 ± 20.98 hrs, respectively, p.>0.05, Table 3). Patients weaned by NIV spent nearly 2 days less in the ICU, in comparison to weaning by PSV (8.47 ± 4.79 vs. 10.80 ± 5.28 days), the difference was not statistically significant (P > 0.05, Table 3). Mortality at discharge from ICU, three (20% mortality) and five deaths (33.33% mortality), respectively, were observed in the NIV and PSV group at the time of ICU discharge. Nevertheless, the difference was not statistically significant (P > 0.05, Table 3). At 30 days, there were lesser number of absolute deaths in the NIV group (5 vs. 9); however, it did not achieve statistical significance (P > 0.05, Table 3) Table 3 Results Group I Group II Duration of ventilation (in days) 6.20 ± 5.20 7.47 ± 6.38 * Duration of weaning (in hours) 35.17 ± 16.98 47.05 ± 20.98 * Duration of ICU stay (in days) 8.47 ± 4.79 10.80 ± 5.28 * Death in ICU 3 (20%) 5 (33.33%) * Death at 30 days 5 (33.33%) 9 (60%) * Nosocomial pneumonia 1(6.66%) 5 (33.33% * ) * P > 0.05 Five patients (33.33%) in the PSV group suffered from VAP where as only one (6.66%) in the NIV group developed pneumonia. Other minor complications noted in the NIV group were claustrophobia (2 patients), skin abrasions (2 patients) and gastric distension (1 patient) [Table 4]. Table 4 Complications of NIV Complications Number of patients Claustrophobia 2 Skin abrasion 2 Gastric distension 1 Pneumonia 1 BODY.DISCUSSION: NIV is recommended strongly in patients of chronic obstructive pulmonary disease with hypercapnic respiratory failure.[12] The requirement of prolonged MV in patients of COPD is due to impaired pulmonary mechanics, increased intrinsic PEEP, increased airway resistance with reduced pressure generating capacity of the muscles and pulmonary hyperinflation.[1314] Whereas muscle fatigue and altered gas exchange are primarily responsible for weaning failure.[15] NIV allows the respiratory muscles to rest, and improves the patients' breathing pattern and gas exchange. Therefore, the patients who are likely to benefit from NIV are those with hypercapnic respiratory failure, a frequent situation in COPD and weaning failure.[1617] NIV improves hypoxemia, hypercapnia and prevents rapid shallow breathing.[18] There is less evidence supporting the efficacy of NIV in weaning. Few case reports and nonrandomized studies reported the beneficial effect of NIV in difficult to wean patients and increasing survival in patients needing prolonged ventilation.[19–21] Recent randomized control studies have shown that NIV decreases the duration of ventilatory support, length of ICU stay, incidence of Nosocomial pneumonia and improves survival when compared to conventional weaning techniques.[6–9] In the present study, we did not observe any significant statistical difference between the two groups regarding the duration of ventilation, weaning hours, ICU stay, VAP and mortality. In spite of small sample size in this study, there were two and four more deaths at discharge and 30 days follow up, respectively, in the group weaned by invasive PSV. The incidence of VAP was also 5 times higher (33.33% vs.6.66%) in the group weaned by PSV. Statistical significance was not achieved because study was under powered. Increase in the duration of MV often leads to increased incidence of nosocomial pneumonia.[22–25] The presence of endotracheal tube predisposes to the development of nosocomial pneumonia by impaired cough reflex and mucociliary clearance. NIV has been one of the strategies to decrease the incidence of VAP.[26] Present study also confirms the same, as the incidence of VAP was 5 times less in the NIV arm of the study. NIV also decreases the mortality in the post extubation phase by avoiding sedation, tracheostomy, and intubation and allows swallowing, thereby decreasing gastro-oesophageal reflex, aspiration to the airways and VAP.[27–30] The overall mortality in the present study is higher than the published data.[68] Nava et al. did not have mortality of any patient at the end of 60 days in the group weaned by NIV. In the study by Girault, the mortality was comparable between the two groups, weaned by PSV and NIV, respectively (2 vs. 0).[8] The higher mortality in our study relates to the severity of underlying disease, as evidenced by higher APACHE scores (≈30), low FEV1(<30%), severe acidosis and lower mean arterial pressures. VAP also contributed significantly to the mortality as all the patients having VAP died. Therefore, the patients included in the present study were not only sick but also had the severest form of COPD. Nava concluded that NIV is more effective than PSV in weaning the patients of COPD from MV.[6] Whereas, Girault acknowledged it as a new and useful systematic approach to weaning.[8] Cochrane review has also demonstrated consistent positive effect on overall mortality in patients of COPD primarily by facilitating early weaning and extubation.[31] It concluded that NIV has a promising net clinical benefit. VENISE trial also supports the role of NIV in the management of difficult to wean patients of acute on chronic respiratory failure.[32] NIV not only reduces the need for ventilation and re-intubations, but also increases the survival.[610] The reason for the success of NIV is that it avoids complications of artificial airways. Esteban et al. also demonstrated the utility of NIV in preventing re-intubations in patients of COPD.[33] In the present study, NIV was as effective as PSV in weaning. It did reduce the complications associated with invasive ventilation and weaning like VAP and death in absolute terms. Present study however had one limitation, i.e. the numbers of patients studied were small. In conclusion, NIV is as effective as PSV in weaning the patients of COPD with acute exacerbations from MV with some minor complications. Therefore, it should be the preferred weaning strategy for patients of COPD on MV in Indian scenario and otherwise. We believe that it is the first of the kind where there is direct comparison of PSV and NIV in weaning failed patients of COPD from India subcontinent.

TITLE: Cardiovascular outcomes during treatment with dabigatran: comprehensive analysis of individual subject data by treatment ABSTRACT.BACKGROUND: Dabigatran 150 mg twice daily was shown to be superior to warfarin in preventing stroke in subjects with nonvalvular atrial fibrillation (SPAF) in the RE-LY (Randomized Evaluation of Long-term anticoagulation therapY) trial. Numerically, more myocardial infarctions occurred in patients receiving dabigatran compared with well-controlled warfarin. This observation prompted a comprehensive analysis of cardiovascular outcomes, including myocardial infarction, in all completed Phase II and III trials of dabigatran etexilate. ABSTRACT.METHODS: The analysis included comparisons of dabigatran with warfarin, enoxaparin, and placebo. Data were analyzed for the occurrence of cardiovascular events from 14 comparative trials (n = 42,484) in five different indications. Individual study data were evaluated, as well as pooled subject-level data grouped by comparator. ABSTRACT.RESULTS: In the pooled analysis of individual patient data comparing dabigatran with warfarin (SPAF and venous thromboembolism treatment indications), myocardial infarction occurrence favored warfarin (odds ratio [OR] 1.30, 95% confidence interval [CI] 0.96–1.76 for dabigatran 110 mg twice daily and OR 1.42, 95% CI 1.07–1.88 for dabigatran 150 mg twice daily). The clinically relevant composite endpoint of myocardial infarction, total stroke, and vascular death demonstrated numerically fewer events in dabigatran 150 mg patients (OR 0.87, 95% CI 0.77–1.00), but was similar for dabigatran 110 mg (OR 0.99, 95% CI 0.87–1.13). Dabigatran had similar myocardial infarction rates when compared with enoxaparin or placebo. ABSTRACT.CONCLUSION: These analyses suggest a more protective effect of well-controlled warfarin, but not enoxaparin, compared with dabigatran in preventing myocardial infarction in multiple clinical settings. Dabigatran showed an overall positive benefit-risk ratio for multiple clinically important cardiovascular composite endpoints in all evaluated clinical indications. In conclusion, these data suggest that myocardial infarction is not an adverse drug reaction associated with use of dabigatran. BODY.INTRODUCTION: Thrombotic cardiovascular disease remains a major public health challenge. Dabigatran etexilate (hereafter referred to as dabigatran) is a direct, reversible thrombin inhibitor that provides a potential alternative to long-established and newer oral and parenteral anticoagulants for the treatment and prevention of various thromboembolic diseases. In the RE-LY (Randomized Evaluation of Long-term anticoagulation therapY) atrial fibrillation study, dabigatran etexilate 150 mg twice daily was associated with significantly lower rates of the primary efficacy composite endpoint of stroke and systemic embolic events, compared with well-controlled warfarin, with similar rates of major bleeding events.1,2 In RE-LY, dabigatran 110 mg twice daily was noninferior to warfarin for prevention of stroke/systemic embolic events, with lower rates of major bleeding than warfarin. The prespecified, treatment-blinded, and independent assessment of all efficacy endpoints in RE-LY identified that there were numerically more myocardial infarctions (MIs) in the dabigatran treatment groups than the group receiving warfarin (target international normalized ratio [INR] 2.0–3.0). Those differences did not reach statistical significance, and the overall annualized MI rates in RE-LY were <1% in all three treatment groups and comparable with those reported in numerous recent atrial fibrillation studies (0.55%–1.4%, Table 1).3–11 No imbalances were seen between dabigatran and well-controlled warfarin (mean time in therapeutic range [TTR] 64.4%, median TTR 67.3%) in the occurrence of the prespecified composite endpoint of MI, sudden/arrhythmic and pump failure deaths, or additional endpoints derived from adverse events of unstable angina, cardiac arrest resulting in death, and post-MI cardiac deaths.12 Further, RE-LY prespecified the composite endpoint of "net clinical benefit" (all strokes, systemic embolic events, MI, pulmonary embolism, major bleeding, and all-cause death), which favored both dabigatran doses versus warfarin.12 A recent pooled analysis of all four Phase III trials of dabigatran versus enoxaparin for the prevention of venous thromboembolism (VTE) in patients undergoing total hip or knee replacement surgery demonstrated no significant difference in the incidence of MI between dabigatran and enoxaparin.13 However, the finding of a numeric increase in MI frequency with dabigatran compared with well-controlled warfarin in RE-LY, coupled with a decrease in the occurrence of other cardiovascular outcome events and overall net clinical benefit, has prompted considerable debate in the medical literature.14–17 The discordance between rates of MI and other outcome events seen in RE-LY, the apparent lack of a consistent MI signal seen with other direct thrombin inhibitors,18,19 and the debate surrounding the publication of a meta-analysis of cardiovascular outcome events in a limited subset (seven) of dabigatran trials14,16 which did not use individual patient data, dictated that we produce definitive data to establish whether patients on dabigatran are at increased risk of cardiovascular events and MI in particular. We hypothesized that the risk of cardiovascular events in patients on dabigatran etexilate was not increased when compared with other anticoagulants. We performed a comprehensive review of all cardiovascular outcome data obtained during the conduct of all Phase II and III dabigatran clinical studies (n = 14) covering five different clinical indications with a variety of comparators administered according to current clinical guidelines. Two trials did not have comparators and so were excluded from these analyses. Additionally, these analyses included all individual patient data within the Boehringer Ingelheim dabigatran database, and since database searches did not identify any published Phase II or III dabigatran studies other than those known to Boehringer Ingelheim, these comprise all known studies as of January 2012. We prespecified a meta-analytic approach pooling the patient-level data from the clinical trials grouped by treatment, to make comparative assessments of the frequency of MI, cardiovascular, and other vascular outcome events, both as individual events and as components of prespecified composite cardiovascular endpoints, due to the known large differences in the risk of cardiovascular outcomes in the different patient populations studied. For the pooled analyses, we focused on the doses of dabigatran and their respective comparators, as approved by numerous health authorities and recommended in multiple clinical and practice guidance documents. BODY.MATERIALS AND METHODS.SUBJECTS AND STUDY SELECTION: The analysis was performed by employees of the market authorization holder of dabigatran etexilate (Boehringer Ingelheim) which resulted in unrestricted access to all patient-level data. Studies included in the analysis were all available Phase II and Phase III trials conducted during the development of dabigatran etexilate that were completed through January 2012. An additional literature search in PubMed, Scopus, and the Web of Science in January 2012 for randomized controlled trials evaluating the safety and efficacy of dabigatran that reported on MI or acute coronary syndrome (ACS) as secondary outcomes, using the search terms "dabigatran" or "dabigatran etexilate" or "BIBR 1048" and "randomized clinical trial" or "randomized trial" or "randomized controlled trial", did not identify further studies. Finally, the analysis was performed on data from four Phase II (n = 4,478) and ten Phase III (n = 38,006) studies, all of which included exactly one of the following comparators: warfarin, enoxaparin, or placebo. A brief overview of these trials is given in Table 2.1,2,20–32 The studies were conducted between November 2002 and June 2011. Results from all but one of these studies were previously published1,2,20–26,30,32 or reported in abstract form;28,29,31 and one set of data remains unpublished.27 As stated earlier, data from two Phase II trials were excluded from these data analyses because they had no active or placebo comparator arm.33,34 The follow-up periods in these 14 studies were between 1 month and 5 years. A summary of the individual studies, including the number of subjects treated, treatment outcomes, and primary safety and/or efficacy endpoints, is provided in Table 3. As expected, the trial populations differed in terms of demographic and baseline characteristics (eg, age and concomitant disease therapy), the prevalence of known coronary disease, active or placebo comparators, and trial durations (Supplementary Table 1). Dabigatran was administered once daily in seven trials and twice daily in ten trials, some of which also had once-daily arms, and some patients received both once-daily and twice-daily doses in one trial. All studies were conducted with the approval of local ethics committees and the relevant governmental health authorities, and followed the most stringent of the relevant guidelines for the protection of human subjects. Trial registration numbers are listed in Table 3. BODY.MATERIALS AND METHODS.OUTCOMES AND DEFINITIONS: For the current analyses, the outcomes of interest were MI (including silent MI), stroke, vascular and cardiovascular death, and three prespecified composite outcomes: MI and cardiovascular death; MI and nonfatal stroke; and MI, stroke, and vascular death. The definitions of an MI event for RE-LY have been previously reported,12 and are more inclusive than those included in the Phase III protocols for the apixaban and rivaroxaban atrial fibrillation studies.10,11 The definitions of MI for the trials included in these analyses can be found in Supplementary Table 2. For all pooled analyses, the best available individual subject data were used to evaluate cardiovascular events, including MI. For studies with independent adjudication of events, positively adjudicated outcome events were considered to be the best available data. If a study did not have a formal cardiovascular event adjudication, then all investigator-reported adverse events, ie, any of the cardiovascular outcome events included in these analyses, were considered outcome events, irrespective of the investigator or company assigned causal relatedness to study treatment. Adverse event selection was based on Medical Dictionary for Regulatory Authorities (MedDRA) preferred terms that were documented in the clinical trial database. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSES: One of the 14 studies did not include a currently approved dose of dabigatran,20 and no cardiovascular outcome events were reported in three studies24,26,27 (Table 2). The pooled safety analyses included only subjects who had received at least one dose of study drug (because the focus was a safety analysis) and the observation period was from the date of randomization to the date of study termination, to be consistent with intention-to-treat principles. Pooling was based on individual patient data, but "study" was retained as a factor in the statistical models. For the pooled analyses, a fixed-effects model based on the Mantel-Haenszel method was used for combining results from individual trials. Because this was planned as a safety analysis, we used the fixed-effects model, which we knew would result in smaller confidence intervals (CIs) than a random-effects model, and as such, between-group differences would be more likely to be considered statistically significant than if a "less conservative" approach using a random-effects model was utilized. However, a random-effects model was also employed and no substantial differences in the CIs compared with the fixed-effects model were noted (data not shown). Odds ratios (ORs) (with a 0.5 continuity correction) and 95% CIs were calculated. A two-sided P-value of <0.05 was considered to be statistically significant. There was no correction for the multiplicity of comparisons. Heterogeneity was assessed using the Chi-square test. A P-value of <0.10 was chosen to denote heterogeneity. All analyses were performed using SAS version 9.2 software (SAS Institute Inc, Cary, NC, USA). BODY.RESULTS: As expected, the patient populations of the 14 identified trials differed in terms of demographic and baseline characteristics (eg, age and concomitant medication use), prevalence of known coronary disease, comparators (active or placebo), and trial duration. Dabigatran was administered once daily in seven trials and twice daily in ten trials, some of which also had once-daily arms, and some patients received both once-daily and twice-daily doses in one trial. These differences were addressed by analyzing the data from studies grouped by different comparators, which also simultaneously addressed the issue of different trial durations (eg, primary VTE prevention in the orthopedic setting for up to 34 days, stroke prevention in atrial fibrillation RE-LY for a mean of 2 years). Nevertheless, the outcome data were consistent when reviewed as results of individual studies or as pooled analyses of individual patient data from studies grouped by comparator. BODY.RESULTS.COMPARISONS WITH WELL-CONTROLLED WARFARIN (TRIALS IN SUBJECTS WITH NONVALVULAR ATRIAL FIBRILLATION AND UNDERGOING ACUTE TREATMENT OR SECONDARY PREVENTION OF VTE).POOLED ANALYSES OF INDIVIDUAL SUBJECT DATA VERSUS WARFARIN: Within the six clinical trials comparing dabigatran 150 mg twice daily versus warfarin, all had a target INR of 2.0–3.0 (Table 2).1,2,26–28,30,31 No cardiovascular events were reported in two studies, so the pooled analysis of individual data is derived from data from four trials.1,2,28,30,31 The occurrence of all stroke (OR 0.68; 95% CI 0.54–0.85) and nonfatal stroke (OR 0.65; 95% CI 0.49–0.86) favored dabigatran (Figure 1A). There was a trend to less risk for vascular death in subjects receiving dabigatran 150 mg twice daily compared with warfarin (OR 0.86; 95% CI 0.73–1.01), occurrence of the composite endpoint of MI, nonfatal stroke, and cardiovascular death (OR 0.91; 95% CI 0.79–1.06), and the composite endpoint of MI, all stroke, and vascular death (OR 0.87; 95% CI 0.77–1.00). The OR for the composite outcome of MI and cardiovascular death, comparing dabigatran 150 mg twice daily versus warfarin, measured an OR of 1.06 (95% CI 0.89–1.26). For MI events alone, there was an elevated rate compared with warfarin, with an OR of 1.42 (95% CI 1.07–1.88, Figure 1A). There was no evidence of statistical heterogeneity. Analyses using different treatment intervals showed similar results. The comparison of individual patient data for dabigatran 110 mg twice daily and warfarin (Figure 1B) included only one trial (RE-LY),1,2 because there were no reported cardiovascular events in the only other study that included this dose and warfarin.27 Therefore, these data were identical to those reported in Table 4 for the RE-LY trial results. For MI alone, the data favored warfarin (OR 1.30; 95% CI 0.96–1.76). BODY.RESULTS.COMPARISONS WITH WELL-CONTROLLED WARFARIN (TRIALS IN SUBJECTS WITH NONVALVULAR ATRIAL FIBRILLATION AND UNDERGOING ACUTE TREATMENT OR SECONDARY PREVENTION OF VTE).SUBJECTS WITH NONVALVULAR ATRIAL FIBRILLATION: INDIVIDUAL TRIAL DATA VERSUS WELL-CONTROLLED WARFARIN: Data are available from the RE-LY trial. As previously reported,1,12 annualized rates of stroke occurrence measured 1.44%, 1.01%, and 1.58% per year for dabigatran 110 mg twice daily, dabigatran 150 mg twice daily, and warfarin, respectively, while there was a small, statistically nonsignificant excess occurrence of MI in dabigatran-treated subjects (Table 4). The rates of MI in RE-LY measured 0.82%, 0.81%, and 0.64% per year for the dabigatran 110 mg twice daily, dabigatran 150 mg twice daily, and warfarin treatment groups, respectively; the differences did not reach statistical significance.1,12 The rates of fatal MI events were all similar and low (0.13%, 0.11%, and 0.10% per year for dabigatran 110 mg, dabigatran 150 mg, and warfarin groups, respectively). Of all the RE-LY subjects who sustained an MI, more than half had a history of coronary artery disease and about one third had a history of prior MI. A subgroup analysis showed that while MI occurred more frequently in subjects with a baseline history of coronary artery disease versus those without in all treatment groups (Table 4), the MI rates were similar in the dabigatran-treated and warfarin-treated subjects for total MI, as well as for subcategories of silent, clinical, and fatal MI. Additionally, when evaluating the effect of INR control in RE-LY, it was noted that those with a mean TTR ≤65% had about a 30% lower annualized MI rate than warfarin-treated individuals with worse INR control (TTR <65%, Table 5). In a post hoc multivariate analysis, the only subgroup to show a statistically significant increase in MI rates with dabigatran versus warfarin was those patients with a baseline history of valvular heart disease (n = 3,955; 21.8% of RE-LY subjects had valvular heart disease without a prosthetic valve or that was not hemodynamically significant or likely to require valve surgery during the conduct of the trial). The identification of such subjects did not require quantification of valvular disease severity (eg, with ultrasound) but was based on investigator-reported medical history only. The MI rates in this group measured 1.06%, 1.08%, and 0.31% per year for dabigatran 110 mg twice daily, dabigatran 150 mg twice daily, and warfarin, respectively (Table 4). In subjects without valvular heart disease, the MI rates were 0.64%, 0.64%, and 0.63% per year for the three treatment groups, respectively (Table 4), showing no difference between those receiving dabigatran and warfarin. The MI rate in the warfarin group with a baseline history of valvular heart disease was low, with very few events having been diagnosed (n = 8), and the rate was about half the MI rate (0.31% per year) observed in warfarin-treated subjects with no baseline history of valvular heart disease (n = 58, with a rate of 0.63% per year), an observation that lacks clinical plausibility. A further evaluation was performed looking at the timing of MI relative to administration or discontinuation of the study drug. This analysis showed that >30% of MI events in RE-LY occurred off study drug in all three treatment groups.12 However, there continued to be a numeric excess of first MI events in subjects randomized to dabigatran compared with warfarin more than 90 days after cessation of treatment, an observation that is not readily explainable (Table 6). Adjudicated vascular death rates were 2.43%, 2.28%, and 2.69% per year for the dabigatran 110 mg, dabigatran 150 mg, and warfarin treatment groups, respectively (Table 4). The risk of vascular death for dabigatran 150 mg twice daily was significantly lower than for well-controlled warfarin (hazard ratio [HR] 0.85, 95% CI 0.72–0.99, P = 0.04). In addition, the protocol-prespecified composite endpoint of stroke, systemic embolic events, MI, pulmonary embolism, and vascular death for dabigatran 150 mg twice daily compared with warfarin had an HR of 0.84(95% CI 0.74–0.96, P = 0.009). There were no significant differences between the groups with regard to rates of cardiovascular death, or the composite endpoints of MI and cardiovascular death or MI and all-cause mortality. The protocol-prespecified composite endpoint of "net clinical benefit" (all strokes, systemic embolic events, MI, pulmonary embolism, major bleeding, and all-cause death) favored both dabigatran doses, with rates of 7.34% per year with dabigatran 110 mg twice daily, 7.11% per year with dabigatran 150 mg twice daily, and 7.91% per year with warfarin (HR 0.92, 95% CI 0.84–1.01, P = 0.09 for dabigatran 110 mg and HR 0.9, 95% CI 0.82–0.99, P = 0.02 for dabigatran 150 mg twice daily).12 BODY.RESULTS.COMPARISONS WITH WELL-CONTROLLED WARFARIN (TRIALS IN SUBJECTS WITH NONVALVULAR ATRIAL FIBRILLATION AND UNDERGOING ACUTE TREATMENT OR SECONDARY PREVENTION OF VTE).SUBJECTS UNDERGOING ACUTE TREATMENT OR SECONDARY PREVENTION OF VTE: INDIVIDUAL TRIAL DATA VERSUS WELL-CONTROLLED WARFARIN: Two acute VTE treatment studies (n = 5,060)30,31 of 6 months' duration compared dabigatran versus warfarin for the treatment of acute VTE, and within each study there was a low incidence of definite ACS events during the treatment period, but there were more events in those treated with dabigatran (0.3%) than in those treated with warfarin (0.1%). In a secondary prevention trial of up to 3 years' duration comparing dabigatran versus warfarin for the prevention of recurrent VTEs,28 nine MI events were reported in the 150 mg twice daily dabigatran group versus one event in the warfarin group (Table 7). There was a higher prevalence of baseline coronary risk factors, including hypertension, diabetes, and established coronary artery disease, in those treated with dabigatran than in those treated with warfarin in these studies. BODY.RESULTS.COMPARISONS WITH ENOXAPARIN OR PLACEBO (IN SUBJECTS UNDERGOING PRIMARY AND SECONDARY PREVENTION OF VTE AND SUBJECTS WITH ACS).POOLED ANALYSES OF INDIVIDUAL SUBJECT DATA VERSUS ENOXAPARIN AND PLACEBO: In four studies comparing dabigatran 220 mg once daily versus enoxaparin21–23,25 (Figure 2) and two studies comparing dabigatran 150 mg twice daily versus placebo29,32 (Figure 3A), point estimates for MI and composite endpoints that included MI were <1.0 and >1.0, respectively, although the CIs were wide. Similarly, conclusions regarding point estimates for cardiovascular and vascular death are limited by wide CIs due to the low number of events. Only one trial32 compared dabigatran 110 mg twice daily versus placebo (Figure 3B). BODY.RESULTS.COMPARISONS WITH ENOXAPARIN OR PLACEBO (IN SUBJECTS UNDERGOING PRIMARY AND SECONDARY PREVENTION OF VTE AND SUBJECTS WITH ACS).SUBJECTS UNDERGOING PRIMARY PREVENTION OF VTE: INDIVIDUAL TRIAL DATA VERSUS ENOXAPARIN: In three VTE primary prevention studies, ie, RE-MODEL (Thromboembolism Prevention After Knee Surgery), RE-NOVATE (Oral dabigatran versus enoxaparin for thromboprophylaxis after primary total hip arthroplasty), and RE-MOBILIZE (prevention of venous thromboembolism after total knee arthroplasty trial) in subjects undergoing elective total hip or knee joint replacement surgery (n = 8,135), rates of adjudicated definite/likely ACS events were low and similar with dabigatran (150 mg or 220 mg once daily groups combined) or enoxaparin (0.8% versus 0.7%, respectively, Table 7).13 Investigator-reported ACS event data are available for all four primary VTE prevention studies, including the RE-NOVATE II study which investigated the 220 mg dose of dabigatran without central adjudication of ACS events. In the pooling of the four trials, ACS events identified by investigators locally occurred in 0.16% of patients in the dabigatran 220 mg once daily group compared with 0.35% in the enoxaparin group.13 BODY.RESULTS.COMPARISONS WITH ENOXAPARIN OR PLACEBO (IN SUBJECTS UNDERGOING PRIMARY AND SECONDARY PREVENTION OF VTE AND SUBJECTS WITH ACS).SUBJECTS UNDERGOING SECONDARY PREVENTION OF VTE: INDIVIDUAL TRIAL DATA VERSUS PLACEBO: In a secondary VTE prevention study comparing dabigatran versus placebo,29 only one MI was reported in each treatment group. All-cause mortality rates were similar with dabigatran and the comparator treatment, predominantly warfarin (Table 7). BODY.RESULTS.COMPARISONS WITH ENOXAPARIN OR PLACEBO (IN SUBJECTS UNDERGOING PRIMARY AND SECONDARY PREVENTION OF VTE AND SUBJECTS WITH ACS).SUBJECTS WITH ACS: INDIVIDUAL TRIAL DATA VERSUS PLACEBO: In the Phase II RE-DEEM (Randomized Dabigatran Etexilate Dose Finding Study In Patients With Acute Coronary Syndromes) dose-ranging ACS study,32 almost all subjects received dual antiplatelet therapy (>95% at 1 month) in addition to dabigatran (over the dose range 50–150 mg twice daily) or placebo (Supplementary Table 3). MIs occurred at a rate of 1.6% for placebo and 3.0%, 2.7%, 1.7%, and 2.3% for dabigatran doses of 50, 75, 110, and 150 mg twice daily, respectively. Two fatal MIs (0.5%) occurred in each of the placebo and dabigatran 50 mg twice daily and 75 mg twice daily groups. There were no fatal MIs in the dabigatran 110 mg twice daily and 150 mg twice daily groups. There were three strokes in the placebo group, one in the dabigatran 75 mg twice daily group, and none in the other dabigatran dosing groups. Cardiovascular and all-cause mortality rates were lowest in the 110 mg twice daily and 150 mg twice daily groups compared with the lower dose dabigatran and placebo groups (Supplementary Table 3). BODY.DISCUSSION: Findings from our review and analyses of all comparative Phase II and III clinical trials of dabigatran etexilate clearly indicate that the rate of MI with well-controlled warfarin (for stroke prevention in patients with atrial fibrillation and acute VTE treatment or secondary VTE prevention) is lower than with dabigatran 150 mg twice daily. This is consistent with the results from RE-LY, which contributed to the majority of the data in these analyses. When comparing MI rates for dabigatran-treated patients with those for patients treated with enoxaparin or placebo, no significant difference could be observed, supporting the conclusion that dabigatran is not causing MI but rather that dabigatran is less effective than well-controlled warfarin at preventing MI;15,35 also supporting the conclusion that warfarin is protective in preventing MI is the observation that warfarin-treated patients in RE-LY with better INR control (TTR ≥65%) had lower rates of MI than those with worse INR control (TTR <65%). Additionally, in all patient populations treated with dabigatran, the MI rates were low and any increase in the rates of MI compared with warfarin were counterbalanced by an improved overall clinical benefit due to a reduction in stroke, and/or lower rates of other cardiovascular outcome events and/or overall cardiovascular mortality in those receiving dabigatran. The rationale for conducting this detailed review of individual and pooled study results was based on the observation of a nonsignificant increase in the number of MI events with dabigatran versus warfarin in RE-LY1 and a recently published meta-analysis that included only selected dabigatran clinical trial data.16 This MI imbalance, although small (1.5 excess events per 1,000 subjects per year), could not be explained by differences in baseline characteristics, including previous coronary artery disease or MI, diabetes, hypertension, or other baseline therapies. The meta-analysis by Uchino and Hernandez16 included trials with different patient types and comparators, did not utilize individual patient data, and did not include analyses according to differences in baseline characteristics and risk factors, which therefore resulted in significant limitations and raised questions about its reliability and validity. The overall MI rates in RE-LY in each treatment group were low (0.6%–0.8% per year) and similar to those observed in other recent trials3–11 in subjects with atrial fibrillation (0.55%–1.4%) receiving warfarin, antiplatelet agents, or other new oral anticoagulants; however, we still believed a thorough and as comprehensive as possible evaluation of MI rates and cardiovascular endpoint data was necessary. Due to unlimited access to the trial databases of Boehringer Ingelheim, we were able to complete this comprehensive and complete evaluation1,2,20–32 addressing the limitations of other recent meta-analyses and publications. The results in different subject populations and with different comparators (warfarin, enoxaparin, and placebo), when reviewed as individual studies or as pooled analyses of individual patient data, were consistent. In individual studies of the treatment of stroke associated with atrial fibrillation and indications other than atrial fibrillation, all stroke (ischemic and hemorrhagic), all-cause, and cardiovascular mortality rates were similar or lower with dabigatran than with warfarin. There was a consistent imbalance in MI rates between dabigatran and warfarin for all evaluated populations, although the absolute risk differences were small. The finding in RE-LY that warfarin-treated patients with better INR control (TTR ≥65%) had approximately 30% lower annual MI rates than warfarin-treated subjects with poorer INR control (TTR <65%) supports the concept that better warfarin INR control is associated with better MI prevention. The different mechanisms of action of warfarin and dabigatran may have a role in the different profiles observed for these drugs. The lack of any negative effect on myocardial ischemic events in the studies comparing dabigatran versus enoxaparin or placebo in over 10,000 patients strongly supports the position that MI is not an adverse effect of dabigatran, but rather that well-controlled warfarin seems to have a greater beneficial effect in preventing MI than dabigatran. Of interest, enoxaparin remains a guideline-recommended therapy for the treatment of acute MI and we observed no differences in the occurrence of MI in patients exposed to dabigatran or enoxaparin. A network meta-analysis of dabigatran versus placebo and aspirin treatment indicated no difference in the risk of MI, which is also supportive of this hypothesis.36 Furthermore, in an evaluation of a factor Xa inhibitor (rivaroxaban) compared with a well-controlled warfarin comparison (TTR was 64% in the North American population), this trend toward a lower potency of the new oral anticoagulant regarding MI prevention was also seen (HR 1.15, 95% CI 0.74–1.80).37 Additionally, a similar trend was recently reported for edoxaban in the HOKUSAI-VTE study.38 Therefore, it is reasonable that our results reflect the beneficial effects of well-controlled warfarin (median TTR 67.3%) for the prevention of myocardial ischemia.35 Several studies have shown that warfarin has a protective effect against MI compared with nonwarfarin anticoagulants in patients with atrial fibrillation who are prescribed anticoagulation for stroke prevention.3,15,39 Other studies have shown that warfarin plus aspirin is useful for the management of patients with coronary artery disease, particularly in the secondary prevention setting.35,40,41 When looking at specific subgroups in the RE-LY trial, interestingly, the imbalance in MI events in the pooled analysis of studies versus warfarin was predominantly driven by the results of RE-LY. The very low MI rate in warfarin-treated RE-LY subjects with baseline valvular heart disease (0.3%) may have skewed the results. Indeed, the rate was half that observed in warfarin-treated subjects without a history of valvular heart disease (0.6%). A lower MI rate in subjects with valvular heart disease who are at high risk for morbidity and mortality does not seem medically plausible42 since the actual MI rate (0.3%) in this subgroup of warfarin subjects was lower than any reported MI rate in all major recent studies of subjects with atrial fibrillation (0.55%–1.4%);3–11 the presence of valvular heart disease is indicative of a greater degree of cardiovascular comorbidity rather than its absence. Additionally, the RE-LY study exclusion criteria did not prespecify quantitative measures to identify nonhemodynamically significant valvular heart disease, making it a subjective, investigator-dependent characterization. Importantly, it should also be noted that there was no difference in MI rates comparing dabigatran versus warfarin in over 14,000 (78%) of the RE-LY subjects without a baseline history of valvular heart disease. Investigation of the timing of myocardial ischemic events in relation to treatment, important in the assessment of causality, showed that a portion of the observed excess MI rates with dabigatran in RE-LY was evident more than 3 months after discontinuation of the study drug. This suggests that this difference may be due to underlying imbalances between the treatment groups rather than the treatments themselves. In support of this, it is notable that >30% of the MI events in the dabigatran groups occurred off treatment, by which time most subjects were receiving oral anticoagulation with a nonstudy drug.12 Despite a shorter half-life of dabigatran compared with warfarin, there was no difference in the number of MI events within the first 30 days of stopping either treatment, indicating lack of any rebound effect. A recently published study investigating whether a rebound effect could be present in patients on dabigatran in RE-MOBILIZE, RE-MODEL, RE-NOVATE, and RE-NOVATE II detected no increased ACS signal with dabigatran etexilate compared with enoxaparin during or after treatment.13 There is no evidence in animal models that dabigatran causes vascular injury. Instead, studies show that dabigatran has antithrombotic and antiatherosclerotic properties and suggest that dabigatran treatment may limit atherosclerosis progression and preserve vessel lumen patency.43–46 The findings of the RE-LY trial need to be considered from both clinical and public health perspectives. While RE-LY indicated a modest, nonsignificant increase in MI compared with warfarin, this was more than counterbalanced by significant beneficial effects on stroke reduction (six fewer events per 1,000 subjects per year) and lower observed rates of cardiovascular mortality (five fewer events per 1,000 subjects per year) and total mortality, compared with warfarin.44 The RE-LY protocol prespecified a composite outcome endpoint of net clinical benefit (all strokes, systemic embolic events, MI, pulmonary embolism, major bleeding, and all-cause death), the occurrence of which favored both dabigatran doses.12 In RE-LY subjects with a prior history of coronary artery disease or previous MI, the relative risk of MI with dabigatran versus warfarin and the net clinical benefit of dabigatran over warfarin were similar compared with the group without a prior history of coronary artery disease at baseline.12 This means that the treatment effects of dabigatran on composite major clinical outcomes (eg, stroke/systemic embolic events, major bleeding, and MI) were consistent in patients at higher (ie, patients with previous MI or coronary artery disease) and lower risk of myocardial ischemic events.12 Additionally, warfarin-treated subjects in RE-LY with better INR control had lower MI rates than warfarin-treated subjects with poorer INR control. In the recently published RELY-ABLE (Long Term Multi-center Extension of Dabigatran Treatment in Patients With Atrial Fibrillation Who Completed RE-LY Trial) safety follow-up of RE-LY, 2.3 years of additional treatment with dabigatran was evaluated (total mean follow-up time of 4.3 years). The annual rates of MI were low and similar between the groups, at 0.69% and 0.72% per year for 150 mg twice daily and 110 mg twice daily, respectively. These low event rates for MI, as well as for all other evaluated outcomes, were consistent with the rates seen in RE-LY even after a long duration of exposure to dabigatran treatment.47 As a class, direct thrombin inhibitors have previously been shown to be effective in the treatment of myocardial ischemia.48 The parenteral direct thrombin inhibitor, bivalirudin, was shown to be superior to heparin for the prevention of death or MI in subjects with ACS.49 Bivalirudin was also shown to be more effective than heparin in subjects with ST-segment elevation MI undergoing percutaneous coronary intervention.50 The first oral direct thrombin inhibitor, ximelagatran, was shown to be effective in reducing the occurrence of new ischemic events when given with aspirin in subjects who had sustained a recent MI.18 The rates for MI were similar comparing ximelagatran and warfarin for the prevention of stroke in subjects with atrial fibrillation.51 We note the limitations inherent in interpretation of this post hoc analysis of the efficacy and safety of dabigatran. First, the included studies were conducted in different study populations with different baseline characteristics (eg, age, concomitant diseases, and treatment) and, for warfarin, varying levels of control measured as TTR. Second, treatment duration and follow-up periods with dabigatran varied between the trials. Third, different processes were used to identify outcome events and adjudication. Finally, trials with a longer duration (eg, RE-LY) have had a higher impact on the results of this analysis. In contrast with a previous meta-analysis,16 we tried to minimize the impact of these variables by only pooling data using the same comparator (ie, warfarin, enoxaparin, or placebo). Thus, the results of the pooled analysis can be considered hypothesis-generating but not confirmatory. Our findings greatly expand the analysis recently reported by Uchino and Hernandez,16 in which summary trial data from only seven dabigatran trials (n = 31,097 subjects) were pooled, and their analyses were conducted pooling all comparators, subject populations, indications, and dabigatran dosage, including some doses that are apparently not clinically effective. In comparison, the current analysis relies on individual subject data and a more robust statistical approach, based on pooling of trials with similar comparators and doses, as well as use of a multifactorial analysis adjusting for differing risk factors concerning various outcome measures. BODY.CONCLUSION: Based on the findings from our analyses and available clinical and preclinical investigations, we conclude that, although MI occurs more frequently in dabigatran-treated subjects than in warfarin-treated subjects, MI is not an adverse drug reaction resulting from administration of dabigatran. Further, dabigatran had an overall positive benefit-risk ratio when the composite endpoints of efficacy and safety are assessed in each of the clinical indications evaluated, including all comparisons with well-controlled warfarin. BODY.SUPPLEMENTARY TABLES: Table S1Patient characteristicsTrialAge, mean (SD)Males, %Concomitant disorders, % (n/N) Prior CADPrior MIDiabetesHypertensionBISTRO II2065.9 (10.6)38.94.1 (80/1,949)1.4 (28/1,949)3.8 (74/1,949)38.0 (740/1,949)RE-MODEL2167.7 (8.9)34.011.9 (247/2,075)1.4 (29/2,076)11.2 (232/2,075)59.4 (1,232/2,075)RE-NOVATE2263.9 (10.8)43.68.9 (309/3,459)0.8 (27/3,463)7.1 (247/3,458)46.3 (1,601/3,460)RE-MOBILIZE2366.1 (9.5)42.311.2 (292/2,596)1.7 (45/2,596)15.8 (410/2,596)62.6 (1,625/2,596)Japanese TKR2471.6 (7.8)17.07.2 (37/512)0 (0/512)15.8 (81/512)59.6 (305/512)RE-NOVATE II2562.0 (11.4)48.26.4 (129/2,011)0.5 (11/2,013)8.4 (168/2,011)46.1 (927/2,012)PETRO2669.7 (8.2)81.961.0 (306/502)NA25.1 (126/502)70.9 (356/502)Japanese AF2768.4 (8.6)88.024.1 (40/166)6.0 (10/166)27.7 (46/166)69.9 (116/166)RE-LY1,271.5 (8.7)63.627.8 (5,010/18,040)16.6 (2,994/18,040)23.3 (4,204/18,040)78.8 (14,221/18,040)RE-MEDY2854.6 (15.2)61.07.2 (207/2,856)1.2 (34/2,856)9.0 (258/2,856)38.6 (1,102/2,856)RE-SONATE2955.8 (15.3)55.56.8 (91/1,343)2.0 (27/1,343)8.0 (107/1,343)38.8 (521/1,343)RE-COVER3054.7 (16.0)58.46.5 (166/2,539)1.4 (35/2,539)8.3 (211/2,539)35.9 (911/2,539)RE-COVER II3154.9 (16.2)60.67.7 (198/2,568)1.3 (33/2,568)9.8 (251/2,568)35.6 (913/2,568)RE-DEEM3261.8 (11.4)75.937.6 (699/1,860)29.7 (5,552/1,860)32.3 (600/1,886)67.8 (1,261/1,860)Note: Demographic data were collected for randomized or safety set, as available.Abbreviations: AF, atrial fibrillation; CAD, coronary artery disease; MI, myocardial infarction; NA, no available data; SD, standard deviation; TKR, total knee replacement; VTE, venous thromboembolism; BISTRO, Boehringer Ingelheim Study in Thrombosis; PETRO, Dabigatran with or without concomitant aspirin compared with warfarin alone in patients with nonvalvular atrial fibrillation; RE-DEEM, RandomizEd Dabigatran Etexilate Dose Finding Study in Patients With Acute Coronary Syndromes Post Index Event With Additional Risk Factors for Cardiovascular Complications Also Receiving Aspirin and Clopidogrel: Multicentre, Prospective, Placebo controlled, Cohort Dose Escalation Study; RE-LY, Randomized Evaluation of Long-term anticoagulation therapY; RE-COVER, A Randomized Trial of Dabigatran Versus Warfarin in the Treatment of Acute Venous Thromboembolism; RE-MODEL, Regulation of coagulation in Orthopedic surgery to pRevent Deep venous thrombosis and pulmonary embolism; RE-NOVATE, Oral dabigatran versus enoxaparin for thromboprophylaxis after primary total hip arthroplasty; RE-MOBILIZE, prevention of venous thromboembolism after total knee arthroplasty trial; RE-MEDY, A Phase III, Randomised, Multicenter, Double-blind, Parallel-group, Active Controlled Study to Evaluate the Efficacy and Safety of Oral Dabigatran Etexilate (150 mg Bid) Compared to Warfarin (INR 2.0–3.0) for the Secondary Prevention of Venous Thromboembolism; RE-SONATE, Twice-daily Oral Direct Thrombin Inhibitor Dabigatran Etexilate in the Long Term Prevention of Recurrent Symptomatic VTE. Table S2Definition of MI/ACS eventsTrialDefinition of MI/ACS eventsBISTRO II20Events were selected as AEs according to sub-SMQ MI. Events were not adjudicatedRE-MODEL21Events were labeled as MI or reinfarction based on cardiac enzyme or ECG evidence in the blinded medical recordACS adjudication using MedDRA lower-level termsRE-NOVATE22Events were labeled as MI or reinfarction based on cardiac enzyme or ECG evidence in the blinded medical recordACS adjudication using MedDRA lower-level termsRE-MOBILIZE23Events were labeled as MI or reinfarction based on cardiac enzyme or ECG evidence in the blinded medical recordACS adjudication using MedDRA lower-level termsJapanese TKR24Events were selected as AEs according to sub-SMQ MIEvents were not adjudicatedRE-NOVATE II25Events were labeled as MI or reinfarction based on cardiac enzyme or ECG evidence in the blinded medical recordPETRO26Events were prespecified outcome events, but were not adjudicatedJapanese AF27Events were prespecified outcome events, but were not adjudicatedRE-LY1,2Events were adjudicated in a blinded mannerClinical MI was defined as the presence of at least two of the following three criteria:• Typical prolonged severe chest pain or related symptoms or signs (eg, ST changes or T-wave inversion in the ECG) suggestive of MI• Elevation of troponin or creatine kinase-MB to more than the upper level of normal, or if creatine kinase-MB was elevated at baseline, re-evaluation to >50% increase above the previous level• Development of significant Q waves in at least two adjacent ECG leadsRE-MEDY28Investigators identified possible ACS events that were then prospectively adjudicated by an independent committee.Adjudicated events were then categorized as definite, likely, unlikely, or not ACSRE-SONATE29Investigators identified possible ACS events that were then prospectively adjudicated by an independent committee.Adjudicated events were then categorized as definite, likely, unlikely, or not ACSRE-COVER30Investigators identified possible ACS events that were then prospectively adjudicated by an independent committee.Adjudicated events were then categorized as definite, likely, unlikely, or not ACSRE-COVER II31Investigators identified possible ACS events that were then prospectively adjudicated by an independent committee.Adjudicated events were then categorized as definite, likely, unlikely, or not ACSRE-DEEM32ACS events were prespecified as adverse events of special interest, and included MI, CV death, vascular death, and unstable anginaAbbreviations: ACS, acute coronary syndrome; AE, adverse event; AF, atrial fibrillation; CV, cardiovascular; ECG, echocardiogram; MedDRA, Medical Dictionary for Regulatory Authorities; MI, myocardial infarction; SMQ, standardized MedDRA query; TKR, total knee replacement; VTE, venous thromboembolism; BISTRO, Boehringer Ingelheim Study in Thrombosis; PETRO, Dabigatran with or without concomitant aspirin compared with warfarin alone in patients with nonvalvular atrial fibrillation; RE-DEEM, RandomizEd Dabigatran Etexilate Dose Finding Study in Patients With Acute Coronary Syndromes Post Index Event With Additional Risk Factors for Cardiovascular Complications Also Receiving Aspirin and Clopidogrel: Multicentre, Prospective, Placebo Controlled, Cohort Dose Escalation Study; RE-LY, Randomized Evaluation of Long-term anticoagulation therapY; RE-COVER, A Randomized Trial of Dabigatran Versus Warfarin in the Treatment of Acute Venous Thromboembolism; RE-MODEL, Regulation of Coagulation in Orthopedic surgery to pRevent Deep venous thrombosis and pulmonary embolism; RE-NOVATE, Oral dabigatran versus enoxaparin for thromboprophylaxis after primary total hip arthroplasty; RE-MOBILIZE, prevention of venous thromboembolism after total knee arthroplasty trial; RE-MEDY, A Phase III, Randomised, Multicenter, Double-blind, Parallel-group, Active Controlled Study to Evaluate the Efficacy and Safety of Oral Dabigatran Etexilate (150 mg Bid) Compared to Warfarin (INR 2.0–3.0) for the Secondary Prevention of Venous Thromboembolism; RE-SONATE, Twice-daily Oral Direct Thrombin inhibitor Dabigatran Etexilate in the Long Term Prevention of Recurrent Symptomatic VTE. Table S3Clinical endpoints in RE-DEEM32 patientsPlacebo (n = 371)Dabigatran 50 mg bid (n = 369)75 mg bid (n = 368)110 mg bid (n = 406)150 mg bid (n = 348)All death, n (%)14 (3.8)8 (2.2)10 (2.7)7 (1.7)7 (2.0)CV death, n (%)9 (2.4)8 (2.2)9 (2.5)5 (1.2)4 (1.2)All MI, n (%)6 (1.6)11 (3.0)10 (2.7)7 (1.7)8 (2.3)Fatal MI, n (%)2 (0.5)2 (0.5)2 (0.5)0 (0.0)0 (0.0)Nonfatal MI, n (%)4 (1.1)9 (2.4)8 (2.2)7 (1.7)8 (2.3)Stroke, n (%)3 (0.8)0 (0.0)1 (0.3)0 (0.0)0 (0.0)Note: Part of this table is adapted from Oldgren J, Budaj A, Granger CB, et al. Dabigatran versus placebo in patients with acute coronary syndromes on dual antiplatelet therapy: a randomized, double-blind, phase II trial. Eur Heart J. 2011;32(22):2781–2789,32 by permission of Oxford University Press and the European Society of Cardiology. Copyright © 2011.Abbreviations: bid, twice daily; CV, cardiovascular; MI, myocardial infarction; RE-DEEM, RandomizEd Dabigatran Etexilate Dose Finding Study in Patients With Acute Coronary Syndromes Post Index Event With Additional Risk Factors for Cardiovascular Complications Also Receiving Aspirin and Clopidogrel: Multicentre, Prospective, Placebo Controlled, Cohort Dose Escalation Study.

TITLE: The analgesic efficacy of continuous presternal bupivacaine infusion through a single catheter after cardiac surgery ABSTRACT.BACKGROUND:: Median sternotomy, sternal spreading, and sternal wiring are the main causes of pain during the early recovery phase following cardiac surgery. ABSTRACT.AIM:: This study was designed to evaluate the analgesic efficacy of continuous presternal bupivacaine infusion through a single catheter after parasternal block following cardiac surgery. ABSTRACT.MATERIALS AND METHODS:: The total of 40 patients (American Society of Anesthesiologist status II, III), 45–60 years old, undergoing coronary – artery bypass grafting were enrolled in this prospective, randomized, double-blind study. A presternal catheter was inserted with continuous infusion of 5 mL/h bupivacaine 0.25% (Group B) or normal saline (Group C) during the first 48 postoperative hrs. Primary outcomes were postoperative morphine requirements and pain scores, secondary outcomes were extubation time, postoperative respiratory parameters, incidence of wound infection, Intensive Care Unit (ICU) and hospital stay duration, and bupivacaine level in blood. ABSTRACT.STATISTICAL METHODS:: Student's t-test was used to analyze the parametric data and Chi-square test for categorical variables. ABSTRACT.RESULTS:: During the postoperative 48 h, there was marked reduction in morphine requirements in Group B compared to Group C, (8.6 ± 0.94 mg vs. 18.83 ± 3.4 mg respectively, P = 0.2), lower postoperative pain scores, shorter extubation time (117 ± 10 min vs. 195 ± 19 min, respectively, P = 0.03), better respiratory parameters (PaO2/FiO2, PaCO2 and pH), with no incidence of wound infection, no differences in ICU or hospital stay duration. The plasma concentration of bupivacaine remained below the toxic threshold (at T24, 1.2 ug/ml ± 0.3 and T48 h 1.7 ± 0.3 ug/ml). ABSTRACT.CONCLUSION:: Continuous presternal bupivacaine infusion has resulted in better postoperative analgesia, reduction in morphine requirements, shorter time to extubation, and better postoperative respiratory parameters than the control group. BODY.INTRODUCTION: Effective pain relief after cardiac surgery has assumed importance with the introduction of fast track discharge protocols that requires early weaning from ventilation. Inadequate pain control reduces the capacity to cough, mobility, increases the frequency of atelectasis, and prolongs recovery.[12345] A major cause of pain after cardiac surgery is the median sternotomy particularly on the first two postoperative days.[6] The most often used analgesics in these patients are parental opioids which can lead to undesirable side-effects as sedation, respiratory depression, nausea, and vomiting[37] Infiltration of local anesthetics near the surgical wound has shown to improve early postoperative pain in various surgical procedures.[891011] This study was designed to examine the efficacy of presternal infusion of bupivacaine 0.25% through a single catheter after parasternal block in controlling postoperative pain after cardiac surgery. Our primary outcome was postoperative morphine requirements and pain scores, the secondary outcomes were extubation time, postoperative respiratory parameters, incidence of wound infection, Intensive Care Unit (ICU) and hospital stay duration, and bupivacaine level in blood. BODY.MATERIALS AND METHODS: After approval of the institutional ethics committee and written informed patients' consent, 40 patients, 45–60 years old, American Society of Anesthesiologist (ASA) status II and III, scheduled for open heart surgery with sternotomy (for coronary – artery bypass grafting with cardiopulmonary bypass [CPB]), were enrolled in this prospective, randomized, controlled, double-blind study. The patients exclusion criteria included; emergency surgery, previous sternotomy, patients with preoperative poor left ventricular function (ejection fraction <40%), preexisting pulmonary or neurological dysfunction, clinically significant kidney or liver disease, patients allergic to local anesthetics, preexisting coagulopathy, patients with prolonged CPB time (>120 min), intraoperative inotropic support (dobutamine >5 μg/kg/min or epinephrine infusion >1 μg/min), patients required intra-aortic balloon pump, postoperative hemodynamic instability (including the occurrence of serious arrhythmia) or bleeding that required surgical re-exploration. Patients were trained to report pain on a visual analog scale (VAS) during the preanesthetic examination; the routine preoperative cardiac medications were continued till the morning of surgery. All patients were premedicated with intramuscular morphine 0.1 mg/kg about 30 min before the operation. On arrival at the operating room, a 16-G intravenous (i.v) cannula and a 20-G radial arterial cannula were inserted; general anesthesia was induced with fentanyl 10 μg/kg, midazolam 0.1 mg/kg and propofol 3–4 mg/kg. Pancuronium 0.15 mg/kg was administered to facilitate endotracheal intubation and was repeated during surgery as required to ensure proper muscle relaxation. Anesthesia was maintained using 0.5–1.5% sevoflurane in an oxygen-air mixture (1:1 ratio). Mechanical ventilation was provided by a Narkomed anesthesia machine (North American Dräger, Telford, PA, USA) using a tidal volume of 10 mL/kg according to weight. A 3-port central jugular venous line was inserted (Certofix Duo, B. Braun Melsungun A.G.) for central venous pressure monitoring. Standard monitoring included an electrocardiogram, invasive blood pressure, pulse oximetry, capnography, temperature (central-distal), urine output, airway volume and pressure. All operations were performed by the same surgical team in a standard method through a median sternotomy incision with saphenous veins and internal thoracic arteries harvesting for coronary artery bypass graft. At the end of the operation and just before sternal wire placement, patients were randomly divided into two equal groups using computer-generated random numbers with closed-sealed envelopes to receive either 0.25% bupivacaine solution (Group B) or 0.9% saline solution, control group (Group C). A series of intercostal blocks was performed by the surgeon just lateral to the sternal border, 2 ml of either bupivacaine or saline for each of the 5 interspaces bilaterally (total 20 ml). The patients were observed for 10 min for any bleeding caused by an inadvertent vascular injury before the closure of the sternum. After sternal wiring, a small diameter multihole soft catheter generally used for epidural analgesia (Portex Epidural Catheter 18-gauge; Smith Medical ASD Inc., Keene, NH) was positioned anteriorly to the sternum above the fascia in the subcutaneous tissue during wound closure. Steri-strips (3M Neuss, Germany) and wound dressing were used to secure the catheters. Before connection of the catheter to the pump, the syringes used for intercostal block and the infusion pump were filled with either bupivacaine or saline solutions prepared by the anesthesiologist in charge of the patient who opened the allocation envelope and who was not part of the study, both solutions looked identical. A bolus of 5 mL of the study solution was injected in the catheter after aspiration test before connection to an elastometric infusion pump (Accufuser plus® P 4003, Korea), this infusion pump consists of a disposable 275 ml elastomeric reservoir that delivers continuous infusion of either solution at a fixed rate of 5 ml/h. Because the filling volume of the pumps for the solution was supposed to provide an infusion for >48 h (expected duration 55 h), pumps were not refilled to avoid the risk of manipulation. To reduce the number of painful interventions, the catheter was concomitantly removed with the chest drain tube removal. At the end of surgery, neuromuscular block was not antagonized, patients were transferred intubated to the ICU where they were mechanically ventilated (Servo 900 D, Siemens, Uppsala, Sweden). The criteria for extubation were hemodynamic stability, absence of arrhythmias, adequate airway reflexes, normothermia, mediastinal drainage(<100 ml/h for 2 h), and acceptable blood gas analysis (pH >7.30, arterial oxygen tension >60 mmHg and arterial carbon dioxide tension <50 mmHg) at an inspired oxygen fraction of 0.4. An anesthesiologist blinded to group assignment recorded pain score postoperatively on patient's arrival at the ICU (T0), every 4 h for 12 h then every 6 h for 48 h using a VAS (0 = no pain, 10-the worst pain imaginable). The patients were asked to take a deep breath and intensity of pain was recorded. If the patients were still intubated, the observer asked whether he or she had a pain score of 10 points, if this was not the case, the observer repeated the question decreasing the score 1 point each time until the patients confirmed the answer by nodding. Intravenous (i.v) morphine 2 mg i.v. bolus was administered if VAS pain score was ≥3 and the total amount of morphine given during the first postoperative 48 h was recorded. Quality of oxygenation was assessed by arterial blood gas (ABG) analysis (baseline [after insertion of arterial cannula and before induction of general anesthesia], at extubation, 6, 12, and 24 h), this consisted of PaCO2, pH, PaO2 with calculation of PaO2/FiO2 ratio. The FiO2 is estimated either from the raw data measures during mechanical ventilation or the estimated FiO2 during spontaneous breathing (if face mask was used at O2 rates 8, 9, and 10 (L/min) then the estimated FiO2 was 0.35, 0.45, and 0.55, respectively, if nasal cannula was used at O2 rates 1, 2, 3, 4, 5 (L/min) then the estimated FiO2 was 0.24, 0.28, 0.32, 0.36, and 0.4, respectively. When no oxygen was administered, FiO2 was 0.21. Extubation time (from arrival at the ICU till tracheal extubation) was recorded, time to discharge from the ICU and hospital were recorded (according to the institution's standardized protocol for all patients undergoing cardiac surgery). Medical administration and data collected were performed in a double-blinded manner such that the patient, the surgeon administering the block, the anesthesiologist providing anesthesia, the ICU staff in the postoperative care unit, were not aware of the group assignment. Randomization and allocation were only revealed for data analysis after the study was completed. The safety outcome of the study was the bupivacaine blood level. Blood samples were drawn 24 h and 48 h after surgery for analysis of the bupivacaine concentrations. Samples were centrifuged at 50,000 revolutions/min for 10 min. Then the bupivacaine concentration in samples was determined by a high-performance liquid chromatography. Only the samples taken from patients who received bupivacaine were analyzed. Plasma concentration of >4 μg/ml was taken as toxic threshold. The occurrence of any postoperative sternal wound infection or delayed wound healing during hospitalization was also recorded. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: The primary outcome of the study was postoperative morphine requirement compared with the Control group. A sample size of 15 patients was needed in each groups to achieve 80% power to detect 50% difference in postoperative morphine consumption between the two groups, 20 patients/group were included to replace any drops and to increase power on secondary outcomes. Statistical analysis was done on a personal computer using the Statistical Package for Social Sciences version 16.0 (SPSS Inc., Chicago Illinois, USA). Data were expressed as mean values ± standard deviation or numbers (%), Student's t-test was used to analyze the parametric data and discrete (categorical) variables were analyzed using the Chi-square test, with P < 0.05 was considered statistically significant. BODY.RESULTS: The total of 40 patients were enrolled in this study; three patients had accidental catheter removal (two patients in the control group and one patient in the bupivacaine group) and were excluded. There were no significant differences between bupivacaine group (Group B) and control group (Group C) with respect to age, sex, body surface area, ASA status, preoperative ejection fraction, duration of surgery, cross-clamping, and CPB times [Table 1]. Table 1 Patients characteristics and intraoperative data Group C ( n =18) Group B ( n =19) P Age (years) 53±5.8 54±4.5 0.06 Sex (male/female) 10/8 11/8 0.057 Body surface area (m 2 ) 2.1±0.4 2.2±0.2 0.39 ASA physical status II/III (number of patients) 9/9 10/9 1 Preoperative EF (%) 55.8±4 53.7±4 0.24 Duration of surgery (min) 217.1±13.5 234.9±15.2 0.68 Cross clamping (min) 63.9±3.4 59.3±3.7 0.75 Total CPB (min) 90.7±20 91.7±19 0.95 Data are presented as mean±SD or ratio ( n ). P >0.05 was considered statistically nonsignificant. CPB: cardiopulmonary bypass, EF: ejection fraction, ASA: American Society of Anesthesiologist, SD: Standard deviation The overall morphine requirements over the first 48 h in the ICU were significantly less in Group B than Group C (8.6 ± 0.94 mg vs. 18.83 ± 3.4 mg, respectively, P = 0.02) [Table 2]. The mean VAS score of pain was significantly less in Group B than Group C at most time points [Figure 1]. Table 2 Morphine consumption and recovery times after surgery Group C ( n =18) Group B ( n =19) P Morphine consumption over the 48 h (mg) 18.83±3.4 8.6±0.94* 0.02 Extubation time (min) 195±19 117±10* 0.03 ICU stay (h) 55.4±22 56.1±30 0.36 Hospital stay (days) 5.7±1.6 5.5±1.2 0.3 Data are presented as mean±SD. * P <0.05; statistically significant difference between the two groups. ICU: intensive Care Unit, SD: Standard deviation Figure 1Visual analog score at different time points (columns are mean, and error bars are the standard deviation) The time to extubation was obviously shorter in Group B compared to Group C (117 ± 10 min vs. 195 ± 19 min, respectively, P = 0.03). However, there were no statistically difference between the two groups in the ICU and the hospital stay duration [Table 2]. The respiratory parameters (PaO2/FiO2, PaCO2 and pH) were better in Group B compared to Group C at extubation (although they were acceptable for extubation in Group C). PaO2/FiO2 was better at 6 h, 12 h and 24 h in Group B compared to Group C, with no significant differences in PaCO2 and pH value at the same times between the two groups [Table 3]. Table 3 Postoperative respiratory outcomes Group C ( n =18) Group B ( n =19) P Baseline (Before induction of anesthesia)  PaO 2 /FiO 2 mmHg 339.9±16.3 337.6±23.1 0.74  PaCo 2 mmHg 39.4±3 38.6±2.9 0.3  pH 7.36±0.04 7.38±0.04 0.6 At extubation  PaO 2 /FiO 2 mmHg 337.2±12.1 351.7±15.5* 0.004  PaCo 2 mmHg 39.4±3 36.6±2.9* 0.03  pH 7.31±0.08 7.36±0.06* 0.04 At 6 h  PaO 2 /FiO 2 mmHg 330.8±16.8 347.7±22* 0.018  PaCO 2 mmHg 39.3±5.4 37.0±4.3 0.67  pH 7.33±0.03 7.32±0.06 0.09 At 12 h  PaO 2 /FiO 2 mmHg 332.9±20.3 347.1±12.3* 0.013  PaC 2 O mmHg 38.4±1.8 37.5±5.3 0.95  pH 7.36±0.02 7.36±0.05 0.09 At 24 h  PaO 2 /FiO 2 mmHg 335±9.3 350±9.5* 0.001  PaCO 2 mmHg 39±2.4 39±2.3 0.54  pH 7.36±0.3 7.4±0.3 0.88 Data are presented as mean±SD. * P <0.05; statistical significant difference between the two groups. SD: Standard deviation There was no incidence of sternal infection or delayed wound healing recorded in either group. The plasma concentration of bupivacaine remained below the toxic threshold (at T24, 1.2 ug/ml ± 0.3 and at T48 h 1.7 ± 0.3 ug/ml), with no signs or symptoms of bupivacaine toxicity observed. BODY.DISCUSSION: The present study demonstrated that parasternal bupivacaine infiltration followed by continuous subcutaneous presternal infusion of bupivacaine 0.25% at 5 ml/h for 48 h in patients undergoing cardiac surgery with median sternotomy, had resulted in better postoperative analgesia, reduction in morphine requirements, shorter time to extubation, better postoperative respiratory parameters, with no incidence of wound infection, no difference in ICU and hospital duration with no signs or symptoms of bupivacaine toxicity compared to control group. Wound infiltration of local anesthetics has been studied for postoperative analgesia in different surgical settings and has proved to reduce postoperative pain effectively.[891011] Although the same method applied directly to the sternotomy incision in cardiac surgery has been studied, results were controversial.[1213141516] In the present study, bupivacaine 0.25% was infiltrated to block the anterior cutaneous branches of the intercostal nerves (close to the sternal border) followed by continuous infusion of bupivacaine in the subcutaneous fascia above the sternum through a multihole catheter, which resulted in better postoperative analgesia and reduction in postoperative morphine requirements. Similar to our results, Chiu, et al. in their study found that bupivacaine 0.15% infused continuously at 2 ml/h initiated at time of wound closure provided uninterrupted analgesia and contributed to short- and long-term pain relief for thoracotomy patients.[13] Four other trials studied different local anesthetic solutions applied directly to the sternal wound through two catheters from an elastomeric pump, the 1st study used bupivacaine 0.5% at 4 ml/h (3), the 2nd and 3rd used ropivacaine 0.2% at 4 ml/h,[1517] the 4th one used ropivacaine 2 ml/h.[18] The results of the previous studies showed a reduction in postoperative pain and opioid consumption with a significant decrease in hospital stay duration. On the contrary, Magnano et al. used bupivacaine 0.5%; 10 ml for wound infiltration followed by continuous infusion of 10 mg/24 h[14] and Agarwal et al. used ropivacaine 0.3% for wound infiltration followed by continuous infusion of 4 ml/h for 64 h,[16] both studies showed that local anesthetic infusion after median sternotomy did not reduce postoperative pain, VAS and time to extubation. Magnano et al. attributed their results that they used a catheter with few holes only at the tip which was ineffective in long surgical incisions as in median sternotomy and that the lower portion of the wound was probably "uncovered" by the anesthetic drug, they recommended to prolong the duration of bupivacaine infusion to be more effective in controlling delayed postoperative pain.[14] Agarwal attributed the failure of their analgesic technique that their study was stopped earlier than planned due to wound infection.[16] In our study, it was possible that placing catheters for sternal wound infusion closer to the anterior branches of the intercostal nerves has improved analgesic efficacy.[15] Patients in the bupivacaine group were extubated earlier with better respiratory parameters (indicated by the ABGs analyses) at time of extubation and at the subsequent readings compared to the control group. Eljezi et al., demonstrated no improvement in the postoperative respiratory functions with improvement of analgesia in their study group.[15] The main explanation for such difference is that they depend on pulmonary function tests to assess oxygenation which needs deep inspiratory maneuvers of the spirometric volumes tests and that sternotomy pain was a minor factor in the respiratory dysfunction observed while we just assessed the ABGs. As regarding ICU and hospital stay duration, White et al. demonstrated no improvement in length of ICU stay probably because there were no attempt by the ICU staff in his study to fast – track cardiac patient.[3] In our study, reducing pain, earlier tracheal extubation, and good pulmonary function in the bupivacaine group did not change the policy of the ICU team as regard ICU and also hospital stay durations (as they follow routine discharge protocols), and hence differences between groups if found are not easy to identify. There were no serious adverse events reported in all the previous studies except in one of them where sternal wound infection with increased incidence of catheter-related problems was noticed which lead to discontinuation of the trial.[16] An increased wound infection could be consistent with a theoretical concern that local anesthetics exert multiple antiinflammatory effects on granulocytes and on release of some inflammatory modulators and may enhance some aspects of immuno-suppression that is more likely to occur with levorotatory stereoisomer, ropivacaine, and its closely related chiral congener, levobupivacaine than racemic anesthetics, like bupivacaine.[1920] In the present study, the plasma levels of bupivacaine remained beneath the mean level observed for occurrence of neurologic symptoms in human (unbound drug 0.6 μg/ml, i.e. 4 μg/ml of total drug), there were no signs or symptoms of local anesthetic toxicity (neurological or cardiovascular) observed in any of our patients, White et al. found that 0.25% or 0.5% bupivacaine infusion at 4 ml/h in the sternotomy wound resulted in safe serum concentration (<2 μg/ml).[3] We chose the lower concentration of bupivacaine (0.25%), which has resulted in effective pain management in the above study and at the same time to avoid any adverse effects of the high concentration. Furthermore, we used a more simple method to infuse bupivacaine 0.25% in the presternal tissue via a single catheter. The bupivacaine infusion was started before sternal wire closure to provide an afferent block as earlier as possible, with no need of refilling which decreased the incidence of manipulation and therefore infection. There were no pump malfunction, disconnection, or breakage of the catheter noted in the study period. There were several limitations in our study; first, our study was performed on relatively cardiac stable patients. We are aiming to assess the efficacy of this block on more complicated cases. Second, although the patients in Group B were extubated earlier than in Group C, the time of assessing the respiratory parameters was at fixed intervals (at extubation, 6, 12, and 24 h) to maintain blind collection of data. BODY.CONCLUSION: Delivering bupivacaine 0.25% through a single catheter embedded anteriorly to the sternum during the wound closure is a simple technique, it provided adequate postoperative analgesia with less morphine requirements.

TITLE: Preoperative Acute Normovolaemic Hemodilution (ANH) in combination with Hypotensive Epidural Anaesthesia (HEA) during knee arthroplasty surgery. No effect on transfusion rate. A randomized controlled trial [ISRCTN87597684] ABSTRACT.BACKGROUND: Hypotensive epidural anaesthesia (HEA) combines a high epidural anaesthesia, performing a sympathetic blockade, with low-dose iv-infusion of epinephrine to stabilize circulation in the conscious patient. Mean artery blood pressure is reduced to 45–50 mmHg and hereby a reduced blood loss. In this study we have combined HEA with preoperative acute normovolaemic hemodilution (ANH) in attempt to further reduce the blood loss and need for blood transfusion in total knee arthroplasty surgery (TKR). ABSTRACT.METHODS: Twenty-eight patients scheduled for TKR are randomised to ANH or no hemodilution (non-ANH). Both groups are anaesthetized with HEA. ANH is established with predonation of 20 % of the total blood volume, and replacement with equal volume of HAES 6 %. Blood re-transfusion is completed within 6 h. ABSTRACT.RESULTS: A mean of 877 ml blood was predonated (19.7 % of the total blood volume). Blood loss was, except from the intraoperative loss, significantly higher in ANH group. The total loss was 1306 mL (ANH) vs. 1026 mL (non-ANH), p < 0.05. Except from the first hour postoperatively, hematocrit was identical in between groups postoperatively. The amount of blood transfusion was identical 386 ml (ANH) vs. 343 ml (non-ANH) (ns). 50 % went through surgery without receiving blood (ANH) vs. 58 % (non-ANH). No renal, neurological or cardiopulmonary complications were registered. ABSTRACT.CONCLUSIONS: These data suggest no benefits in combining HEA and ANH in TKR surgery. Probably because of the reduced viscosity of the blood after ANH, there is an increased postoperative blood loss. The need for homologous blood transfusion was identical. BODY.BACKGROUND: The risks associated with banked homologous blood products are well known. These risks include among others: ABO incompatibilities, viral or bacterial infections, and immunosuppression [1]. Immunomodulation may have long lasting effects on the patient who has received a single unit of homologous blood, and may alter the immune response in a way that may render the recipient vulnerable to infection [2]. Several techniques for management of surgical patients without homologous blood transfusion are available. Acute normovolaemic hemodilution (ANH) is the simultaneous exchange of whole blood with an identical volume of an iso-oncotic colloid. The resulting dilutional anaemia is compensated for by an increase in cardiac output and enhanced arterial oxygen extraction. Although bleeding during surgery remains essentially unchanged, blood lost during the surgical procedure contains fewer red cells because the patient's blood has been diluted. At the conclusion of surgery or when needed, donated blood may be returned to the patient [3]. ANH is found to be effective in surgical cases with a high-expected blood loss [4]. The concept of decreasing the arterial blood pressure to hypotensive levels during anaesthesia and surgery has been used for decades in attempt to reduce intraoperative blood loss and to improve conditions in the surgical field. A mean arterial blood pressure (MAP) of 55–60 mmHg has been regarded as the lowest safe level in young healthy patients, one of the reasons being that this range represents the lowest MAP at which auto regulation of the cerebral blood flow is still active. Sharrock introduced the hypotensive epidural anaesthesia (HEA) in 1989 [5]. Epidural anaesthesia was used to establish a sympathetic blockade, causing a hypotensive state. Low dose of epinephrine was infused to maintain and stabilize cardiac output and heart rate. During HEA, a MAP of 45–50 mmHg was allowed since the cerebral condition could be monitored through the patient's ability to communicate, as only slight sedation was used. HEA is a safe technique with a low complication – and mortality rate, even in patients with left ventricular dysfunction [6-8]. This method reduces blood loss and need for transfusions significantly in hip arthroplasty surgery [9]. We have in a previous paper demonstrated a reduced perioperative blood loss at 47 % in knee arthroplasty surgery (TKR) during HEA [10]. The aim of this study was to evaluate the potential benefits of a reduced need for homologous blood transfusion by combining HEA and ANH in TKR surgery. BODY.METHODS: The study was approved by the regional scientific ethics committee and corresponds with the Helsinki II declaration. In all patients written informed consent was obtained. Twenty-eight patients scheduled for primary cemented TKR surgery were included in the study period. Exclusion criteria were: Patients underage (<18 years), hematocrit < 0.30, recent myocardial infarction (<6 months), unstable angina, diagnosed and clinical symptomatic aortic- or mitral valve stenosis (>30 mmHg), previous stroke, unmedicated hypertension and treatment with beta-antagonists or anticoagulants. No patient was medicated with salicylate or nonsteroidal anti-inflammatory drugs in the two weeks prior to surgery. On the day of surgery patients were consecutive and blindly randomised to HEA with or without ANH using computerised randomisation to two treatments (MedStat®). ANH was performed by drawing 20 % of the total blood volume through a venous cannula immediately before anaesthesia. This volume was simultaneously replaced with an equal volume of Hydroxy Ethyl Starch 6 % (HAES 6 %). The following formula was used: Total blood volume = body weight (kg) × 60 mL per kg. The donated volume of blood was collected in transfusion bags together with a stabilizer. Until re-transfusion in the recovery room, the blood was storaged in a cooler box. Retransfusion was terminated within 6 hours. Both groups were anaesthetized with the hypotensive epidural anaesthesia technique. BODY.METHODS.HEA: To establish the sympathetic blockade, epidural anaesthesia was performed at the Th12-L1/L1-L2 interspace with ropivacaine 1%, 25–35 ml to achieve analgesia at Th1-2 dermatome. For epidural infusion, ropivacaine 1% 6–8 ml/h was used until end of surgery. The infused dose varied according to the patient's age, height and weight. The arterial pressure decreased progressively over 10 minutes after the epidural bolus injection to a MAP of 45–50 mmHg. In case of insufficient decrease in blood pressure, bolus ropivacaine 5 ml epidurally was used. Infusion of epinephrine 0.01–0.07 μg/kg/min was started immediately after insertion of the central vein catheter; in order to prime the catheter before the sympathetic blockade was established. During surgery sedation with propofol was adjusted to a level where communication was possible. Intraoperative infused fluid volume was in both groups isotonic saline 5 mL/kg/h as basic need and Isotonic saline three times the amount of blood loss as substitution for intraoperative blood loss. Oxygen was delivered at 3 l/min on a nasal catheter. All patients were kept warm, using a Bair-hugger. No knee tourniquet was used. Monitoring: 5-lead ECG, pulse oximetry, respiratory rate, end-tidal CO2, radial- and central venous pressures. All data were continuously recorded on a computer, using the Datex Collect (software (Datex, Denmark). Infusion of epinephrine was continued postoperatively until MAP reached 75 mmHg, hereafter epinephrine was reduced gradually until removal. In both groups, the intraoperative blood loss was determined by measurement of the volume in the suction apparatus, and by weighing of towels. The postoperative blood loss was determined by measuring the drainage for 48 hours after operation. Drains were removed after 48 hours. Postoperative analgesia was performed as patient controlled epidural analgesia using ropivacaine 0.2 % and morphine 0.05 mg/ml for three days, according to our normal routine. The epidural catheter was removed on the third postoperative day. Hereafter the patients were treated orally with morphine and acetaminophen. Fragmin® was given as thrombo-prophylaxis. All patients used a continuous passive knee motion apparatus the first two postoperative days. The patients were anaesthetized by one of the three anaesthesiologist authors and operated by two surgeons. Demographics, hematocrit pre- and post ANH, at arrival at recovery room, and at 1., 2., 3., and 5. postoperative day were recorded. Intra- and postoperative fluid- and blood transfusions were also registered. Blood transfusion was indicated when the hematocrit value was below 28 %. BODY.METHODS.STATISTICS: After passing through the exclusion criteria patients were consecutive and blindly randomised to HEA with or without ANH using computerised random numbers for 2 treatments (MedStat®). The result of the randomisation was known immediately before anaesthesia. Data are presented as means (SD) or means (range). Comparison between groups was performed with a Student's t-test. For ASA-score a Mann Whitney's test was used. Chi-test was applied on gender and the fraction of transfused patients in the two groups. A p < 0.05 was chosen as significance level. The sample size of this study was determined by an expected effect of ANH on blood transfusion rate of 50 % (MIREDIF). α = 5 %, β = 10 %, SD = 400 n1 = n2 = 2(t2( + tβ)2SD2/MIREDIF2 Power = (1-β) = 1-0.10 = 0.90 This calculation resulted in 13 patients in each group. We decided to enrol 14 in each group. BODY.RESULTS: In all 33 patients were screened to enter this study. Three patients were excluded because of treatment with beta-antagonists and 2 patients because of previous stroke. Twenty-eight patients, 14 ANH and 14 non-ANH were enrolled in the study. Patient characteristics and preoperative blood measurements are shown in Table 1. Except from the preoperative mean artery blood pressure (measured before blood donation) there was no significant difference in between groups. Intraoperative values are shown in Table 2. The ANH group was given ropivacaine 1 %, mean 288 ± 32 mg and non-ANH ropivacaine 1 %, 297 ± 22 mg (ns). The intraoperative mean artery blood pressure was identical between groups. MAP demonstrated a reduction of 53.4 % (ANH) and of 58.4 % (non-NAH), compared to preoperative blood pressure (ns). Table 1 Demographics and preoperative measurements of the patients. ANH Non-ANH P n 14 14 Sex, F/M 10/4 11/3 1.00 Age, yr 75.8 (6.4) 70.1 (9.6) 0.08 Weight, kg 75 (11.5) 76 (13.2) 0.81 Height, cm 166 (8) 166 (10) 0.98 ASA (median (range)) 2 (1–2) 2 (1–2) 0.75 Mean arterial blood pressure, mmHg 103 (12) 118 (19) 0.02 * Total blood volume, mL 4471 (809) 4446 (736) 0.93 ANH: Acute normovolaemic hemodilution; non-ANH: No acute normovolaemic hemodilution; Mean (SD) * significance Table 2 Perioperative values for the patients. ANH 14 Non-ANH 14 P Mean arterial blood pressure, mmHg 47.9 (1.8) 49.0 (3.6) 0.31 Central vein pressure, mmHg 4.5 (2) 6.1 (4) 0.21 Duration of surgery, min 93 (13) 100 (20) 0.26 Duration of anaesthesia, min 182 (15) 193 (23) 0.16 Duration of hypotension, min 102 (20) 110 (22) 0.34 Core temperature after surgery, C 36.5 (0.4) 36.7 (0.7) 0.20 Postoperative ICU-time, hrs 6.8 (1.2) 7.1 (0.8) 0.36 ANH: Acute normovolaemic hemodilution; non-ANH: No acute normovolaemic hemodilution; Mean (SD) * significance Postoperatively in the recovery room, MAP was below 75 mmHg for 38 min ± 40 (ANH) vs. 52 min ± 77 (non-ANH) (p = 0.56). Time used for anaesthesia (from patient enters the operating theatre to entering the recovery room) and duration of surgery was identical between groups. Core temperature on admission to the recovery room was also without a significant difference. The preoperative donated blood volume was mean 877 mL ± 175, or 19.7 % of the total blood volume. All blood was retransfused to ANH patients in the recovery room. Perioperative blood loss and substitution are shown in Table 3. At almost all registrations there was a reduced blood loss in non-ANH group. Colloid substitution was in ANH group 679 mL and blood transfusion 386 mL, without including the infusion of HAES used in the ANH procedure and the predonated amount of blood. Fifty percent in the ANH group received homologous blood transfusion during the hospital stay (771 mL pr. patient transfused) vs. 42 % in non-ANH group (800 mL pr. patient transfused) (ns). Numbers of units packed red cells are shown in Table 4. No patients received blood components other than erythrocytes. Table 3 Blood loss and substitution for the patients. ANH 14 Non-ANH 14 P Blood loss intraoperative, mL 131 (78) 111 (56) 0.45 Blood loss 1. postop. Hour, mL 295 (129) 204 (77) 0.03 * Blood loss 2. postop. Hour, mL 192 (98) 118 (72) 0.03 * Blood loss 3. postop. Hour, mL 132 (85) 111 (61) 0.47 Blood loss 24. postop hours, mL 1057 (292) 836 (280) 0.05 * Blood loss 2. postop day, mL 109 (78) 83 (67) 0.36 Total blood loss, mL 1306 (300) 1026 (294) 0.02 * Homologous blood transfusion (PRC), mL 386 (720) 343 (454) 0.85 Crystalloid first 24 hours, mL 3229 (582) 3312 (731) 0.75 Colloid first 24 hours, mL 679 (575) 377 (361) 0.12 Donated amount of blood, mL 877 (175) ANH: Acute normovolaemic hemodilution; non-ANH: No acute normovolaemic hemodilution; Mean (SD) * significance Table 4 Patients receiving blood substitution (units of packed red cells). ANH 14 Non-ANH 14 P Zero 7 8 1 unit 4 0 2 Units 1 4 3 Units 1 0 >3 Units 1 2 Average transfused, mL 386 343 0.85 ANH: Acute normovolaemic hemodilution; non-ANH: No acute normovolaemic hemodilution The perioperative blood analysis demonstrated, except from hematocrit at start and conclusion of surgery, no differences (Table 5). Table 5 Perioperative blood measurements. ANH 14 Non-ANH 14 P Hematocrit preoperative, % 38.7 (3) 38.1 (5) 0.71 Hematocrit at start of surgery, % 30.4 (5) 38.1 (5) 0.001 * Hematocrit 1. hour postop, % 28.7 (3) 32 (4) 0.04 * Hematocrit 1. postop day, % 29.8 (3) 29.9 (4) 0.91 Hematocrit 2. postop day, % 30.7 (3) 29.9 (4) 0.63 Hematocrit 3. postop day, % 30.4 (3) 28.8 (2) 0.14 Hematocrit 5. postop day, % 29.4 (3) 30.3 (4) 0.53 Platelet count preoperative, 10 6 /l 257 (72) 265 (63) 0.35 Platelet count 1. postop day, 10 6 /l 190 (60) 185 (42) 0.81 Platelet count 5. postop day, 10 6 /l 322 (85) 337 (77) 0.38 APTT preoperative, sec 32 (3) 30 (3) 0.16 Creatinine preoperative, mol/L 83 (21) 85 (23) 0.79 Creatinine 1. postop.day, mol/L 82 (20) 80 (17) 0.58 Creatinine 5. postop day, mol/L 80 (22) 74 (13) 0.39 ANH: Acute normovolaemic hemodilution; non-ANH: No acute normovolaemic hemodilution; Mean (SD) * significance There was no difference in time spent in hospital between the groups. At the 7'Th postoperative day all patients in this study were transferred to a hotel department for further mobilisation. During the hospital stay we did not observe any cases of renal failure according to the Creatinine measurements. All patients were discharged from the hospital alive. BODY.DISCUSSION: In both groups there was a remarkable low total blood loss, due to the anaesthetic technique used (HEA). The blood loss was, especially the non-ANH group, in a range identical with the result of our previous study comparing HEA with normotensive regional anaesthesia in TKR surgery [10]. ANH failed in the present study to demonstrate a reduced need for blood transfusion with homologous blood products during TKR surgery in HEA anaesthesia. We here use a moderate hemodilution (20 % of total blood volume) to a hematocrit near 30% agreeing with the recommendations for patients at this age [11]. Despite of this 50% in the ANH group received homologous blood. In a previous ANH study without the use of HEA anaesthesia, 45 % of TKR patients received homologous blood [12]. A case study analysis of patients who had undergone moderate ANH (15 % of patients blood volume) estimated that only 0.5 unit of blood is "saved" [13]. Mathematical models concur that savings are small unless profound hemodilution is accompanied by large blood loss (> 2 L) [14]. Another study postulates that the safety and efficacy of more extensive hemodilution is controversial because it is not possible for all patients and may provide only little additional blood conservation [15]. At almost all registrations the ANH group demonstrated an increased postoperative blood loss compared to the non-ANH group. Dilution of the blood after ANH is the most reasonable explanation to this. TKR surgery is known to be associated with extensive postoperative blood loss, probably because of difficulties in performing postoperative compression and haemostasis of the tissue in the knee [10]. This fact combined with the reduced viscosity of the blood could increase the postoperative blood loss. We did not register any difference in time spent in surgery or postoperative hypotension that could explain differences in the blood loss. In a previous study mild hypothermia was found to be correlated to a significantly increased blood loss in hip arthroplasty surgery [16]. We did not register differences in postoperative core temperature. Another possible explanation for the difference in blood loss could be alternation of the coagulation system because of the hemodilution. ANH is found to cause a slight coagulopathy including a decrease in platelet count, prothrombin time, fibrinogen, AT-III and plasminogen. Furthermore APTT is increased. The combination of ANH and prostaglandin E1 induced hypotension to 55 mmHg did not alter the coagulation system any further [17]. Pre- and postoperative platelet count and APTT were in our study without significant differences. The registered difference in the mean age between the groups must be without significant influence on the perioperative blood loss. We found no complications related to the brain or the heart during the hospital stay. In a recently published work, HEA demonstrates identical rates of cardiac and thromboembolic complications in elderly patients compared to normotensive epidural anaesthesia. Furthermore, there were no differences in early or long-term cognitive dysfunction [18]. None of the patients died in the perioperative period, in agreement with the very low mortality described by Sharrock [7]. BODY.CONCLUSIONS: HEA is found to reduce the perioperative blood loss and the need for transfusion compared to conventional anaesthesia [9,10], but the combination of HEA and ANH seems in this study to be without any further benefits regarding the blood loss and need for transfusion. BODY.COMPETING INTERESTS: None declared. BODY.AUTHOR CONTRIBUTORSHIP: All three authors carried out the protocol, inclusion of patients, anaesthesia and data collections. PJ wrote the primary manuscript and performed the statistical analysis. MBM and UTL were rewriting the primary 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: Vitamin A Supplementation at Birth Might Prime the Response to Subsequent Vitamin A Supplements in Girls. Three Year Follow-Up of a Randomized Trial ABSTRACT.OBJECTIVES: Within a randomised trial of neonatal vitamin A supplementation (VAS) in Guinea-Bissau, neonatal VAS did not affect overall infant mortality. We conducted a post-hoc analysis to test the hypothesis that neonatal VAS primes the response to subsequent vitamin A. ABSTRACT.METHODS: All trial children were offered VAS after follow-up ended at 1 year of age (FU-VAS). We compared mortality between 1 and 3 years of age according to initial randomization to neonatal VAS or placebo in Cox-regression models; we expected that children randomized to neonatal VAS compared with those randomized to placebo would have lower mortality after reception of FU-VAS. ABSTRACT.RESULTS: Of 4345 infants enrolled in the original trial, 3646 lived in the study area at 1 year of age and 2958 received FU-VAS. Between 1 and 3 years of age, 112 children died. After FU-VAS, neonatal VAS was associated with lower mortality than placebo: Mortality Rate Ratio (MRR) = 0.54 (95%CI: 0.31–0.94). The effect was more pronounced in girls (MRR = 0.37 (0.16–0.89)) than boys (MRR = 0.73 (0.35–1.51)). The beneficial effect of neonatal VAS may have been particularly strong for girls who received both VAS in a campaign and FU-VAS (MRR = 0.15 (0.03–0.67)). Among children who had not received FU-VAS, mortality in the second and third year of life did not differ according to reception of neonatal VAS or placebo. Hence, in the second and third year of life the effect of neonatal VAS versus placebo was different in girls who had or had not received FU-VAS (p for homogeneity = 0.01). ABSTRACT.CONCLUSIONS: The present results suggest that neonatal VAS primes the response in girls such that they get a beneficial effect after a subsequent dose of VAS. ABSTRACT.TRIAL REGISTRATION: Clinicaltrials.gov NCT00168597 BODY.INTRODUCTION: Meta-analyses have estimated that vitamin A supplementation (VAS) after 6 months of age reduces all-cause mortality by 23–30% in low-income countries [1], [2]. The WHO therefore recommends VAS at vaccination contacts after 6 months of age and at national immunization days when routine coverage with VAS is less than 80% [3]. Hence, children may receive VAS on several occasions. The original VAS trials provided several doses of VAS with 4- or 6-monthly [4]–[8] intervals. Three trials presented the mortality reduction by dose [7]–[9] and in two of the trials, the beneficial effect of vitamin A seemed most pronounced after a second dose [8], [9]. In a randomized trial comparing two different doses of VAS given with oral polio vaccines (OPV) during national immunization days in Guinea-Bissau, a low dose was better for girls, whereas the dose difference mattered little for boys [10]. When we repeated the trial, more children had received VAS previously, and the high dose of VAS tended to be more beneficial in these children (submitted paper). This made us hypothesize that a first dose of VAS may prime a more beneficial response to subsequent VAS. Between 2002 and 2005, we conducted a randomized trial of VAS provided with Bacille Calmette-Guérin vaccine (BCG) at birth to study the effect on infant mortality [11]. We found no overall effect of VAS, the mortality rate ratio for VAS versus placebo at birth being 1.07 (0.79–1.44) [11]. All children were offered VAS (100,000 IU) at a follow-up visit after 1 year of age. This provided an opportunity to study the hypothesis that neonatal VAS primed the response to subsequent VAS, leading to improved survival in children who received VAS at birth and at follow-up. BODY.METHODS.SETTING: The Bandim Health Project (BHP) keeps a health and demographic surveillance system covering approximately 100,000 inhabitants in six suburban districts of the capital of Guinea-Bissau. All children in the study area are followed with home visits every three months until the age of three years to register vaccinations, hospitalizations, and survival. Guinea-Bissau is classified as having sub-clinical vitamin A deficiency by UNICEF [12]. Within the present cohort 27% had low retinol binding protein (corresponding to <0.70 μM retinol) at 6 weeks and 9% at 4 months of age, controlled for acute infection [13]. Vitamin A campaigns for children aged 6 months to 5 years were conducted in November 2003, November 2004, November 2005, and in May and November 2006. The BHP registered all children in the study area who received VAS during these campaigns. Within the VAS-at-birth trial we found at 12 months of age that 23% of the children were stunted and 8% underweight (length- and weight-for-age z-score <−2) [14]. BODY.METHODS.ENROLMENT: The VAS-at-birth trial has been described in details elsewhere [11]. Mothers of normal birth weight infants (> = 2500 g) without signs of overt illness or malformations were invited to participate when the child was due to be BCG-vaccinated, either shortly after giving birth at the maternity wards, or when the mother brought the child for BCG vaccination at the health centers after a home delivery. Mothers were informed by a trained field worker about the study and asked if they wanted their child to participate. Since the majority of the mothers are illiterate verbal consent was obtained (specified in the approved protocol). Written consent was obtained if the mother knew how to write her own name. If not, a statement that she had understood the information given and agreed to participate was signed by the assistant. Provided consent, the mother drew a lot from an envelope prepared by the study supervisor. Each envelope contained 100 lots: 50 marked "1" and 50 marked "2", indicating from which of two numbered bottles, "1" or "2," the child should receive the supplement. The lots were folded, making it impossible to tell what was written on them before they were opened. A new envelope was not taken into use before the previous envelope had been completely emptied. VAS was 1⁄2 ml of oil containing 50,000 IU vitamin A as retinyl palmitate and 10 IU vitamin E and placebo was 1⁄2 ml of oil containing only 10 IU vitamin E. The code was broken 12 months after the last child was enrolled. We found no adverse effects of 50,000 IU vitamin A with BCG vaccine at birth [15]. BODY.METHODS.VAS AT FOLLOW-UP VISITS: All trial children were visited at home by a special team after turning 1 year of age. Repeated visits were made to children who were absent or travelling. Children who had moved within the urban study area were localized if possible and visited at the new address. Field workers conducting the 1-year visit were unaware of the treatment allocation at enrolment. At the 1-year visit an interview on previous VAS doses and hospitalizations was conducted and the child was offered 100,000 IU vitamin A (FU-VAS) if it had not received campaign VAS in the preceding month. The dose of 100,000 IU rather than 200,000 IU was used because previous results had indicated that the lower dose was associated with better survival [10]. The initiation of the 1-year visits was delayed for logistical reasons. Hence, children enrolled in the first year had later follow-up visits than children enrolled later. All children were followed for mortality through the demographic surveillance system until the age of 3 years. Deaths were registered at the 3-monthly home visits. The registration of a death is followed by a short interview about the cause of death conducted by a local physician or midwife. BODY.METHODS.GENERATION OF THE “PRIMING HYPOTHESIS”: We did not plan to study how neonatal VAS affects the response to a later dose of VAS when we designed the trial of neonatal VAS. However, contrasting effects of two previous randomized trials comparing the recommended dose of VAS and half this dose conducted during VAS campaigns in 2002 and 2004, made us hypothesize that the effect of VAS depends on prior dosing: In the 2002-trial, the low dose (half of the recommended 100,000/200,000 IU for children 6–11/>12 months respectively) was more beneficial than the high dose for girls [10]. In the 2004-trial, conducted to test this observation, there was no beneficial effect of the low dose (submitted). More children had received VAS previously in the 2004-trial, and children who had received VAS previously tended to benefit from the high dose. We therefore hypothesized that the effect of VAS may depend on prior supplementation. Since the neonatal VAS trial had randomized the children to VAS or placebo at enrolment and had provided a follow-up dose of VAS (FU-VAS) to all participants, we used the data set to test whether mortality after FU-VAS depended on the prior randomization to VAS or placebo. The longitudinal registration of undertaken by the BHP made this study possible in spite of not being planned. BODY.METHODS.TESTING THE “PRIMING HYPOTHESIS”: Had the study been planned, we would have randomized to VAS or placebo at birth (as done) and randomized the children again at 12 months of age to FU-VAS or placebo (not done) to get groups A, B, C and D (Figure 1). In this prospective follow-up study, our groups A and B consist of children who were met at home at the follow-up visit and received FU-VAS before 3 years of age. Children still living in the study area at 12 months of age, who had not yet received FU-VAS formed groups C and D; some of these children never received FU-VAS and some received FU-VAS later and then changed to groups A and B. 10.1371/journal.pone.0023265.g001Figure 1Trial profile.# Note: children in "A" contribute survival time in group C between age 12 months and date of reception of FU-VAS. ¤ Note: children in "B" contribute survival time in group D between age 12 months and date of reception of FU-VAS. * Among absent and travelling children 11 deaths occurred before 3 years of age (Vitamin A: 6; placebo: 5), bringing the total number of deaths up to 112. To examine whether VAS-at-birth primed the response to FU-VAS, we compared survival of children who had received FU-VAS according to randomization to VAS or placebo at birth, i.e. comparing groups A and B (trial profile, Figure 1). For priming to be important group A should have lower mortality than group B even though both groups had received VAS after one year of age. Additionally, we investigated whether the lower mortality of A than B was due to the initial profiling of the immune system by VAS by comparing the effect of VAS versus placebo at birth both during the first year of life and during the second/third year of life among those who had not received FU-VAS (groups C and D). BODY.METHODS.ETHICS STATEMENT: The protocol for the neonatal VAS trial was approved by the Ministry of Health in Guinea-Bissau ('Núcleo de Coordenação de Pesquisa, Ministerio da Saude Publica') and 'The Danish National Committee on Biomedical Ethics' gave its consultative approval. The original trial was registered at clinicaltrials.gov, number NCT00168597. BODY.METHODS.STATISTICAL METHODS: Survival was assessed in Cox proportional hazards models with age as underlying time, comparing the mortality rates in the two groups originally randomized to VAS or placebo at birth. Hence, to compare the two groups we provided mortality rate ratios (MRR) controlled for age. Follow-up time was censored at 3 years of age, or date of moving out of the area, whichever came first. Accidental deaths were censored at the date of death. Our primary analysis was an analysis comparing those randomized to VAS or placebo at birth after reception of FU-VAS (groups A and B). In the secondary analyses we compared the effect of VAS versus placebo at birth both during the first year of life and during the second/third year of life among those who had not received FU-VAS (groups C and D). Furthermore, we studied whether additional VAS in campaigns before FU-VAS modified the effect of neonatal VAS versus placebo. We used FU-VAS rather than VAS in campaigns in our primary analysis of priming since the information on FU-VAS was available for all enrolled children. Since the children were followed to 3 years of age, we also assessed the long-term effect of neonatal VAS between birth and 3 years of age regardless of subsequent doses of VAS. Hence, all comparisons were based on the comparison of the two groups of children who were originally randomized to neonatal VAS or placebo. All analyses were stratified by sex as VAS may have sex-differential effects [10], [16]; estimates are reported for boys and girls separately as well as combined. Sub-group analyses examined whether the effect of neonatal VAS was limited to certain sub-groups; we stratified by nutritional status at FU-VAS, timing of follow-up, maternal education, and place of enrolment. Effect modification was analyzed by investigating the homogeneity of the effect of neonatal VAS in the different categories of the suspected modifier using Wald test statistics. The proportional hazard assumption was assessed using Schoenfeldt's residuals and by visual inspection of log-log plots. Visual inspection of log-log plots suggested that the proportional Hazards assumption was violated the first months after FU-VAS, though this was not confirmed by Schoenfeldt's residuals. We tested whether the estimate changed, if we excluded the first 2 months after FU-VAS (during which 12 deaths occurred). This was not the case and all results have therefore been presented for the whole period. All analyses were conducted using Stata 10.1. BODY.RESULTS.STUDY POPULATION: The study profile is presented in Figure 1. Between November 13, 2002, and November 28, 2004, we enrolled 4345 children of whom 3646 (all: 84%; vitamin A: 87%, Placebo: 81%) were still living in the study area at 12 months of age. Of these, 2958 (vitamin A: 81%; placebo: 82%) were met at home at the follow-up visit and received FU-VAS before 3 years of age (groups A and B). Among those still living in the study area at 12 months of age, 688 never received FU-VAS and formed groups C and D along with children who had not yet received FU-VAS. Children were followed to 3 years of age, thus the follow-up ended November 28, 2007. We registered a total of 112 deaths (vitamin A at birth: 47, placebo at birth: 65) in the second and third year of life. Based on simple death interviews 108 deaths were due to infectious disease, whereas 4 deaths were accidents and censored at the date of death (vitamin A: 1 (intoxication), placebo: 3 (1 drowning, 2 burns)). The infant mortality rate was 47 per 1000 person-years-at-risk (PYR) [11]; and declined to 14 and 12 per 1000 PYR in the second and third year of life. BODY.RESULTS.BASELINE CHARACTERISTICS: There were no baseline differences between the children who had received neonatal VAS or placebo, continued to live in the study area and were met at follow-up (Table 1). There were no differences between the children met at follow-up (groups A and B) and those not met (parts of groups C and D) with regard to treatment, sex or anthropometric measurements at enrolment. Children living in the area at 1 year of age and met at follow-up were more likely to have mothers with some schooling, to belong to the ethnic group Pepel, and to live in households with electricity, but the distribution did not differ between the neonatal vitamin A and placebo groups. 10.1371/journal.pone.0023265.t001 Table 1 Baseline characteristics of all enrolled and children who received vitamin A supplementation at the 12-month follow-up visit. All enrolled Registered in the area at 1 year Received VAS at FU visit Vitamin A Placebo Vitamin A (A+C) Placebo (B+D) Vitamin A (A) Placebo (B) N (%) N (%) N (%) N (%) N (%) N (%) Number of children 2145 (49) 2200 (51) 1787 (49) 1859 (51) 1440 (49) 1518 (51) Boys 1 1075 (50) 1125 (51) 893 (50) 944 (51) 722 (50) 776 (51) Maternal schooling 2 No 624 (29) 638 (29) 513 (29) 541 (29) 384 (27) 415 (27) Yes 1290 (60) 1362 (62) 1134 (63) 1186 (64) 962 (67) 1005 (66) Maternal ethnicity 2 Pepel 605 (28) 606 (28) 548 (31) 545 (29) 469 (33) 465 (31) Other 1458 (68) 1536 (70) 1219 (68) 1300 (70) 958(67) 1046 (69) Electricity 2 No electricity 1379 (64) 1416 (64) 1171 (66) 1219 (66) 925 (64) 992 (65) Electricity available 686 (32) 726 (33) 600 (34) 624 (34) 505(35) 518 (34) Enrolment in rainy season 1 1047 (49) 1107 (50) 862 (48) 955 (52) 714 (50) 785 (52) Enrolment at National Hospital 1 1220 (56) 1266 (58) 1000 (56) 1064 (57) 815 (56) 883 (58) Registered VAS given in campaign before follow-up visit 820 (57) 861 (57) Hospitalized before follow-up visit 2 Yes 99 (7) 114 (8) No 1337 (93) 1400 (92) Median age at follow up in months (10–90%) 15 (12–24) 15 (12–25) Mean MUAC at follow-up visit in mm (SD) 148 (13) 148 (12) 1 Variables with two levels (e.g. included at national hospital/elsewhere) are presented by one of the levels if there is full information on all participants. 2 Numbers do not add up due to a few having missing information. BODY.RESULTS.PRIMARY ANALYSIS: THE EFFECT OF NEONATAL VAS ON MORTALITY AFTER FU-VAS: After FU-VAS, mortality was significantly lower for children who had received neonatal VAS (group A) than for children who had received placebo (group B) (MRR: 0.54 (0.31–0.94)) (Table 2, Figure 2). This was due to a marked effect in girls (MRR: 0.37 (0.16–0.89)), whereas there was no statistical significant effect of neonatal VAS versus placebo on survival in boys after FU-VAS: MRR: 0.73 (0.35–1.51). 10.1371/journal.pone.0023265.g002Figure 2Cumulative mortality in the second and third year of life.Cumulative mortality depending on whether the children had received neonatal vitamin A (50,000 IU) or placebo and vitamin A (100,000 IU) at follow-up after 1 year of age (FU-VAS). Graphs by sex. 10.1371/journal.pone.0023265.t002 Table 2 The effect of neonatal vitamin A/placebo on mortality in the 1 st 1 and 2 nd /3 rd 2 year of life depending on whether the children had received neonatal vitamin A (50,000 IU) or placebo and vitamin A (100,000 IU) at follow-up after 1 year of age. Age group Neonatal VAS Mortality per 1000 PYRS (deaths/PYRS 3 ) Neonatal Placebo Mortality per 1000 PYRS (deaths/PYRS 3 ) Mortality Rate Ratio (VAS/placebo) Ratio of effect of neonatal VAS after FU-VAS vs. effect of neonatal VAS after No FU-VAS P for homogeneity of effect of neonatal VAS in the strata FU-VAS and No FU-VAS All children 0–11 months 48.7 (91/1870) 44.9 (88/1960) 1.08 (0.80–1.45) 12–35 months FU-VAS 5 8.9 (19/2145) 16.5 (37/2245) 0.54 (0.31–0.94) No FU-VAS 6 26.6 (27/1016) 24.0 (25/1043) 1.10 (0.64–1.90) 0.48 (0.22–1.05) 0.07 Boys 0–11 months 43.8 (41/936) 49.9 (50/1001) 0.87 (0.57–1.31) 12–35 months FU-VAS 4 , 5 11.3 (12/1062) 15.7 (18/1147) 0.73 (0.35–1.51) No FU-VAS 4 , 6 14.1 (7/496) 24.6 (13/527) 0.57 (0.23–1.42) 1.28 (0.40–4.15) 0.68 Girls 0–11 months 53.5 (50/934) 39.6 (38/959) 1.35 (0.89–2.06) 12–35 months FU-VAS 4 , 5 6.5 (7/1084) 17.3 (19/1098) 0.37 (0.16–0.89) No FU-VAS 4 , 6 38.5 (20/520) 23.3 (12/515) 1.67 (0.81–3.42) 0.22 (0.07–0.69) 0.009 Stratified by sex. 1 Minor deviations from the previously reported estimate (11) as risk time was not censored at time of vitamin A supplementation in a campaign between 6 and 11 months. 2 Four deaths due to accidents not included. 3 PYRS = Person-years (of observation). 4 The effect of neonatal VAS differed for boys and girls depending on whether they have received FU-VAS or not; p for interaction between sex, neonatal VAS and FU-VAS: 0.035. 5 Groups A and B. 6 Groups C and D. BODY.RESULTS.SECONDARY ANALYSES: COMPARING THE EFFECT OF NEONATAL VAS VERSUS PLACEBO AFTER FU-VAS WITH THE EFFECT OF NEONATAL VAS VERSUS PLACEBO BEFORE 12 MONTHS OF AGE AND THE EFFECT OF NEONATAL VAS VERSUS PLACEBO IN CHILDREN WHO HAD NOT RECEIVED FU-VAS: The effect of neonatal VAS versus placebo during the first year of life, prior to FU-VAS, was different from the effect after FU-VAS for girls but not for boys (Table 2). Neonatal VAS versus placebo had no significant effect on mortality in the second and third year of life in children who had received no FU-VAS (group C versus group D). In girls who had received no FU-VAS the MRR was 1.67 (0.81–3.41) and in boys it was 0.57 (0.23–1.42) similar to the estimates in the first year of life. Hence, the effect of neonatal VAS versus placebo was significantly different in girls who had received FU-VAS (group A versus group B) and girls who had not yet received FU-VAS (group C versus group D) (ratio of effects: 0.22 (0.07–0.69), p = 0.01 for homogeneity) (Table 2). In contrast, the effect of neonatal VAS versus placebo did not differ for boys (ratio of effects: 1.28 (0.40–4.15)) (Table 2). Thus the priming effect during the second and third year of life was only seen among girls and was statistically significant from the effect among boys (p = 0.04 for interaction between sex, neonatal VAS and FU-VAS). Fifty-seven percent of the children who received FU-VAS had received VAS in a campaign between enrolment at birth and the follow-up-visit. The participation rate was the same for boys in the VAS (58%) and the placebo (58%) group and girls in the VAS (55%) and the placebo (55%) group (p for same rate of participation = 0.43) (Table 3). After FU-VAS, children who had also received campaign VAS tended to benefit more from having received neonatal VAS compared with children who had not received VAS in a campaign. This was due to a differential effect in girls; girls who had received both campaign VAS and FU-VAS had a strong beneficial effect of neonatal VAS (MRR = 0.15 (0.03–0.67)) whereas girls who had not received campaign VAS prior to FU-VAS had no significant effect of neonatal VAS (MRR = 0.87 (0.27–2.86)) (test for interaction, p = 0.07). There was no difference for boys. 10.1371/journal.pone.0023265.t003 Table 3 The effect of neonatal vitamin A versus placebo on mortality following 100,000 IU of vitamin A given in the 2nd/3rd year of life stratified by vitamin A received in campaign before FU-VAS 1 . Potential effect modifier Neonatal VAS Mortality per 1000 PYRS (deaths/PYRS 2 ) Neonatal Placebo Mortality per 1000 PYRS (deaths/PYRS 2 ) Mortality Rate Ratio (Neonatal VAS/placebo) P for homogeneity of effect of neonatal VAS in the strata Campaign VAS and No Campaign VAS Vitamin A received in Campaign before FU-VAS All  Campaign VAS 6.8 (8/1181) 17.9 (22/1228) 0.38 (0.17–0.85)  No Campaign VAS 11.4 (11/965) 14.8 (15/1016) 0.78 (0.36–1.69) 0.21 Boys  Campaign VAS 10.0 (6/598) 13.7 (9/658) 0.74 (0.26–2.08)  No Campaign VAS 12.9 (6/463) 18.4 (9/489) 0.71 (0.25–2.00) 0.97 Girls  Campaign VAS 3.4 (2/582) 22.8 (13/570) 0.15 (0.03–0.67)  No Campaign VAS 10.0 (5/502) 11.4 (6/528) 0.87 (0.27–2.86) 0.07 1 Four deaths due to accidents not included. 2 PYRS = Person-years (of observation). BODY.RESULTS.SUBGROUP ANALYSES: The effect of neonatal VAS versus placebo on survival after FU-VAS did not depend on timing of follow-up visit (data not shown), maternal education (p for same effect in children who received neonatal VAS versus placebo = 0.40/p = 0.97 for boys/girls), place of enrolment (health centre vs. hospital: p = 0.76/p = 0.22) or mid upper arm circumference (MUAC) (lowest quartile vs. other: p = 0.64/p = 0.63). BODY.RESULTS.OVERALL EFFECT OF NEONATAL VAS: Since the children were followed to 3 years of age, we measured the long-term effect of neonatal VAS versus placebo between birth and 3 years of age regardless of subsequent doses of VAS. The effect tended to be beneficial for boys, the MRR being 0.79 (0.56–1.10). In spite of the beneficial effect of boosting with FU-VAS for girls, the overall effect of neonatal VAS tended to be slightly negative for girls, the MRR being 1.14 (0.82–1.58). In the combined analysis for boys and girls, the MRR was 0.95 (0.76–1.20). BODY.DISCUSSION.MAIN OBSERVATIONS: Long-term follow-up in our randomized trial of neonatal VAS versus placebo showed that children who received VAS after 12 months of age (FU-VAS) had significantly lower mortality if they had received neonatal VAS and not placebo, due to a beneficial effect in girls. The beneficial effect of neonatal VAS may have been particularly strong for girls who received several doses of subsequent VAS (campaign VAS, FU-VAS). This is in strong contrast to the lack of effect or maybe even negative effect of neonatal VAS versus placebo on infant mortality [11], an effect which continued in the second and third year of life in girls who did not receive FU-VAS. Neonatal VAS had no strong effect on survival in boys who received FU-VAS. Taken together these data suggest that priming with neonatal VAS was important, but only among girls. BODY.DISCUSSION.STRENGTHS AND WEAKNESSES: Ideally children should have been randomized to VAS or placebo at birth and at 12 months to get groups A, B, C and D. This was not the case as the study was not planned. All non-randomized comparisons, i.e. comparisons between groups A and B on one hand and groups C and D on the other hand, should be interpreted with caution. However, it should be noted that the main comparison of group A versus group B as well as the comparison of group C versus group D, represent comparisons of subgroups within randomized groups and as discussed below there is no indication that the generation of these subgroups introduced bias. As hypothesized group A had lower mortality than group B, but only for girls. It could be speculated that the negative effect of neonatal VAS for infant girls [11] left only the healthier girls likely to survive in the second and third year of life. However, there was no difference in MUAC or previous hospitalizations between VAS and placebo girls at follow-up. Importantly, we also examined the mortality of recipients of neonatal VAS and placebo during the second and third year of life among those who had not yet received FU-VAS (groups C and D). Since the effects were totally different, the lower mortality of group A versus group B was not due to neonatal VAS per se. Though groups C and D were the result of selection bias (not being home to receive FU-VAS) rather than initial randomization, it seems unlikely that the comparison of C versus D, and the contrast with the comparison of A versus B has been seriously biased. C versus D essentially continued the effect of neonatal VAS versus placebo on infant mortality for both boys and girls. Therefore the lower mortality of A compared with B among girls is likely to be a priming effect of neonatal VAS. This priming effect may be strongest after several doses of VAS since the beneficial effect was most pronounced in girls who had also received campaign VAS before receiving FU-VAS (Table 3). BODY.DISCUSSION.CONSISTENCY WITH OTHER STUDIES: The effect of repeated dosing on mortality has not been studied thoroughly. However, the first trials of VAS may suggest a similar positive effect after subsequent doses of VAS. In Ghana, the mortality ratio after the first dose of VAS versus placebo [9] was 0.99 (0.78–1.67) but declined to 0.68 (0.55–0.82) after subsequent doses. In Nepal, the mortality ratio was 0.76 (0.50–1.15) in the first 4 months of the study and 0.68 (0.45–1.00) and 0.67 (0.45–0.99) in the subsequent rounds [8]. No study has examined possible differential effect for boys and girls in detail. In India, two supplements separated by six months were given to children aged 1–5 years. The study reports no statistically significant difference between the vitamin A and placebo group after 0, 1 or 2 doses of VAS/placebo, but it appears that the female/male mortality ratio changed depending on the number of doses [7]. The pattern is compatible with a particular beneficial effect of the subsequent doses of VAS in girls as observed in the present trial. A more beneficial effect for girls than boys was also seen in a study of weekly low-dose supplements from India [17]. These studies seem to support a priming effect in other settings as well. BODY.DISCUSSION.POTENTIAL BIOLOGICAL MECHANISMS: We can only speculate about possible mechanisms, but the explanation may lie in priming of the naïve immune system. Binding of retinoic acid, the active metabolite of retinol, to its receptors has been shown to alter interactions with proteins that induce epigenetic changes [18]. Hence, a high dose of vitamin A in early life may cause epigenetic alterations, which could lead to immediate effects on the immune system, but also prime the response to subsequent high doses of VAS. Also, vitamin A has been shown to have an up-regulating effect on the dendritic cells in the gut, enhancing their capacity to generate retinoic acid from vitamin A via positive feed-back loop [19]. Hence, if the enzymatic apparatus is stimulated by high doses of VAS in early life, this may alter the processing of subsequent doses of VAS. We have no explanation for the observed sex-differences, but sex-differential imprinting effects of neonatal VAS have been shown in rats; VAS affected later hormone content in immune cells (monocytes-macrophages-granulocytes and T-lymphocytes) in a way that differs for males and females [20]. BODY.DISCUSSION.IMPLICATIONS AND CONCLUSIONS: Long-term follow-up in this randomized trial suggests that neonatal VAS compared with placebo primed the response to subsequent VAS in girls. If this is true, the dosing regime may influence the effect of VAS on child mortality. It should be noted that for girls neonatal VAS had a negative effect on survival until 3 years of age if no FU-VAS was given. Though we observed a beneficial effect of re-exposure to VAS in the second and third year of life this was not enough to reverse the trend towards a negative effect seen in infancy. First and foremost, it is essential to study if a beneficial effect of priming in girls can be achieved without a negative effect of the first dose; for example, is there beneficial priming when the first dose is given after 6 months of age as is currently recommended? Our studies and the trials documenting a particularly beneficial effect of the second dose of VAS were designed to study the overall effect of VAS on mortality rather than a priming effect. However, the consistent pattern suggests that there are reasons to conduct additional randomized trials to determine the optimal interval between doses and the optimal timing of the first dose.

TITLE: Early biomarkers of brain injury and cerebral hypo- and hyperoxia in the SafeBoosC II trial ABSTRACT.BACKGROUND: The randomized clinical trial, SafeBoosC II, examined the effect of monitoring of cerebral oxygenation by near-infrared spectroscopy combined with a guideline on treatment when cerebral oxygenation was out of the target range. Data on cerebral oxygenation was collected in both the intervention and the control group. The primary outcome was the reduction in the burden of cerebral hypo- and hyperoxia between the two groups. In this study we describe the associations between the burden of cerebral hypo- and hyperoxia, regardless of allocation to intervention or control group, and the biomarkers of brain injury from birth till term equivalent age that was collected as secondary and explorative outcomes in the SafeBoosC II trial. ABSTRACT.METHODS: Cerebral oxygenation was continuously monitored during the first 72h of life in 166 extremely preterm infants. Cranial ultrasound was performed at day 1,4,7,14, and 35 and at term. Electroencephalogram (EEG) was recorded at 64h. Blood-samples taken at 6 and 64 hours were analysed for the brain injury biomarkers; S100beta, brain-fatty-acid-binding-protein, and neuroketal. All analyses were conducted post hoc. ABSTRACT.RESULTS: Significantly more infants with a cerebral burden of hypoxia within the 4th quartile versus infants within quartile 1–3 were diagnosed with severe intracranial haemorrhage (11/39 versus 11/117, p = 0.003), had low burst rate on EEG (12/28 versus 21/103, p = 0.015), or died (14/41 versus 18/123, p = 0.006), whereas none of these events were significantly associated with cerebral hyperoxia. The blood biomarkers were not significantly associated with the burden of cerebral hypo- or hyperoxia. ABSTRACT.CONCLUSIONS: The explorative analysis showed that early burden of cerebral hypoxia, but not hyperoxia was significantly associated with low brain electrical activity and severe intracranial haemorrhage while none of the three blood biomarkers were associated with the burden of either cerebral hypo- or hyperoxia. BODY.INTRODUCTION: Extremely preterm infants have an immature cardiorespiratory system and cerebral autoregulation can be impaired, especially during the first days of life [1,2]. This makes the developing brain of the preterm infant susceptible to fluctuations in the cerebral blood flow (CBF) [3] and may cause episodes of cerebral hypo- and hyperoxia. Near-infrared spectroscopy (NIRS) is a non-invasive method for estimating tissue oxygenation. NIRS measures the ratio of the concentrations of oxygenated haemoglobin to total haemoglobin on an absolute scale with a range of 0% to 100% [4]. Changes in cerebral NIRS-values are correlated to CBF [5]. A number of biomarkers–characteristics that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention [6]—may be present if the brain is suffering from hypo- or hyperoxia. Severe intraventricular haemorrhage (IVH grade III) and periventricular haemorrhagic infarction (PVHI) mainly develop within the first 3 days of life [7], during the period of transition from intra- to extra-uterine life when the brain is especially vulnerable. Low cerebral oxygenation, as estimated by NIRS, during this transition has been associated with higher grades of intraventricular haemorrhage and lower 2-year developmental quotients [8–10]. In addition high values of NIRS in animal studies are associated with brain injury [11], as confirmed in human asphyxiated term new-borns [12]. Cerebral NIRS monitoring is currently used in some neonatal intensive care units as part of the standard of care for extremely preterm infants and infants with hypoxic-ischemic encephalopathy. Yet it remains to be determined if monitoring cerebral oxygenation combined with clinical interventions when cerebral oxygenation levels are out of range actually prevents cerebral injury, improves neurological outcome, and/or increases the survival of the extremely preterm infants [13]. The phase II randomized clinical trial, SafeBoosC II, demonstrated that it is possible to reduce the burden of cerebral hypoxia during the first 72 hours of life [14]. The SafeBoosC II study was, however, not powered to detect differences in clinical outcomes [15]. In the present post hoc analysis, we use the SafeBoosC II data to explore the association between the burden of cerebral hypo- and hyperoxia regardless of trial allocation to intervention or control group, and the secondary and explorative outcomes of the trial (all being potential biomarkers of brain injury or death): namely serial cranial ultrasound (cUS), electroencephalographic (EEG) measures, and blood molecular markers. BODY.PATIENTS AND METHODS.INFANT CHARACTERISTICS AND STUDY DESIGN: SafeBoosC II is a multicentre randomised clinical feasibility trial [15]. A total of 166 extremely preterm infants were included in the SafeBoosC II study before 3 hours of age: 86 infants were randomised to the experimental group (cerebral NIRS monitoring in combination with an evidence based intervention guideline [16] for NIRS values out of range (55–85%)) and 80 infants to the control group (blinded collection of NIRS values combined with treatment as usual). The inclusion criteria were infants born more than 12 weeks before term (gestational age <27 weeks and six days) with a decision to provide full life support and the possibility to start cerebral NIRS monitoring within three hours after birth. Written informed consent from the parents was mandatory before inclusion and randomisation. The randomisation was web based and handled by The Copenhagen Trial Unit. The generated allocation sequence of 1:1 with block sizes 4 and 6 in random order concealed for the investigators. The intervention period was 72 hours. The 2y-followup of the infants is on going. The infants were recruited from 8 European countries each represented by one neonatal intensive care unit (June 2012 to December 2013). The trial is registered at ClinicalTrial.gov, NCT01590316, the protocol is available in full at http://www.safeboosc.eu. BODY.PATIENTS AND METHODS.THE BURDEN OF CEREBRAL HYPO- AND HYPEROXIA: The primary outcome of the SafeBoosC II trial was the burden of hypo- and hyperoxia. This was calculated as the time spent below or above the target limits multiplied by the mean deviation from the lower (55%) or the upper limit (85%) during the first 72 hours of life, expressed in percentage hours (%hours). The burden was computed from un-edited NIRS-values and extrapolated to 72 hours, without knowledge of any other outcomes of the trial [14]. BODY.PATIENTS AND METHODS.CRANIAL ULTRASOUND: On day 1 (anytime during the first 24 hours of life), 4 (± 1), 7 (± 1), 14 (± 1), and 35 (± 1) and at term equivalent age (week 38 to 44) standardized cUS (6 coronal and 5 sagittal images through the anterior fontanel and one through the mastoid window) was performed. The images were anonymised and uploaded to a central server. The images were centrally analysed by two experts (CH and MB) using the software program OsiriX version 6.0 (Pixmeo, Geneva, Switzerland). The process of the central scoring is described in detail elsewhere [17]. IVH grade III, PVHI, post haemorrhagic ventricular dilatation, porencephalic cysts, cystic periventricular leukomalacia, cerebral atrophy at term, stroke and cerebellar haemorrhage at one or more of the scans were classified as severe brain injury, and thereby as severe adverse outcome. BODY.PATIENTS AND METHODS.ELECTROENCEPHALOGRAM: EEG was analysed in 133 infants, the median age at EEG-recording was 65 hours postnatal and the median time of recording was 2 hours [18]. Electrodes were placed at P3 and P4 position according to the international 10-20-system. Needle, disc or hydrogel electrodes were used according to local practice. The electrode impedance was less than 20kΩ during the recording. If the child was treated with morphine, other opioids, or sedative medications this was documented. All EEG analysis was performed in Matlab version R2014b using custom build programmes (MathWorks, Natick, Massachusetts, USA), without knowledge of the medical history of the infant. The analysis of the EEGs is described in detail elsewhere [18]. The raw EEG was band pass filtered (0.5–30 Hz) using a zero phase filter and converted into range-EEG (rEEG) [19]. Artefacts in the rEEG were independently visually identified by two of the authors (GG and AMP) blinded to the clinical history of the infant. The EEG analysis was conducted on the remaining artefact-free data. Burst rate was calculated as the number of bursts per minute. A burst was defined as nested (high frequency) oscillations within large slow-wave depolarisations using an extraction algorithm based on the co-occurrence of a slow (0.5–2 Hz) wave and higher (8–22 Hz) frequency oscillation, as described by Hartley et al. [20]. Consecutive events occurring within 0.5 seconds of one another were counted as one and events of duration less than 4/22 of a second were discounted [20]. Burst rates were significantly affected (decreased burst rate) by the use of morphine and EEG-recordings with an online filtration at 2–15 Hz (decreased burst rate) [18]. Therefore the burst rates were adjusted for these variables. Adjusted burst rate within the 1st quartile was considered an adverse outcome. Spectral-analyses were conducted using Matlab routines (Neurospec 2.0, Neurospec.org). The EEG data was segmented into epochs of 2 seconds with an overlap of 50% (1 second). After fast Fourier transformation the 95% spectral edge frequency (SEF95) for each infant was defined as the frequency between 0.5 and 30 Hz, below which 95% of the power was present. SEF95 was significantly affected by the EEG sampling frequency (high sampling frequency higher SEF95) [18]. Therefore the SEF95 was adjusted for this. Adjusted SEF95 within the 1st quartile was considered an adverse outcome. BODY.PATIENTS AND METHODS.BLOOD BIOMARKERS: At the age of 6 and 64 hours (±1 h) 1 ml of blood was drawn from 123 infants with an indwelling arterial or venous line [18]. After inclusion of the last patient, the samples were shipped and analysed centrally (HaemoScan, Groningen, The Netherlands) without knowledge of the medical history of the infant. S100beta (50 μl) was assessed by ELISA: clone 1B2 monoclonal antibody (Abnova, Taipei, Taiwan) and biotinylated clone 8B10 (Hytest, Turku, Fi). Intra-assay variance is 4.6% and the lower level of quantification (LLOQ) is 39 pg/ml. Brain fatty acid binding protein (BFABP) (50 μl) was determined by ELISA with BFABP polyclonal capture antibodies and monoclonal detection antibody (HaemoScan). Intra-assay variance is 6.4% and the LLOQ is 150 pg/ml. Neuroketal (60 μl) was performed by competitive enzyme immunoassay (HaemoScan). Intra-assay variance is 10% and the LLOQ is 4.1 pg/ml. The laboratory analyses are described in detail elsewhere [18]. An increase in the biomarker concentration from 6 to 64 hours was considered as a marker of potential cerebral injury during the intervention period, therefore and adverse outcome was defined as an increase in the absolute value of the biomarker concentration from 6 hours to 64 hours within the 4th quartile. BODY.STATISTICS: The median and inter quartile range of burden of cerebral hypo- and hyperoxia was determined. The infants were divided in groups according to a burden within or below the 4th quartile of the burden of cerebral hypo- and hyperoxia, respectively. The infant characteristics were compared between the burden-groups using the chi-square test or independent t-test as appropriate. Odds ratios with 95% confidence intervals were determined for adverse outcomes for infants within the 4th quartile of the burden of hypo- or hyperoxia versus infants in quartile 1–3. Thereafter univariate correlation analysis was conducted to determine the patient characteristics associated with the composite outcome of severe brain injury or death. Finally, a multiple logistic regression was used with the composite outcome as dependent variable, centre, gestational age above or below 26 weeks (the stratification variables used in the randomized SafeBoosC II trial), and intervention as forced entry independent variables, and the patient characteristics which had significant correlation to the composite outcome as independent variables in a backward stepwise elimination procedure (P-out 0.1). The between twins inter-cluster correlation for the burden of hypo-and hyperoxia was low (0.02) [14]. Therefore, there was no need to exclude one infant from each twin-cluster thus data from all infants were included in the analysis. None of the analyses reported here were specified in the SafeBoosC-II study protocol. The dichotomisation between one quartile and the three other quartiles was copied from previous work on biomarkers in our group [21], and chosen before any correlations were calculated. The statistics was performed using IBM SPSS Statistics for Windows Version 20.0 (Armonk, New York, USA). BODY.ETHICS: The SafeBoosC phase II trial was approved by each hospital's research ethics committee (Hopital Femme Mere Enfants, Lyon, France; Rigshospitalet, Copenhagen, Denmark; La Paz University Hospital, Madrid, Spain; Cork University Maternity Hospital, Cork, Ireland; Wilhelmina Children's Hospital, Utrecht, The Netherlands; Medical University of Graz, Graz, Austria; Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; and Rosie Hospital, Cambridge University Hospitals, United Kingdom), and where required (Austria, Denmark, and France) by the competent authority responsible for medical devices. Written informed parental consent was mandatory before inclusion in the trial. The trial was conducted in compliance with the guidelines of the Declaration of Helsinki in its latest form and the International Conference on Harmonisation good clinical practice guidelines. BODY.RESULTS: One-hundred-and-sixty-six infants were included in the SafeBoosC II trial, Fig 1. Cerebral oximetry data was missing for two infants due to technical issues (n = 1) and withdrawal of consent (n = 1). 10.1371/journal.pone.0173440.g001Fig 1Flowchart.Flow of participants through the SafeBoosC II study. The median (min—max) burden of cerebral hypoxia was 30.6%hours (0.7–803.9). The limits for hypoxia quartile 4 were 99.3–803.9%hours and for quartile 1–3; 0.7–99.2%hours. The median (min—max) burden of cerebral hyperoxia was 1.2%hours (0.0–223.0). The limits for quartile 4 were 14.2–223.0%hours and for quartile 1–3; 0.0–14.1%hours. As expected according to the primary result of the SafeBoosC II trial [14], there were significantly fewer infants form the intervention group with a burden of hypoxia in the 4th quartile, than in quartile 1–3, whereas the burden of cerebral hyperoxia within the 4th quartile versus quartile 1–3, was unaffected by allocation in the SafeBoosC trial, Table 1. Male sex was associated with less cerebral hypoxia and more cerebral hyperoxia. Furthermore, gestational age was positively associated with cerebral hyperoxia whereas the other baseline characteristics were not associated with either cerebral hypo- or hyperoxia. 10.1371/journal.pone.0173440.t001 Table 1 Baseline characteristics and treatment during the first 72 hours of life according to burden of cerebral hypo- or hyperoxia split in the three lowest quartiles and the highest quartile. Burden of hypoxia Burden of hyperoxia Quartile 1 to 3 Quartile 4 Quartile 1 to 3 Quartile 4 n = 123 n = 41 P-value n = 123 n = 41 P-value Baseline characteristics   Gestational age (week), mean (SD) 26.4 (1.2) 26.5 (1.5) 0.78 26.3 (1.3) 27.7 (1–0) 0.35 Gestational age below 26 weeks 38 (31) 14 (34) 0.7 42 (34) 10 (24) 0.025 Birth weight (gram), mean (SD) 847 (211) 875 (207) 0.47 849 (208) 872 (216) 0.54 Male sex 65 (53) 13 (32) 0.02 53 (43) 25 (61) 0.047 Twins 21 (17) 12 (29) 0.09 23 (19) 10 (24) 0.43 Antenatal steroids full course 82 (67) 31 (78) 0.2 88 (72) 25 (61) 0.18 Prolonged rupture of membranes 40 (33) 17 (36) 0.21 42 (34) 15 (38) 0.72 Maternal chorioamnionitis 6 (5) 5 (13) 0.1 9 (8) 2 (5) 0.59 APGAR-score <5 points at 5 minutes 21 (17) 8 (20) 0.69 20 (16) 9 (23) 0.38 Umbilical arterial pH, mean (SD) 7.32 (0.1) 7.31 (0.1) 0.62 7.32 (0.1) 7.29 (0.1) 0.13 SafeBoosC II intervention group 74 (60) 12 (29) 0.001 67 (54) 19 (46) 0.37 Treatment during the first 72h of life Surfactant treatment 90 (73) 35 (85) 0.11 90 (73) 35 (85) 0.11 Mechanical ventilation 79 (64) 30 (73) 0.29 80 (65) 29 (70) 0.5 Patent ductus arteriosus treatment 17 (14) 4 (10) 0.52 15 (12) 6 (15) 0.71 Use of vasopressors/inotropes 22 (18) 16 (40) 0.004 29 (24) 9 (22) 0.79 Any red blood cell transfusion 31 (26) 18 (45) 0.025 41 (35) 8 (20) 0.07 Corticosteroids 4 (3) 4 (10) 0.1 6 (5) 2 (5) 0.99 Values are numbers (percentages) unless stated otherwise. P-values have not been corrected for multiple comparisons. Serial ultrasound scans of 155 infants were available for evaluation by central reading. Twenty-seven (27/155) had severe brain injury, of which 22 had IVH grade III or PVHI. The remaining five infants had stroke (n = 2), cerebral atrophy (n = 2), or cerebellar haemorrhage (n = 1). Cerebral hypoxia was significantly associated with severe brain injury and death, Table 2. There was no significant association between cerebral hyperoxia and severe brain injury and death, Table 3. Cerebral hypoxia was significantly associated with early occurrence (day 1 to 4) of severe intracranial haemorrhages whereas there was no association between cerebral hypoxia and later occurrence (day 5 to day 14) of severe intracranial haemorrhages, Table 2. Cerebral hypoxia was also significantly associated with low EEG burst rates at 64 hours of age whereas there was no association between cerebral hypoxia and SEF95 at 64 hours of age. Of the 14 infants with early severe IVH ten (10) infants had EEG measurement and of these five (5) infants had burst rates within the 1st quartile, for the infants without severe haemorrhages the number of recorded EEGs was 115 and hereof 26 had burst rates within the 1st quartile (chi-square analysis between groups p = 0.12). The three plasma-biomarker-levels were not associated with cerebral hypoxia, Table 2. Cerebral hyperoxia was not associated with EEG burst rate, SEF95 or plasma biomarkers, Table 3. The odds ratios (ORs) and 95% confidence interval for adverse outcomes are illustrated in Figs 2 and 3. 10.1371/journal.pone.0173440.g002Fig 2Risk for adverse outcomes for infants with a burden of cerebral hypoxia within or below the 4th quartile.Odds ratio (OR) and 95% confidence interval. 10.1371/journal.pone.0173440.g003Fig 3Risk for adverse outcomes for infants with a burden of cerebral hyperoxia within or below the 4th quartile.Odds ratio (OR) and 95% confidence interval. 10.1371/journal.pone.0173440.t002 Table 2 Distributions of early and late adverse outcomes of cranial ultrasound, EEG variables, blood biomarkers, term diagnoses, and death according to the burden of cerebral hypoxia within or below the 4 th quartile. Burden of hypoxia Quartile 1–3 Quartile 4   n = 123 n = 41 P-value * P-value OR (95% CI) Cranial ultrasound IVH 3–4 day 1–4 6/117 8/38 0.003 0.045 4.93 (1.59–15.30) IVH 3–4 day 5–14 5/111 3/31 0.63 >0.95 2.27 (0.51–10.09) IVH 3–4 at any time 11/117 11/39 0.003 0.045 3.77 (1.49–9.63) Severe brain injury—any time 14/116 13/39 0.002 0.03 3.64 (1.53–8.69) EEG variables time 64h Burst-rate in the 1 st quartile 21/103 12/28 0.015 0.23 2.93 (1.20–7.12) Spectral edge frequency in the 1 st quartile 15/99 5/27 0.67 >0.95 1.27 (0.42–3.88) Plasma biomarkers difference between 6 and 64h S100beta increase in 4 th quartile 25/92 6/30 0.43 >0.95 0.67 (0.25–1.83) BFABP increase in 4 th quartile 24/89 8/31 0.90 >0.95 0.94 (0.37–2.39) Neuroketal increase in 4 th quartile 22/92 9/30 0.51 >0.95 1.36 (0.55–3.41) Other major adverse outcome at term age Necrotising enterocolitis 13/123 6/41 0.48 >0.95 1.45 (0.51–4.10) Retinopathy of prematurity 18/123 4/41 0.43 >0.95 0.63 (0.20–1.98) Bronchopulmonary dysplasia 52/104 14/27 0.86 >0.95 1.08 (0.46–2.51) Death 18/123 14/41 0.006 0.09 3.03 (1.34–6.84) Combined adverse outcome Death or severe brain injury 28/117 20/41 0.003 0.045 3.03 (1.44–6.38) Death or IVH 3 or 4 25/118 19/41 0.002 0.03 3.21 (1.51–6.84) Values are given as numbers of events / numbers of infants investigated for event. * P-value after Bonferoni correction for multiple comparisons. 10.1371/journal.pone.0173440.t003 Table 3 Distributions of early and late adverse outcomes of cranial ultrasound, EEG variables, blood biomarkers, term diagnoses, and death according to the burden of cerebral hyperoxia within or below the 4 th quartile. Burden of hyperoxia Quartile 1–3 Quartile 4   n = 123 n = 41 P-value * P-value OR (95% CI) Cranial ultrasound IVH 3–4 day 1–4 11/116 3/39 0.74 >0.95 0.80 (0.21–3.01) IVH 3–4 day 5–14 6/100 2/36 0.98 >0.95 0.92 (0.18–4.79) IVH 3–4 at any time 17/117 5/39 0.79 >0.95 0.87 (0.30–3.52) Severe brain injury—any time 21/117 6/38 0.76 >0.95 0.86 (0.32–2.31) EEG variables time 64h Burst-rate in the 1 st quartile 27/95 3/36 0.17 >0.95 0.50 (0.19–1.35) Spectral edge frequency in the 1 st quartile 13/91 7/35 0.43 >0.95 1.50 (0.54–4.14) Plasma biomarkers difference between 6 and 64h S100beta increase in 4 th quartile 24/93 7/29 0.86 >0.95 0.92 (0.35–2.41) BFABP increase in 4 th quartile 23/92 9/28 0.45 >0.95 1.42 (0.57–3.58) Neuroketal increase in 4 th quartile 25/93 6/29 0.5 >0.95 0.71 (0.26–1.95) Other major adverse outcome at term age Necrotising enterocolitis 17/123 2/41 0.12 >0.95 0.32 (0,07–1,45) Retinopathy of prematurity 20/123 2/41 0.064 >0.95 0.26 (0.06–1.18) Bronchopulmonary dysplasia 55/97 11/34 0.015 0.23 0.37 (0.16–0.83) Death 26/123 6/41 0.36 >0.95 0.64 (0.24–1.68) Combined adverse outcome Death or severe brain injury 38/120 10/38 0.53 >0.95 0.77 (0.34–1.75) Death or IVH 3 or 4 35/120 9/39 0.46 >0.95 0.73 (0.31–1.69) Values are given as numbers of events / numbers of infants investigated for event. * P-value after Bonferoni correction for multiple comparisons. Correlation analyses of the following variables showed a significant correlation (p<0.05) between gestational age, birth weight, clinical chorioamnionitis, surfactant, mechanical ventilation, use of vasopressors, blood transfusions, and the burden of cerebral hypoxia within the 4th quartile on one hand and the composite outcome of severe brain injury or death on the other. These variables were included in the multiple logistic regression model with backward stepwise elimination. As described in the methods, centre, gestational age below 26 weeks, and intervention were forced-entry variables in the model. The following variables remained statistically significant: intervention (OR (95% CI) 0.29 (0.12–0.69; p = 0.003,)), gestational age below 26 weeks (3.33 (1.38–8.06; p = 0.007)), use of vasopressors (3.26 (1.26–8.44; p = 0.014)), and blood transfusion (2.97 (1.22–7.23; p = 0.016)). There was no centre-effect. BODY.DISCUSSION: This post hoc analysis shows that early burden of cerebral hypoxia, but not hyperoxia, is associated with a reduction of brain electrical activity, severe brain injury (especially early IVH grade III and PVHI), and death. There were no significant associations between the burden of cerebral hypo- and hyperoxia and the three blood biomarkers. Multiple logistic regressions showed significant associations between intervention, gestational age below 26 weeks, use of vasopressors, and blood transfusion on one hand and the composite adverse outcome severe brain injury or death on the other. Low cerebral oxygenation has previously been associated with IVH and lower developmental quotients at 2-year follow up [8–10]. In a study by Noori et al, involving 22 extremely preterm infants, lower levels of cerebral oxygenation, cardiac output, and cerebral hypoperfusion were found prior to the development of IVH II and PVHI [8]. In term piglet models, however, while low cerebral oxygenation when accompanied by low cerebral blood flow caused permanent brain damage, prolonged cerebral hypoxia alone seemed to be of less importance [22,23]. Piglets had to be exposed to cerebral hypoxia as low as 30–35% for several hours before significant histological damage appeared [23]. In the present analysis the burden of cerebral hypoxia was associated with decreased EEG burst rates. This is in agreement with a previous demonstration of an association between low cerebral blood flow and suppressed EEG in preterm infants [24]. EEG burst rate is decreased in infants with severe IVH [25,26] and suppressed EEG is correlated to adverse developmental outcome [27,28]. In the present analysis we did not find significantly more infants with low burst rates and early severe IVH grade III or PVHI. The three blood biomarkers measured in the SafeBoosC II trial were not significantly associated with either cerebral hypo- or hyperoxia. We did not register whether the samples were drawn from arterial or venous line, therefore we are unable to determine whether the concentrations of the blood biomarkers differ between arterial and venous blood. S100beta is a calcium binding protein present in high concentration in Schwann cells and astrocytes [29] and is released to the systemic circulation after cerebral damage. S100beta is an established marker of brain injury in adult trauma patients [30] and the levels of S100beta in blood and urine from term and preterm infants has been associated with the severity of both hypoxic ischemia and IVH [31–33]. And in a study including 64 term and late preterm infants the levels of S100beta was significantly negatively correlated to NIRS-values [34]. However a study by Rogers et al [35] including 130 extremely preterm infants reporting no associations between the S100beta levels and severe intracranial haemorrhages. S100Beta has also been investigated as a potential biomarker of brain injury in paediatric patients undergoing cardiac surgery–the infants with unfavourable neurological outcome 12 months after surgery had significantly lower cerebral NIRS values during surgery, whereas the levels of S100Beta did not differ [36]. We evaluated the effects of the accumulated burden of hypoxia during the first 72 hours of life rather than correlating specific NIRS-values to the levels of S100beta, and therefore we cannot determine whether the S100beta levels measured in the SafeBoosC II study were correlated to the NIRS-levels. We found no differences between the levels of S100beta between the 4th quartile of cerebral hypoxia where the proportion of infants with severe IVH was highest vesus the three lowest quartiles of cerebral hypoxia. The conflicting results of the associations between S100beta and severe intracranial haemorrhages make the clinical value of this biomarker questionable and further research is needed to evaluate whether S100beta is suitable as an early biomarker of brain injury in extremely preterm infants. BFABP, a brain-specific marker, is rapidly released from astrocytes as a response to ischemia, mechanical and oxidative brain damage [37]. BFABP was chosen as a potential biomarker for brain injury in the SafeBoosC II trial, as it is known to rapidly increase in hypoxic-ischaemic stroke patients, and the high levels persist for several days after an event [37]. BFABP might be a more sensitive marker of minor traumatic brain injury than S100beta [38] and is elevated in patients with various neurodegenerative diseases[39]. A study involving 57 patients undergoing cardiac surgery reports that neither S100beta nor BFABP had any clear prognostic value for postoperative cognitive dysfunction [40]. We found no association between BFABP and the burden of cerebral hypo- or hyperoxia–this might be because of the different nature of the acute and severe local hypoxic-ischemia occurring in stroke patients compared to the global relative cerebral hypoxia measured in the SafeBoosC II trial. Our study ads information to the current litterateur of BFABP and brain injury–suggesting that there is no associations between the burden of cerebral hypoxia at 72h of age and the increase of the levels of BFABP from 6 to 64h of life. Further research on the associations between BFABP and brain injury in extremely preterm infants is needed. Neuroketals are compounds produced by free radical induced peroxidation of docosahexenoic acid, have been associated with white matter damage on MRI in preterm infants [41] and are solely present in the brain [42]. Neuroketal is mainly released as a reaction to cerebral oxidative stress, and may due to their reactivity be involved in formation of protein cross-links, which is a common feature in neurodegenerative diseases, where neuroketal is known to be increased [43]. Neuroketal has been reported elevated in cerebrospinal fluid during the first 3 weeks of life infants with white matter damage on MRI performed at term equivalent age [41]. We expected that the levels of neuroketal would be increased in the infants with cerebral hyperoxia in the 4th quartile, but that was not the case. This might be explained by the early measure of neuroketal– 6 to 64 hours of life and the fact that the level of cerebral hyperoxia in the SafeBoosC phase II trial was low [14]. In addition neuroketal is still only an experimental biomarker of cerebral injury and might not be a good marker of early acute brain injury in extremely preterm infants. Whether neuroketal in the future can serve as a marker of later white matter injury in extremely preterm infants remains to be determined. BODY.DISCUSSION.LIMITATIONS: The explorative post hoc analysis of the SafeBoosC II trial data presents the association between the early cerebral oxygenation and short-term adverse cerebral outcomes and death in extremely preterm infants. However, this study has some limitations. Most importantly, cranial ultrasound was conducted at pre-specified days of life, but the exact timing of IVH grade III and PVHI and other severe brain damages is not available and therefore we cannot know if cerebral hypoxia preceded IVH or vice versa. However, one small study in very preterm infants recently identified a significantly lower regional cerebral oxygenation during the early transition in infants who later developed IVH versus the infants who did not [44]. Similarly, EEG was only recorded once. While the timing at 64 hours of age was expected to assess the potential effects of the accumulated burden over the intervention period, finer details of preceding or concurrent cerebral hypoxia could not be extracted, such as the relative significance of longer periods of moderate hypoxia versus peaks of severe hypoxia. We did not collect data on continuous blood pressure, arterial oxygen saturation, or CBF, which may have contributed to both low cerebral NIRS values and severe brain damage [8,22,23]. The analyses are based on a dataset from our randomised clinical trial; therefore, the multiple logistic regression models were adjusted for the stratification variables (centre and gestational age below 26 weeks), as well as the randomisation indicator (experimental vs. control group). As the burden of hypoxia was reduced by 50% in the experimental group compared with the control group [14], it was therefore not surprising that adjusting for the randomisation indicator did reduce the statistical significance of the burden of hypoxia. We do not think that this changes the main conclusion of the study. On the other hand, the statistical significance of the randomisation indicator means that the risk of severe brain injury or death in the experimental group of the SafeBoosC-II trial was less in the experimental group than in the control group when adjusted for a number of other factors. However, neither the composite outcome nor the particular statistical analyses were specified in the trial protocol. This post-hoc finding must therefore be interpreted conservatively. Finally, the 2-year follow up of the infants included in the SafeBoosC II trial will further explore if cerebral hypoxia and/or the intervention is related to patient-relevant outcomes such as psychomotor deficit. Larger randomised clinical trials investigating possible patient-relevant benefits of continuous NIRS monitoring in extremely preterm infants is needed before the method is implemented as standard care in this population. BODY.DISCUSSION.CONCLUSIONS: Our analysis is currently the largest dataset published on cerebral oxygenation in extremely preterm infants providing data on short-term neurological outcomes. The results support the previous findings of associations between low cerebral oxygenation in extremely preterm infants during the first days of life and EEG suppression, severe intracranial haemorrhage, and death. However, our analyses are exploratory and we were unable to determine which came first: cerebral hypoxia or severe intracranial haemorrhage. We did not find any evidence that cerebral hyperoxia is associated with either death or severe brain damage. We found no associations between the burden of cerebral hypo- or hyperoxia and the three blood biomarkers (S100beta, BFABP, and Neuroketal). BODY.SUPPORTING INFORMATION: S1 AppendixThe SafeBoosC Phase II protocol.(PDF)Click here for additional data file. S2 AppendixConsort 2010 Checklist.(DOC)Click here for additional data file.

TITLE: Automatic Tube Compensation versus Pressure Support Ventilation and Extubation Outcome in Children: A Randomized Controlled Study ABSTRACT: Background. Automatic tube compensation (ATC) has been developed to overcome the imposed work of breathing due to artificial airways during spontaneous breathing trials (SBTs). Objectives. This study aimed to assess extubation outcome after an SBT (spontaneous breathing trial) with ATC compared with pressure support ventilation (PSV) and to determine the risk factors for extubation failure. Methods. Patients ready for extubation were randomly assigned to two-hour spontaneous breathing trial with either ATC or pressure support ventilation. Results. In the ATC group (n = 17), 11 (65%) patients passed the SBT with subsequent extubation failure (9%). While in PSV group (n = 19), 10 (53%) patients passed the SBT with subsequent extubation failure (10%). This represented a positive predictive value for ATC of 91% and PSV of 90% (P = 0.52). Five (83%) of the patients who failed the SBT in ATC group were reintubated. This represented a higher negative predictive value for ATC of 83% than for PSV which was 56%. None of the assessed risk factors were independently associated with extubation failure including failed trial. Conclusion. ATC was equivalent to PSV in predicting patients with successful extubation. A trial failure in ATC group is associated with but does not definitely predict extubation failure. BODY.1. INTRODUCTION: Prolonged and unnecessary delay in tracheal extubation result in increased complication rates for patients receiving mechanical ventilation including airway trauma, chronic lung disease, ventilator associated pneumonia, and increased hospital costs [1]. On the other hand-premature discontinuation carries a set of problems involving difficulty in establishing airways and compromised blood gas exchange [2]. Different methods, including clinical trials and calculated indices, have been developed to evaluate patients on mechanical ventilation and predict the optimum time to make the weaning decision [3]. These methods include, tolerances of spontaneous breathing trials (SBTs), counting the respiratory rate, observation of work of breathing, and many other calculated indices such as the oxygenation index, measurement of the tidal volume and dynamic compliance, and the commonly used rapid shallow breathing index. However, some of these indices may be misleading, cost-effective, and requiring highly sophisticated equipments [4]. Recently, a tolerance of a spontaneous breathing trial while the patient receives varying levels of ventilatory support including continuous positive airway pressure (CPAP), low-level pressure support ventilation (PSV), or very recently automatic tube compensation (ATC) is a new clinical test that has been considered an evidence-based strategy to predict successful weaning from assisted ventilation [5]. The level of support may be relevant to whether the breathing trial is tolerated, because it has been argued that, for some patients, weaning failure may be attributable to the respiratory load imposed by the endotracheal tube [6]. Automatic tube compensation (ATC) is a recent weaning mode of mechanical ventilation that has been developed to overcome the imposed work of breathing due to artificial airways. It delivers the exact amount of resistive load of the endotracheal tube for the flow measured at time, without affecting the patient's breathing pattern [7]. It potentially simulates spontaneous breathing without endotracheal tube, so it has been designated as "electronic extubation". This mode of ventilation thus seems ideally suited for use during the weaning period [8]. PSV has been also widely used in the performance of a spontaneous breathing trial and has been shown to compensate for the additional work of breathing imposed by the endotracheal tube [9]. However, some studies have shown that compared with PSV, ATC was more effective in overcoming the work of breathing necessary to overcome endotracheal resistance and resulted in more significant predictive values for successful weaning and extubation [10]. Pediatric and adult studies evaluating the efficacy of SBTs have not systematically extubated patients who failed the breathing trial. Therefore, the ability of a failed SBT to predict the need for ventilator support was not formally assessed, except in a previous study by Chavez et al. in pediatric population [11]. In our study, we assessed the sensitivity of both ATC and PSV in predicting extubation outcome, and we also assessed the outcome of failed SBTs. BODY.2. PATIENTS AND METHOD.2.1. POPULATION AND SETTING: The study was conducted in Cairo University Pediatric Hospital; pediatric intensive care unit, 9-bed capacity. The study period extended from May 2011 to February 2012. In this prospective, randomized, controlled study, we screened 47 mechanically ventilated patients. BODY.2. PATIENTS AND METHOD.2.1. POPULATION AND SETTING.2.1.1. INCLUSION CRITERIA: Patients were eligible for enrolment in the study if they met the following criteria judged by the intensive care doctors: (1) required mechanical ventilation for more than 24 hours; (2) fulfilling weaning criteria, which was defined in our PICU as follows: low ventilator rate [6–8] or less; fraction of inspired oxygen (FIO2) ≤ 40; level of positive end expiratory pressure (PEEP) [3, 4]; Improvement of the cause of respiratory failure; oxygenation index (OI) ([mean air way pressure × FIO2]/PaO2) < 5; the need for bronchial suction is ≤2 for the last 8 hours; with stable vital, neurological and metabolic status. BODY.2. PATIENTS AND METHOD.2.1. POPULATION AND SETTING.2.1.2. EXCLUSION CRITERIA: Included the following: (1) duration of mechanical ventilation before enrolment is 24 hours or less; (2) patients receiving high doses of sedations or vasoactive drugs; (3) patients with disturbed conscious level despite improvement of lung pathology; (4) patients who developed laryngeal edema after extubation; (5) patients with pulse oxygen saturation < 90%, PH < 7.3 and PaCO2 > 50 mm Hg during the trial. Full history and data analysis including sex, age, weight, cause of mechanical ventilation, period of mechanical ventilation, length of stay in the PICU, bronchodilators, pediatric risk of mortality score (PRISM III) [12] on day 1 admission, blood gases, pretrial oxygenation index, and ventilator setting parameters including: level of positive end expiratory pressure (PEEP); pretrial ventilator rate; pretrial FIO2 were recorded. BODY.2. PATIENTS AND METHOD.2.2. STUDY PROTOCOL AND WEANING PROCEDURES: Patients screened were randomly assigned in a blinded fashion with the use of opaque, sealed envelopes, to undergo two-hours spontaneous breathing trial with ATC (patients breathed through the ventilator circuit using continuous positive airway pressure of 5 cmH2O, FIO2 less than 0.5 with the addition of ATC 100%; the ATC group) or PSV (patients breathed through the ventilator circuit using flow triggering and continuous positive airway pressure of 5 cmH2O, FIO2 less than 0.5, PS adjusted for endotracheal tube size (ETT) (ETT size 3.0–3.5 = PS of 10 cmH2O; ETT size 4.0–4.5 = PS of 8 cmH2O; ETT size ≥ 5.0 = PS of 6 cmH2O) [13]; the PSV group). These parameters were maintained throughout the trial. The spontaneous breathing trial was performed using the Puritan-Bennett 840 ventilator which compensate automatically for air leaks (we do not use cuffed ETTs) and was newly introduced in our PICU; previously used ventilator was Newport E150. The SBT was conducted by a respiratory therapist and nurse in the absence of the attending or other intensive care staff. Physical signs including heart rate; respiratory rate; mean arterial blood pressure; spontaneous expiratory tidal volume (mL/kg/sec); evidence of work of breathing; increased frequency of suction; pulse oxygen saturation blood gases were recorded during the trial. Features of poor tolerance and weaning failure included respiratory rate outside the acceptable range for their age [14]; increase in heart rate of more than 20% with respect to baseline on mechanical ventilation; increase or decrease in mean blood pressure of more than 20% of baseline; signs of increased respiratory work (i.e., retractions, use of accessory respiratory muscles, paradoxical breathing); pulse oxygen saturation < 90% and/or PH < 7.3 and/ or PCO2 > 50 mm Hg. When one of these findings occurred during the trial, the respiratory therapist terminated the trial to the previous ventilator settings. For patients with metabolic or respiratory acidosis during the trial, weaning criteria was revised with ICU doctors, and these patients were excluded from the study. Patients who passed the 2 hr trial were extubated by the respiratory therapist, and patients who failed the trial and were included in our study were recorded to be extubated within the next 24 hr by the intensive care doctors who were blinded to the results of the trial and the study aims. Weaning was considered successful if reintubation was not required within 48 hr of extubation (successful extubation group). Failure to wean was defined as reintubation within 48 hr of extubation (extubation failure group). BODY.2. PATIENTS AND METHOD.2.3. ETHICS: Informed consent was obtained from at least one parent or legal guardian for each patient before enrollment. The study design conformed to the Revised Helsinki Declaration of Bioethics [15] and was approved by the Scientific Ethics Committee of Department of Pediatrics, Faculty of Medicine of Cairo University. BODY.2. PATIENTS AND METHOD.2.4. STATISTICAL ANALYSIS: Data was analyzed using Statistical Package for Special Science software computer program version 16.0 (SPSS Inc., Chicago, Illinois, USA). Continuous variables were expressed as median, minimum, and maximum. Categorical variables were expressed as number (n), percent (%) and were compared using the chi-square test or Fisher's exact test, as indicated. Continuous variables were compared using Mann-Whitney test and Kruskal-Wallis ANOVA, as indicated. Multivariate regression analysis was used to test the association between multiple quantitative and qualitative independent variables with the dependent variable. P value less than or equal 0.05 was considered statistically significant. BODY.3. RESULTS: Out of 47 screened patients, only 36 were enrolled in the study, 6 patients were excluded as they were reintubated due to laryngeal edema, and in the other 5 trial was terminated due to metabolic or respiratory acidosis during the trial. Of the 36 patients enrolled in the study, 17 were weaned from mechanical ventilation on ATC (ATC group) and 19 were weaned on PSV (PSV group). Admission diagnosis of patients enrolled was as follows: lower respiratory tract infections (n = 9); interstitial lung disease (n = 3); postoperative (n = 5); status epileptics (n = 2); encephalitis (n = 2); after arrest (n = 1); history of poison intake (n = 2); autoimmune diseases (n = 2); Guillain-Barré syndrome (n = 2); endocrinal disorder (n = 1); septic shock (n = 3); gastroenteritis and shock (n = 2); myocarditis (n = 1); immunodeficiency (n = 1). Baseline characteristics are shown in Table 1. Respiratory and hemodynamic characteristics during the spontaneous breathing trial are shown in Table 2. There were no significant differences between the ATC and PSV groups in any of the compared items. The course and outcome of the study population are summarized in Figure 1. In the ATC group 11 of 17 (65%) passed the SBT compared with 10 of 19 (53%) in the PSV group, but this difference was not statistically significant, (P = 0.69). Out of 36 patients 12 (33.3%) were reintubated within the first 48 hours after extubation. Failed extubation was equal in both groups (P = 0.9). Causes of reintubation were hypoxemia (n = 5), disturbed conscious level (n = 3), and new sepsis and pneumonia (n = 4). There were no significant difference in the causes of reintubation between the two groups (P = 0.46). Mean length of stay in ATC group was 20.9 ± 14.4, while in PSV group was 21.35 ± 11.8 with no significant difference (P = 0.78). Our study showed that successful completion of the SBT had a greater predictive value for successful extubation than the predictive value of failed trial for extubation failure; successful completion of the SBT on ATC showed a 91%, sensitivity with a positive predictive value of 91% and specificity was 83% with a negative predictive value of 83% and accuracy 88%. While successful completion of the SBT on PS showed a 69% sensitivity for predicting successful extubation with a positive predictive value of 90% and specificity was 83% with a negative predictive value of 56% and accuracy 74% with no significant difference between the two groups (P = 0.52). Table 3 shows the univariate analyses comparing patients who were successfully extubated and patients who failed extubation and reintubated there was a statistically significant association between reintubation and the following risk factors during the breathing trial: tachypnea, tachycardia, increased work of breathing, and failing ATC trial. By stepwise multivariate logistic regression analysis of significant risk factors among all the study group (No = 36), none of the estimated significant risk factors were independently associated with extubation failure. BODY.4. DISCUSSION: In the current study, we compared extubation outcome using 100% ATC versus PSV during a spontaneous breathing trial for two hours. The baseline characteristics in both groups were similar. We found that 9 of 19 (47%) patients in the PSV group failed the breathing trial compared to only 6 of 17 (35%) patients in the ATC group. This observed difference of 12% between both groups however did not reach statistical significance (P = 0.69). The positive predictive values were nearly similar in both groups; 9 of 10 patients in the PSV (90%) passed the SBT and successfully extubated (maintained extubation for >48 hours) compared with 10 of 11 (91%) patients in the ATC group. These findings are similar to a study conducted by Cohen and his colleagues [6] comparing ATC with PSV during a spontaneous breathing trial in adults. They found that patients who failed SBT in PSV group were higher than those in ATC group; however the difference was not significant. They found that PSV had a higher PPV predicting patients with successful extubation than ATC (PSV, 85% versus 80%); however, the difference was not significant (P = 0.87). In another study comparing ATC with PSV, the author found no significant difference in extubation outcome between the two groups; however, he did find that half of the patients who failed a breathing trial with PSV tolerated a subsequent trial with ATC and were successfully extubated [7]. These findings can be explained by the fact that ATC may provide more complete support. This is supported by a previous study in which the authors assessed the accuracy of the compensation provided by PSV and ATC relative to the endotracheal tube-related pressure dissipation. They found that the difference between the theoretical pressure required to overcome the endotracheal tube resistive properties and the actual pressure delivered by the ventilator was lower and negligible when ATC was applied during a spontaneous breathing trial when compared with PSV [10]. In our study, we found that rate of patients who failed the SBT in ATC group and reintubated was higher than that of PSV group. The negative predictive values for successful extubation were 83% for ATC versus 55% for PSV; however, this difference was not statistically significant (P = 0.52). This may be attributed to small number of cases in each group in addition to the nearly similar accuracy of the two groups (74% for PSV and 88% for ATC). The reason for this low negative predictive value in PSV group may be secondary to mechanical factors such as endotracheal tube discomfort, increased work of breathing caused by the augmented resistive force imposed by a small endotracheal tube, and inability to overcome this load due to in part the use of a relative low-level pressure in the PSV group used in this study. Pressure support level for each ETT size used in this study defined by Randolph et al. [13] may need to be reevaluated in another study with a large population size. These findings were similar to a study conducted in pediatric patients by Chavez et al. [11], who found that failed SBTs, using a flow-inflating bag to provide a low constant pressure of 5 mmHg, did not accurately predict extubation failure; however, this study did not compare the outcome of different pressure supported breathing trials. The reintubation rate for the whole studied patient was 33.3% which was higher than recent suggestions, where extubation rate of 15 to 29% implies an acceptable balance between performing premature extubation and unsuccessfully prolonged mechanical ventilation [16–18]. The finding that a significant number of patients who successfully passed the SBTs and extubated subsequently required reintubation merits further considerations. Patients with higher severity scores of illness at admission and those with higher incidence of nosocomial infections are at increased risk of extubation failure. Our relatively high incidence of reintubation rate could be in part due to the high incidence of nosocomial pneumonia and the development of new sepsis among patients under study, a finding which is consistent with other similar studies [19–22]. A potential limitation to our study was the small number of the study population which was related to the low turnover rate due to prolonged length of stay of our patients; median length of stay in the PICU in PSV group was 19 days (4–52 days), and median length of stay in the PICU in ATC group was 20 days (4–50 days). Finally, the use of either ATC or PSV for prediction of extubation outcome in general ICU populations was, reliable and did not require special monitoring or complex data collections. Both have accepted positive and negative predictive values for successful extubation.

TITLE: Comparison of Functional Outcomes of Tibial Plateau Fractures Treated with Nonlocking and Locking Plate Fixations: A Nonrandomized Clinical Trial ABSTRACT: Fixation of tibial plateau fractures with plate has been widely used. This prospective study was planned to compare locking plate fixation of tibial plateau fracture with nonlocking methods in terms of their functional outcomes. The subjects of the study were selected from consecutive patients suffering from tibial plateau fractures referred to Kashani Hospital in Isfahan, Iran, between 2012 and 2013 and were candidate for surgery. The final included patients were assigned to two groups, those who were treated with locking plate (n = 20) and those who were treated with nonlocking plates (N = 21). The mean duration of follow-up was 13.4 months (ranging between 10 and 17 months). The mean of knee scores was significantly higher in locking plate group than in nonlocking plate group at the follow-up time (80.20 ± 10.21 versus 72.52 ± 14.75, P = 0.039). Also, the mean VAS pain severity score was significantly lower in locking plate group compared with nonlocking plate group (4.45 ± 2.50 versus 6.00 ± 2.59, P = 0.046). This study confirmed superiority of the locking plate method over nonlocking plate method with regard to knee score as well as VAS pain score. BODY.1. INTRODUCTION: Proximal tibial fractures are important fractures involving one of the main weight-bearing joints whose serious injury results in movement and ability dysfunctions [1]. The main goal of the treatment of these fractures is to maintain the normal function of the knee joint, improve the joint instability, prevent lower limb malalignment and deformity, and prevent knee osteoarthritis [2–4]. Applying effective preventive approaches can lead to maintained articular surface, uniform plateau level, and a near normal range of knee joint motion. The main defined criteria in functional assessment of patients with proximal plateau fractures of tibia include knee range of motion, time to achieve union, patient's ability to walk, patient's ability to climb stairs, the pain severity while walking as well as at rest, muscle strength, severity of instability in the knee, and loss of active extension of the knee [5–8]. Unfortunately, there is no gold standard treatment approach for various types of tibial plateau fractures; therefore, different methods have been employed depending on the type of fracture. Tibial plateau fixation with plate especially nonlocking plates has been widely used in recent years [9, 10]. One of the commonly applied types of these plates is the locking compression plate that provides greater stability in these unstable fractures and creates a stronger connection between the articular components [11]. Stabilizing the joint surface by this method, due to its less invasiveness, not only seems to cause a significant decrease in side effects but also reduces the length of hospital stay and hospital costs [12, 13]. The present prospective follow-up study was to compare locking plate fixation of tibial plateau fractures and nonlocking methods in terms of long-term articular functional outcomes. BODY.2. METHODS: This prospective nonrandomized clinical trial with a parallel design and allocation ratio of 1 : 1 was conducted to compare success rate and outcomes of locking and nonlocking plate fixation of tibial plateau fractures. The subjects were selected from consecutive patients suffering from tibial plateau fractures and who were referred to Kashani Hospital in Isfahan, Iran, between January 2011 and January 2013 and were candidates for surgery (n = 110). All the participants had unilateral closed fractures without open wound or neuromuscular complications. Exclusion criteria in this study were patients younger than 19 years or older than 60 years, history of diabetes mellitus, Ipsilateral fractures of the femur and tibia, pathological fractures, and open fracture (Figure 1). Forty one patients were thus selected for the study. On admission, written consent was acquired from all patients. The selected patients were assigned to two groups; group one was treated with locking plate fixation (n = 20) and group two with nonlocking plate fixation (N = 21). The patients' assignment to one of the two interventional groups was based on discretion of the physician and also the special condition of the patients for selecting one of the two methods. All patients underwent control radiography after surgery and reduction in all study subjects were near anatomic. Within the treatment schedule and due to intraoperative bone defect, bone graft was used for 12 patients (3 in locking plate and 9 in nonlocking plate). Also, 2 patients received double plate (nonlocking plate type) in both medial and lateral sides with concomitant bone graft. Postoperatively, all patients received supportive care and were discharged, if possible. The splint was used for 3 to 7 days and knee motion started within 2 weeks. The patients were followed up for 10 to 17 months. The status of the tibial plateau fractures was classified according to the Schatzker and AO classifications systems using available preoperative X-rays or CT scans. The two parameters of step-off and widening of articular surface were assessed before and 6 months after surgery. Postoperative X-rays were assessed according to Freedman and Johnson's description for determining the alignment of the tibial plateau, both on coronal plane (medial proximal tibial angle or MPTA) and sagittal plane (posterior proximal tibial angle or PPTA) [14]. Functional outcomes in ten months were assessed using the Knee Society knee score that considers a clinical score (including pain, stability, range of motion, flexion contracture, extension lag, and malalignment) and a functional score (that assesses walking distance and stair climbing) [15]. This score was graded as excellent (80 to 100), good (70 to 79), fair (60 to 69), and poor (below 60) [16]. The severity of pain was assessed using a visual analogue scale (VAS). For all the patients, the knee range of motion (ROM) was measured using a large goniometer with 25 cm movable arms, marked with one-degree increments. The sample size was determined at 95% confidence interval (CI), 10% precision, and was based on mean ROM indices between the locking plate and nonlocking plate groups in the previous studies and was found to consist of at least 20 patients in each study groups. In this regard, the study power was also determined at 85.5%. For statistical analysis, categorized variables were compared using chi-square or Fisher exact tests as required. Continuous variables were compared using independent t-test and Mann-Whitney U test. We used multivariate logistic regression analysis to investigate the potential confounding effects of patients' characteristics and clinical data on the difference in outcomes of surgical protocols. The significance of the results was determined at P values of 0.05 or less. All the statistical analyses were performed using SPSS version 19.0 (SPSS Inc., Chicago, IL, USA). BODY.3. RESULTS: In this study 20 patients were treated with locking plates and 21 were treated with nonlocking plates. The two intervention groups were similar for mean age (34.50 ± 7.92 years, ranging from 20 to 54 years, versus 34.55 ± 10.34 years, ranging from 21 to 55 years, P = 0.986) and male gender distribution (85.0% versus 90.5%, respectively, P = 0.663). The average duration of follow-up was a total of 13.4 months (ranging from 10 to 17 months). No difference was observed in the duration of follow-up between the locking plate and nonlocking plate groups (10.61 ± 3.05 months versus 12.95 ± 2.63 months, P = 0.158). As shown in Table 1, no differences were noticed in the types and patterns of fractures between nonlocking and locking plate fixation methods based on both Schatzker and AO classifications. In this regard, the most common type of fractures based on the Schatzker system was tibial plateau fracture with spilt depression or type II (52.4% in nonlocking and 45.0% in locking methods) followed by tibial plateau fracture with diaphyseal discontinuity or type VI (19.0% in nonlocking and 25.0% in locking methods) with no significant difference between the two groups (P = 0.556). No difference was observed in the mean age of the patients with different types of fractures based on the two classification systems, and the most common mechanisms for tibial plateau fracture were motor accident (MA) (41.5%) and motor to car accident (MTCA) (22.0%), respectively. Regarding postoperative complications, the overall complication rate was 17.1% (7 out of 41 patients). One case had a deep wound breakdown that received double-plate and was managed and improved by reoperation, irrigation, wound debridement, and antibiotic therapy. Superficial infection was revealed in 6 patients (1 in the locking plate and 5 in the nonlocking plate groups) who were all successfully treated with antibiotic therapy. There were no cases of compartment syndrome, deep vein thrombosis, or nonunion. No significant differences were found between the two groups in the severity of the tibial plateau fractures according to the Schatzker (P = 0.556) or AO (P = 0.257) classifications systems (Figures 2 and 3). At the final follow-up, a total of 11 cases had step-off of more than 2 mm (4 cases in the locking plate and 7 cases in the nonlocking plate groups). Also, widening of articular surface of more than 2 mm was found in 5 cases (1 case in the locking plate and 4 cases in the nonlocking plate groups). As presented in Table 2, no significant association was noted between postoperative step-off or widening statuses with the type of fracture according to the two fracture classification systems. The MPTA showed a range of 75–100° (mean = 88.73 ± 5.26°). Eleven cases (26.8%) were outside the normal range (82–92°) and were considered malaligned, of whom 11 (9 nonlocking and 2 locking) were with a valgus angulation (i.e., >92°) and one with a varus angulation (i.e., <82°). In the sagittal plane the PPTA demonstrated a range of 1° to 22° (mean = 7.87 ± 5.14°). Seventeen cases (41.5%) were outside the normal range (4–14°) and considered malaligned in the sagittal plane. There were 3 cases with a PPTA > 14° and 14 cases with a PPTA < 4°. Four of these cases were treated with locking plates and 13 cases treated with nonlocking plates. The average range of motion (ROM) was 118.95 ± 17.13° (range of 70–150°). Among the 41 subjects, 9 (22.0%) had a ROM of less than or equal to 100. No difference was observed in the mean of ROM between the locking plate and nonlocking plate groups (122.35 ± 12.93° versus 115.71 ± 20.14°, P = 0.219). The mean of knee scores was significantly higher in the locking plate group as compared with the nonlocking plate group at the most recent follow-up (80.20 ± 10.21 versus 72.52 ± 14.75, P = 0.039). In this regard, excellent grade of knee score was shown in 65.0% in the locking plate group and in 33.3% in the nonlocking plate group (Figure 4). Also, the mean of functional score was also higher in the locking plate group than in the nonlocking plate group at the follow-up (77.26 ± 9.95 versus 69.55 ± 10.22, P = 0.026). In this regard, excellent grade of functional score was also found in 70.0% in the locking plate group and in 38.1% in the nonlocking plate group. The mean VAS pain score was significantly lower in the locking plate group compared with the nonlocking plate group (4.45 ± 2.50 versus 6.00 ± 2.59, P = 0.046). Postoperative VAS scores positively correlated with both preoperative and postoperative step-off and widening statuses as is shown in Table 3. In this regard, both knee scores and ROM adversely correlated with postoperative step-off and widening status. The inverse relationship between postoperative step-off and widening and the level of knee scores is also shown in Table 4. BODY.4. DISCUSSION: The advent and development of locking compression plate method has effectively improved tibial plateau fractures as common complex fractures. Few published studies have compared long-term results of this procedure especially with respect to functional outcomes with nonlocking methods. The present study showed superiority of the locking plate method to the nonlocking plate with regard to knee score and AS pain scores. On the other hand, to improve the knee functional score and minimize postoperative pain, considering locking plate is preferable to nonlocking plate. Although this superiority seems to be preserved in terms of other parameters including ROM index, function score, bone-graft need, and even postoperative complications, because of employing small sample size in our study, no statistically significant differences were witnessed. Other studies with similar sample sizes obtained similar success in the use of locking plates. Stannard and colleagues [17] collected data from a series of 39 tibial plateau fractures, all of which healed without further intervention and with only two superficial wound infections as complications. Cole et al. [18] reported the results of 42 consecutive tibial plateau fractures with 91% union, 9% malalignment, and 4% infection rate. A study by Ricci et al. [19] reported that 37 of 38 fractures healed without complication and with acceptable alignment employing this treatment method. Most recently, a study by Lee et al. [20] also demonstrated results with no loss of reduction, nonunion, and infection developing in only two of 35 fractures. To compare the outcomes of open reduction and locked plating versus fine-wire external fixation of 58 consecutive bicondylar tibial plateau fractures, Krupp et al. [21] found that locked plating was associated with a decreased time to union, decreased incidence of articular malunion, decreased knee stiffness, and decreased overall complications. Moreover, Biggi [22] discovered that internal fixation with locking plates, following the principles of minimally invasive percutaneous osteosynthesis, could provide satisfactory fracture reduction with good results regarding the midterm clinical outcome. Contrarily, Littlechild et al. [23] noticed that no definite advantage was associated with the use of locked plating for high-energy tibial plateau fractures. Usually a locking plate is inserted providing a raft of proximal locking screws to support the articular surface, buttressing the lateral wall of the proximal tibia and extending distally to adequately support the construct. Because the main goal of the treatment is to restore the congruence of the articular surface supporting the tibial plateau cartilage which is usually depressed, to fix the fracture with a stable device, and finally to allow early rehabilitation, the locking plate method can result in achieving main therapeutic goals with appropriate long-term surgical outcome. A positive point of the study was adjusting age distribution as a potential confounder in the use of locking and nonlocking plates. However, the main limitations of the study included nonrandomized trial, small sample size, and thus partially low study vigor. To confirm advantages of this therapeutic option, further studies should be conducted and its outcome should be compared with other traditional therapeutic methods. BODY.5. CONCLUSION: The present study showed superiority of locking plate to nonlocking plate methods with regard to knee scores and VAS pain scores indicating more improvement in knee functional score and minimizing postoperative pain using the locking plate method.

TITLE: Randomized Controlled Study on Safety and Feasibility of Transfusion Trigger Score of Emergency Operations ABSTRACT.BACKGROUND:: Due to the floating of the guideline, there is no evidence-based evaluation index on when to start the blood transfusion for patients with hemoglobin (Hb) level between 7 and 10 g/dl. As a result, the trigger point of blood transfusion may be different in the emergency use of the existing transfusion guidelines. The present study was designed to evaluate whether the scheme can be safely and effectively used for emergency patients, so as to be supported by multicenter and large sample data in the future. ABSTRACT.METHODS:: From June 2013 to June 2014, patients were randomly divided into the experimental group (Peri-operative Transfusion Trigger Score of Emergency [POTTS-E] group) and the control group (control group). The between-group differences in the patients' demography and baseline information, mortality and blood transfusion-related complications, heart rate, resting arterial pressure, body temperature, and Hb values were compared. The consistency of red blood cell (RBC) transfusion standards of the two groups of patients with the current blood transfusion guideline, namely the compliance of the guidelines, utilization rate, and per-capita consumption of autologous RBC were analyzed. ABSTRACT.RESULTS:: During the study period, a total of 72 patients were recorded, and 65 of them met the inclusion criteria, which included 33 males and 32 females with a mean age of (34.8 ± 14.6) years. 50 underwent abdomen surgery, 4 underwent chest surgery, 11 underwent arms and legs surgery. There was no statistical difference between the two groups for demography and baseline information. There was also no statistical differences between the two groups in anesthesia time, intraoperative rehydration, staying time in postanesthetic care unit, emergency hospitalization, postoperative 72 h Acute Physiologic Assessment and Chronic Health Evaluation II scores, blood transfusion-related complications and mortality. Only the POTTS-E group on the 1st postoperative day Hb was lower than group control, P < 0.05. POTTS-E group was totally (100%) conformed to the requirements of the transfusion guideline to RBC infusion, which was higher than that of the control group (81.25%), P < 0.01. There were no statistical differences in utilization rates of autologous blood of the two groups; the utilization rates of allogeneic RBC, total allogeneic RBC and total RBC were 48.48%, 51.5%, and 75.7% in POTTS-E group, which were lower than those of the control group (84.3%, 84.3%, and 96.8%) P < 0.05 or P < 0.01. Per capita consumption of intraoperative allogeneic RBC, total allogeneic RBC and total RBC were 0 (0, 3.0), 2.0 (0, 4.0), and 3.1 (0.81, 6.0) in POTTS-E groups were all lower than those of control group (4.0 [2.0, 4.0], 4.0 [2.0, 6.0] and 5.8 [2.7, 8.2]), P < 0.05 or P < 0.001. ABSTRACT.CONCLUSIONS:: Peri-operative Transfusion Trigger Score-E evaluation scheme is used to guide the application of RBC. There are no differences in the recent prognosis of patients with the traditional transfusion guidelines. This scheme is safe; Compared with doctor experience-based subjective assessment, the scoring scheme was closer to patient physiological needs for transfusion and more reasonable; Utilization rate and the per capita consumption of RBC are obviously declined, which has clinical significance and is feasible. Based on the abovementioned three points, POTTS-E scores scheme is safe, reasonable, and practicable and has the value for carrying out multicenter and large sample clinical researches. BODY.I: Clinical transfusion of red blood cell (RBC) can be implemented through relatively standardized guidelines, namely blood transfusion guidelines. However, now, there exists no emergency versions of blood transfusion guidelines according to patients' instant hospitalization state, that is to say, current blood transfusion in emergency still need to be operated based on the ordinary blood transfusion guidelines. Because the above transfusion guidelines are mostly used for critically ill patients or patients with elective surgical procedures, it has not been determined whether the emergency patients should be transfused according to the minimum restrictive blood transfusion (6 or 7 g/dl). As the use of blood and blood components in China can be largely by the supply or shortage of blood, due to the floating of the guideline, there is no bevidence-based evaluation index on when to start the blood transfusion for patients with hemoglobin (Hb) between 7 and 10 g/dl. Patients of elective operation have preoperative preparation for a certain period of time. Except the primary diseases, the patients are close to healthy people, vital signs, and physiological status; Second, surgical process is destructive, namely there are the possibilities of blood loss during the whole operation process until the end of surgery. Compared with elective surgery, damage exists prior to most of the emergency operation, even shock and internal environment disorder caused by preoperative blood loss.[12] The patients' operation is a process of repairing, controlling damage, and saving lives. But patients of emergency surgery are most healthy in advance without previous medical history, so along with the advancement of surgery, the patients' bleeding is reduced until the end of the surgery.[3] As a result, the trigger point of blood transfusion may be different in the emergency use of the existing transfusion guidelines, for the same patient facing different doctors or different patients facing the same doctor. With marking scheme to guide the RBC transfusion at the right time and right amount, this study was designed to evaluate whether the scheme can be safely and effectively used for emergency patients, so as to be supported by multicenter and large sample data in the future. BODY.M.PATIENT SELECTION: This study has been registered in Chinese Clinical Trial Register, and the Registration No. was ChiCTR-TRC-14004135. This study was approved by the ethics committee of Affiliated Hospital of Zunyi Medical College (20121115.001). The subjects and their relatives were informed, and informed consent was signed. All patients were randomly divided into the experimental group (Peri-operative Transfusion Trigger Score of Emergency [POTTS-E] group) and the control group (control group) after screening by inclusion and exclusion criteria. BODY.M.INCLUSION CRITERIA: (1) Preoperative Hb <10 g/dl, or intraoperative Hb may be less than 10 g/dl; (2) Age ≥44 years old and ≤55 years old; (3) Permanent resistant area was <2500 m; (4) American Society of Anesthesiologists (ASA) classification: I–IV level; (5) Emergency surgery. BODY.M.EXCLUSION CRITERIA: (1) History of blood system diseases; (2) Multiple tumors and/or tumor metastasis; (3) Clear history of coronary heart disease preoperatively; (4) Nerve surgery and obstetrics patients; (5) Patients must be given hypervolemic hemodilution; (6) Patients had other test drugs within 3 months before the selected research or participated in other clinical trials. BODY.M.ELIMINATION CRITERIA: (1) Intraoperative uncontrollable and life-threatening bleeding, which needs a large number of blood transfusion; (2) Re-emergency operation is needed during hospitalization after an emergency operation. BODY.M.TREATMENT: Intraoperative monitoring was implemented on electrocardiogram, blood pressure (BP), SpO2, temperature, invasive BP (art), and central venous pressure; preoperative and intraoperative medications were executed according to the current standard daily work in clinical; endotracheal intubation anesthesia was selected and the researchers do not interfere in selection and medication of compound anesthesia. BODY.M.BLOOD TRANSFUSION FOR PERI-OPERATIVE TRANSFUSION TRIGGER SCORE OF EMERGENCY GROUP: Through POTTS-E marking scheme, transfusion requirement of the patients and the amount of RBC transfusion were evaluated based on the current transfusion guidelines issued by the China's Ministry of Health. Patients with Hb of 7–10 g/dl were especially refined for scoring and were guided for blood transfusion. Circulation condition, blood oxygenation situations, oxygen consumption, and compensatory state were respectively evaluated by whether the patients needed adrenaline to maintain normal BP, whether higher oxygen concentration was needed to maintain the oxygenation, whether there was high metabolism as a result of high body temperature, and whether there was high shock index. The scheme referred to the POTTS of Western China Hospital of Sichuan University. The scheme had passed the authorization, and there was no conflict of interest [Table 1]. Table 1 Peri-operative transfusion trigger score of emergency Score Epinephrine ( µ g kg −1 min −1 ) a FiO 2 (%) b Temperature (°C) c d Shock index 0 0 ≤35 <38 <1.5 1 0–0.05 36–49 38–40 1.5–2 2 ≥0.06 ≥50 >40 >2 a The amount of Epinephrine for maintain normal blood pressure; b Fraction of inspiration O 2 to sustain the SpO 2 95% or higher; c Nasopharyngeal temperature. For all the patients of the POTTS-E group, the baseline point was 7, POTTS-E was 0–8 points, and blood transfusion indications were 7 + POTTS-E scores. If the total score was still higher than 7.5, however, RBC infusion was still needed to be continued until the Hb value was equal to or greater than the immediate total score. When the total score was more than 10 and Hb >10 g/dl, RBC infusion was no longer needed. BODY.M.BLOOD TRANSFUSION FOR THE CONTROL GROUP: According to the transfusion guidelines (2000) issued by the China's Ministry of Health, when Hb >10 g/dl, RBC transfusion was not needed; while when Hb <7 g/dl, transfusion should be considered; when Hb between 1–9 g/dl, the RBC transfusion should be considered according to patients' cardiopulmonary compensatory function, body metabolism, and oxygen consumption. Doctor in charge should judge the time and amount of RBC infusion according to the guideline of blood transfusion subjectively. Calculation method of autologous blood: The volume of autologous blood was recorded, and autologous hematocrit (HCT) was examined. The volume that was equivalent to 45% HCT was calculated. And each 200 ml of autologous blood with 45% HCT was recorded as 1 U. BODY.M.POSTOPERATIVE FOLLOW-UP: With the end of the surgery as the starting point, the patients were followed-up in the hospital at 24 h, 72 h after the operation and during emergency hospitalization (discharge, transfer or second selective surgery). The patients were recorded for their mortality, blood transfusion-related complications, and vital signs. POTTS-E group was rated for the requirement of postoperative RBC transfusion. And CON group was based on the doctors' experience to decide whether to give RBC transfusion. Postoperative Hb review results and RBC application of the two groups of patients were recorded; the patients were followed up outside the hospital every 4 weeks in the 3 months postoperatively to record blood transfusion-related complications. BODY.M.MEASUREMENTS: The patients were collected for their age, gender, and body mass index; American Society of Anesthesia (ASA) score, operation area, Acute Physiologic Assessment and Chronic Health Evaluation II (APACHE II) scores and vital signs (temperature, heart rate, breathing, BP and SpO2); operation time, intraoperative rehydration, staying time in postanesthetic care unit (PACU) and postoperative emergency hospitalization; mortality and blood transfusion-related complications; heart rate, resting arterial pressure, body temperature, and Hb values during hospital follow-up; the consistency of RBC transfusion standards of the two groups of patients with the current blood transfusion guideline, namely the compliance of the guidelines; utilization rate and per-capita consumption of autologous RBC, intraoperative allogeneic RBC, postoperative allogeneic RBC, perioperative (intraoperative and postoperative) allogeneic RBC, and overall RBC (perioperative allogeneic RBC + autologous blood). BODY.M.STATISTICAL ANALYSIS: Statistical analysis was performed with SPSS 17.0 software package (SPSS Inc., Chicago, IL, USA). In normal distribution, measurement data were represented as mean ± standard deviation (± s), and t-test of independent sample was used in comparison between the two groups; In nonnormal distribution, the data were represented as M (P25, P75), and Wilcoxon test was used in comparison between the two groups; categorical data were represented as rate and constituent ratio, and Chi-square test or Fisher's exact test were used in comparison between groups, and pearmen linear correlation analysis was adopted for data analysis. P ≤ 0.05 referred as the statistically significant difference. BODY.R: A total of 72 cases were included from June 2013 to June 2014 with 7 cases excluded. The excluded cases include 2 cases whose postoperative RBC infusion was not according to the experiment scheme; 2 cases who were given large amount of blood transfusion due to intraoperative vascular rupture; 3 cases were lost to postoperative follow-up because of transferred to other hospitals immediately after surgery. A total of 65 patients completed the data, where 33 cases were in the POTTS-E group of 33 cases, and 32 cases were in the control group, and no mortality occurred in two groups. BODY.R.BASELINE INFORMATION: Peri-operative Transfusion Trigger Score of Emergency group and control group were compared in age, gender, body mass index, ASA score, and operation area; Heart rate, mean arterial pressure, SpO2, body temperature, and shock index when entering into the operation room; there was no statistical difference between the two groups for Hb values and APACHE II scores after entering into the operation room [Table 2]. Table 2 Demography and baseline information Variables POTTS-E ( n = 33) Control ( n = 32) χ 2 / t P Age (years) 35.00 ± 16.80 34.59 ± 12.29 0.111 0.912 Sex (male, n ) 19 14 0.746 0.388 BMI (kg/m 2 ) 21.46 ± 2.32 21.71 ± 3.47 −0.330 0.743 ASA ( n )  II 9 10 0.124 0.724  III 20 19 0.010 0.919  IV 4 3 0.128 0.721 Surgery area ( n )  Abdomen 27 23 0.905 0.341  Chest 2 2 0 1.000  Arms and legs 4 7 1.099 0.294 Vital signs (entering the operation room)  Temperature (°C) 36.51 ± 0.60 36.31 ± 0.63 1.304 0.197  HR (/min) 110.93 ± 23.26 110.68 ± 21.93 0.045 0.964  RR (/min) 25.18 ± 5.45 24.59 ± 4.81 0.647 0.460  MAP (mmHg) 69.94 ± 16.13 73.92 ± 21.18 −0.853 0.397  SpO 2 (%) 96.48 ± 1.17 96.15 ± 10.5 1.187 0.240  Shock index 1.16 ± 0.12 1.17 ± 0.62 −0.076 0.939 Hb (g/dl) 7.87 ± 2.22 7.69 ± 1.98 0.603 0.736 APACHE II 8.69 ± 4.28 8.09 ± 4.65 0.544 0.588 POTTS-E: Peri-operative transfusion trigger score of emergency; BMI: Body mass index; ASA: American society of anesthesiologists; HR: Heart rate; MBP: Mean blood pressure; RR: Relative risk; SpO 2 : Oxygen saturation; Hb: Hemoglobin; APACHE II: Acute physiology and chronic health evaluation II. BODY.R.TREATMENT AND PROGNOSIS OF THE PATIENTS: There were no statistical differences between the two groups in anesthesia time, intraoperative rehydration, staying time in PACU, emergency hospitalization, postoperative 72 h APACHE II scores, blood transfusion-related complications and mortality [Table 3]. Table 3 Operation information and prognosis Variables POTTS-E ( n = 33) Control ( n = 32) t P Operation time (min) 143.81 ± 91.50 166.21 ± 105.81 −0.914 0.364 Intraoperative rehydration (ml) 2275.75 ± 756.73 2763.1250 ± 1324.19 −1.814 0.072 PACU stays (min) 85.09 ± 51.16 90.12 ± 51.25 −0.368 0.714 Emergency hospital stays (days) 10.57 ± 6.68 9.81 ± 6.25 0.475 0.636 Postoperation 72 h APACHE 2.78 ± 2.26 2.96 ± 4.06 −0.222 0.825 Complications (%) 1 (3.03) 1 (3.12) 0 0.982 POTTS-E: Peri-operative transfusion trigger score of emergency; PACU: Postanesthetic care unit; APACHE: Acute physiology and chronic health evaluation. There were no statistical differences in the comparison of vital signs at the 1st and 3rd postoperative day and at the end of the emergency hospitalization. There were no statistical differences in the comparison of Hb values at the time when entering into the operation room, at the 3rd postoperative day and at the end of the emergency hospitalization. Hb values of the two groups were both higher than the time when entering into the operation room at the 1st and 3rd postoperative day and at discharge [Table 4]. Table 4 The two groups of patients are compared in vital signs and Hb (mean ± SD) Vital signs HR (/min) MAP (mmHg) Temperature 1-day 3 days End of hospitalization 1-day 3 days End of hospitalization 1-day 3 days End of hospitalization POTTS-E ( n = 33) 91.78 ± 19.11 88.06 ± 11.92 85.51 ± 12.62 80.23 ± 14.00 81.16 ± 10.68 83.19 ± 7.18 37.22 ± 0.85 36.82 ± 0.53 36.62 ± 0.32 Control ( n = 32) 89.53 ± 17.36 88.93 ± 17.35 83.46 ± 10.63 81.66 ± 10.12 82.17 ± 11.45 84.05 ± 11.16 37.02 ± 0.71 36.69 ± 0.57 36.65 ± 0.56 Hb values Entering into the operation room (g/dl) 1-day (g/dl) 3-day (g/dl) End of hospitalization (g/dl) POTTS-E ( n = 33) 7.87 ± 2.22* 9.13 ± 1.25 † 9.30 ± 2.30 9.54 ± 1.29 Control ( n = 32) 7.69 ± 1.98* 9.89 ± 1.68 9.67 ± 2.36 9.82 ± 1.55 *Compared with the same group, 1-day, 3 days and end of hospitalization P <0.05; † Compared with postoperative day P <0.05. Hb: Hemoglobin; SD: Standard deviation; MAP: Mean arterial pressure; HR: Heart rate. BODY.R.COMPLIANCE OF BLOOD TRANSFUSION GUIDELINES: There were no patients of POTTS-E group that were not transfused with Hb <7 g/dl or were transfused with Hb >10 g/dl; 5 patients of CON group were not transfused with Hb <7 g/dl and 1 case was still transfused with Hb >10 g/dl; POTTS-E group was totally (100%) conformed to the requirements of the transfusion guideline to RBC infusion, which was higher than that of CON group (81.25%) [Table 5]. Table 5 The two groups of patients are compared in compliance of blood transfusion guidelines (%) Variables POTTS-E ( n = 33) Control ( n = 32) χ 2 P <7 g/dl no transfusion 0 (0) 5 (15.62) 7.517 0.006 >10 g/dl still transfusion 0 (0) 1 (3.12) 1.433 0.231 Compliance rate 100 (100) 26 (81.25) 9.135 0.003 POTTS-E: Peri-operative transfusion trigger score of emergency. BODY.R.ANALYSIS ON CORRELATION OF INTRAOPERATIVE TOTAL AMOUNT OF RED BLOOD CELLS WITH PATIENT SERUM IRON, ACUTE PHYSIOLOGIC ASSESSMENT AND CHRONIC HEALTH EVALUATION II SCORES AND HEMOGLOBIN RESULTS WHEN ENTERING THE OPERATION ROOM: In the POTTS-E group, the intraoperative total amount of RBCs showed a linear positive correlation with serum iron (SI) at the time of entering the operation room, with a correlation coefficient of r = 0.609 (P < 0.05); in the control group, the intraoperative total amount of RBCs showed a linear positive correlation with SI at the time of entering the operation room, with a correlation coefficient of r = 0.509 (P < 0.05). In the POTTS-E group, the intraoperative total amount of RBCs showed a linear positive correlation with APACHE II scores at the time of entering the operation room, with a correlation coefficient of r = 0.670 (P < 0.05); in the control group, the intraoperative total amount of RBCs showed a linear positive correlation with APACHE II scores at the time of entering the operation room, with a correlation coefficient of r = 0.523 (P < 0.05). In the POTTS-E group, the intraoperative total amount of RBCs showed a linear negative correlation with Hb value at the time of entering the operation room, with a correlation coefficient of r = −0.360 (P < 0.05); in the control group, the intraoperative total amount of RBCs showed a linear negative correlation with Hb value at the time of entering the operation room, with a correlation coefficient of r = −0.604 (P < 0.05) [Table 6]. Table 6 Correlation of intraoperative total amount of RBCs with SI, APACHE II and Hb Variables RBC of group POTTS-E RBC of group control Correlation coefficient P Correlation coefficient P Serum iron 0.609 0.000 0.509 0.003 APACHE II 0.670 0.000 0.523 0.002 Hb −0.360 0.040 −0.604 0.000 APACHE II: Acute physiology and chronic health evaluation II; Hb: Hemoglobin; RBCs: Red blood cells; POTTS-E: Peri-operative transfusion trigger score of emergency. BODY.R.DIFFERENCES OF RED BLOOD CELL APPLICATION: Utilization rate of RBC: There were no statistical differences in utilization rates of autologous blood of the two groups; the utilization rates of allogeneic RBC, total allogeneic RBC and total RBC were 48.48%, 51.5%, and 75.7% in POTTS-E group, which were lower than those of the CON group (84.3%, 84.3%, and 96.8%) [Table 7]. Table 7 The two groups of patients are compared in utilization rate of RBC (%) Variables Autologous RBC Operation allogeneic RBC Postoperation allogeneic RBC Total allogeneic RBC Total RBC POTTS-E ( n = 33) 14 (42.4) 16 (48.4) 4 (12.1) 17 (51.5) 25 (75.7) CON ( n = 32) 16 (50.0) 27 (84.3) 7 (21.8) 27 (84.3) 31 (96.8) χ 2 0.375 9.346 1.099 8.021 5.143 P 0.54 0.002 0.294 0.005 0.023 POTTS-E: Peri-operative transfusion trigger score of emergency; RBC: Red blood cell. Amount of red blood cell: Per capita consumption of autologous blood, intraoperative allogeneic RBC, total allogeneic RBC and total RBC in POTTS-E groups were all lower than those of CON group [Table 8]. Table 8 The two groups of patients are compared in per capita consumption of RBC (M (P25, P75)) Variables Autologous RBC Operation allogeneic RBC Postoperation allogeneic RBC Total allogeneic RBC Total RBC POTTS-E ( n = 33) 0 (0, 2.6) 0 (0, 3.0) 0 (0, 0) 2.0 (0, 4.0) 3.1 (0.81, 6.0) CON ( n = 32) 0 (0, 2.7) 4.0 (2.0, 4.0) 0 (0, 0) 4.0 (2, 6.0) 5.8 (2.7, 8.2) Z −4.506 −2.236 −4.619 −2.499 −4.864 P 0.012 0.025 0.000 0.000 0.000 POTTS-E: Peri-operative transfusion trigger score of emergency; RBC: Red blood cell. BODY.D: Over years, researchers have tried to find an alternative for allogeneic blood, in order to eliminate the negative impacts of blood transfusion and the tensions of blood application. But there is not a drug or product that can completely replace the human red blood cells based on the current study results and application conclusion.[45] Failure to be replaced does not mean that it is perfect. Modern medical research has shown that allogeneic blood transfusion has a risk.[6789] At the same time, the growing contradiction between supply and demand of blood products has affected the normal medical order.[10] As policy makers of medical action, judging the time and amount of infusion in the face of anemia patients who need RBC infusion is a question needed to be carefully considered.[11] Different blood transfusion guidelines are introduced in various parts of the world, to specific transfusion of Hb for patients with different levels. Perioperative blood transfusion and adjuvant treatment guidelines in 2006 of ASA suggest that: Patients with Hb <6 g/dl should be given RBC infusion, and should not be transfused with Hb >10 g/dl; if the patients' Hb lies in the range of 6–10 g/dl, the RBC transfusion should be determined according to the speed and levels of ischemia, and the risks such as the subsequent complications due to insufficient blood oxygenation capacity, low cardiopulmonary reserves and high oxygen consumption, etc.;[12] British Society of Hematology defines that blood transfusion starts at Hb <8 g/dl;[13] Scottish perioperative blood transfusion guidelines was defined at Hb <7 g/dl;[14] and Spanish Society of blood transfusion defines that blood transfusion starts at Hb <7 g/dl and at Hb <5 g/dl in patients with chronic anemia.[15] Though there are differences in the concrete values in the above blood transfusions guidelines, the general direction is roughly the same-the restrictive transfusion strategy. This is because that the most important reference for such guidelines is a comparative research on open blood transfusion and restrictive blood transfusion (Hébert et al., 1999). The results showed that the critically ill patients whose blood transfusion was started at Hb <7 g/dl with Hb was maintained in 7–9 g/dl had obviously fewer amount of blood transfusion and lower incidence of related complications; And the most important thing was that mortality within 30 days of hospitalization was declined.[16] In a follow-up study, Carson et al. also supported that restrictive blood transfusion can decrease the amount of transfusion but not increase the incidence of complications and mortality of the patients with experiment results.[1718] Determination on the requirement of RBC transfusion needs the quick analysis results of patients' pulmonary function, oxygen partial pressure, oxygenation index, oxygen uptake rate;[1920] Heart rate, cardiac output and vascular resistance;[21] Mixed venous oxygen saturation, arterial lactic acid concentration, base defect, pH (pHi) and CO2 partial pressure in gastric mucosa;[222324] capacity state, etc. Under the emergency state, the above evaluation indexes would deviate from the normal physiological condition because of the stress response, and body compensatory changes. In clinical work, in the face of emergency patients, it is very difficult to require all medical institutions to judge whether patients need RBC infusion according to the evaluation on patients' cardiopulmonary compensatory ability, metabolism, and oxygen consumption.[25] In this study, we referenced to the "POTTS" which was put forward by Professor Liu Jin (Anesthesia Department, Western China Hospital of Sichuan University), to replace the required subjective judgment in the original blood transfusion indications when patients' Hb in 7 ~ 10 g/dl. The evaluation scheme is composed of four parts: Circulation condition, blood oxygenation situations, oxygen consumption, and compensatory state namely whether the patients need adrenaline to maintain normal BP, whether higher oxygen concentration is needed to maintain the oxygenation, whether there is high metabolism with high body temperature and whether there is high shock index. The two groups of actually included patients are consistent at preoperative baseline, and the data are comparable. In the hospital follow-up, surgical operation time, staying time of PACU, and emergency hospitalization time of POTTS-E group did not increase; And there are no differences between the two groups in intraoperative rehydration, APACHE II scores on the 3rd days after operation, and postoperative vital signs; except the 1st day after operation, there are no differences between the POTTS-E group and the control group in Hb on the 3rd day after operation and at the end of emergency hospitalization. Compared with posthospitalization follow-up, there are no differences between the two groups in mortality and blood transfusion-related complications till 6 months after the operation. Due to the own reason of the research center, this study does not involve nerve surgery and obstetrics patients; affected by the local medical policy, the hospitalization condition of ICU is not counted. Since it is the first time to introduce marking scheme into the blood transfusion research of emergency patients; risk control is carried out on the included patients, and some critically ill patients are excluded to ensure safety.[2627282930] The lower limit is 7 points, and the upper limit is 10 points, which are consistent with the currently executed blood transfusion guidelines in China. This study does not appear deaths; due to the differences of the case range, the results do not tally with the study of Heeney et al.[31] But the results still hint that according to the blood transfusion scheme guided by POTTS-E, complications and death risk are not increased, and there are no significant differences in the postoperative vital signs, suggesting the safety of POTTS-E scheme. But limited by the number of cases and the research time, this study fails to complete the comparison in the postoperative quality of life and longer follow-up. In the comparison of transfusion in all included patients, we found that the traditional doctor experience-based transfusion strategy subject to blood transfusion guidelines appeared to be not completely consistent with the requirements of "guidelines" in the perioperative RBC transfusion. The results of the study showed that in the control group, 5 patients did not undergo intraoperative RBC transfusion when the Hb value was less than 7 g/dl, while one patient still underwent allogeneic RBC transfusion when the Hb value was higher than 10 g/dl. Only 81.25% of the patients met the requirements of "guidelines," and this ratio was lower than the 100% in the ETS scoring group. According to the reason analysis, as the experimental group was in strict accordance with the experiment plan and the scoring rules of the experimental group were established within the framework of blood transfusion guidelines, the patients had to undergo transfusion when the Hb value was less than 7 g/dl. Throughout the experiment stages, the number of patients with extra points in the ETS group was small, and no patient achieved 3 points regarding ETS scores, that is, no patient was reported to undergo blood transfusion with an Hb value up to 10 g/dl. Meanwhile, the experiment in the ETS group required its patients to undergo transfusion when Hb value was less than 7 g/dl. As a result, the Hb values of all patients in the ETS group were maintained between 7 and 10 g/dl in the operation stage. In contrast, in the control group, the blood transfusion guidelines allowed doctors to decide the maintained Hb value range only when they had evaluated patient conditions including cardiopulmonary compensatory ability, body metabolism, and oxygen consumption. Although the recommended maintained range of the guidelines was also 7–10 g/dl, the subjective assessment of doctors may be insufficient considering the emergency conditions, resulting in insufficient transfusion for patients with such need while excessive transfusion for patients without such need. Consequently, 6 patients in the control group were out of the requirements of "guidelines". At the same time, we speculated that the rest patients in the control group, whose Hb values ranged from 7 to 10 g/dl might still have these shortcomings above. In the results of this group, we conducted an analysis on the linear correlation of the patient intraoperative total amount of RBCs with shock index, APACHE II scoring, and Hb value when entering the operation room. The results suggested that there was a linear correlation between the blood amount of patients in two groups and above three indicators. However, in the POTTS-E group, the correlation coefficient of blood amount with shock index and APACHE II scores was higher than that in the control group; the correlation coefficient with Hb values in the control group was higher than that in the POTTS-E group. According to the reason analysis, the transfusion need of emergency patients could be estimated preoperatively via their signs, inspection, and testing results, but theoretically, the more objective indicators included by an assessment standard, the greater its correlation with body hypoxia. Therefore, for the transfusion assessment of emergency patients especially patients with hemorrhagic shock, the accuracy of APACHE II scores would be higher than shock index, which appeared to be higher than Hb values. It can be seen that the control group was most closely related to Hb values at the time of entering into the operation room, suggesting that the assessment mixed with experience was easy to be misled by some key indicators, while the POTTS-E group was more closely associated with shock index and APACHE II, suggesting that POTTS-E scheme was closer to patient physiological status and more consistent with their transfusion need. Consequently, it is more reasonable to guide blood transfusion using the POTTS-E scores-based transfusion strategy. Compared with the POTTS-E group, more RBC is infused in the control group; thus, Hb of the POTTS-E group is lower in the control group on the follow-up of the 1st postoperative day. But the mean value is not greater than 1 g/dl. At the same time, as the patients' recovery, there are no differences of Hb on the third postoperative day and at the end of hospitalization. Reason analysis: The control group was given RBC transfusion not completely according to the regulation of blood transfusion guidelines. Some patients needing RBC transfusion fail to strictly perform certain rule. And the average level of Hb after blood transfusion is pulled down. We have reason to believe that, if the control group can complete reference to the guideline, the difference of Hb at follow-up point should be even greater or may be affected for a longer time. Only a minority of patients with POTTS-E >0 during the entire study period, namely most of the patients are given allogeneic RBC transfusion according to the lower limit of Hb <7 g/dl in most of the scoring, so it is not difficult to understand the intraoperative and perioperative utilization rate and per capita consumption of allogeneic RBC in POTTS-E group are lower than those of the control group; autologous blood utilization rate in POTTS-E group is the same as the control group, but the per capita consumption is lower than the control group, and the difference is from the small sample size. But there is reason to believe that if the sample size is enlarged, but the utilization rates of autologous blood in the two groups are not changed, per capita consumption of autologous blood in the control group is the same as the POTTS-E group, and more allogeneic RBC infusion is needed in the control group. Therefore, the intraoperative and perioperative utilization rate and the per capita consumption of total RBC in POTTS-E group should be lower than those of the control group. The results suggest that with the evidence-based method of classification to guide transfusion for emergency patients, utilization rate and the per capita consumption of RBC can effectively be reduced. In summary, POTTS-E scores scheme is safe, reasonable, and practicable and has the value for carrying out multicenter and large sample clinical researches.

TITLE: TREATMENT OF ACUTE AND TRANSIENT PSYCHOTIC DISORDERS WITH LOW AND HIGH DOSES OF ORAL HALOPERIDOL ABSTRACT: The apparent rationale for the popular use of high doses of neuroleptics in psychotic patients is to increase the degree and speed of therapeutic response .However, several recent reports have questioned these claims. The present study was undertaken with the aim to compare the efficacy of high and low oral doses of haloperidol in the treatment of acute and transient psychotic disorders. The sample comprised of forty patients of both sexes diagnosed as acute and transient psychotic disorder who were randomly assigned to high dose (20 mg/day) and low dose (5 mg /day) haloperidol groups with equal number of subjects (n=20) in both groups. Weekly assessment was done on Brief Psychiatric Rating Scale and Haloperidol Side-effects Check List (day 7, 14, 21, 28, 35 & 42). Both groups showed significant improvement in BPRS from baseline scores on all assessments. Comparison of the improvement rate in both study groups revealed no significant difference.

TITLE: Three -Agent Preemptive Analgesia, Pregabalin-Acetaminophen-Naproxen, in Laparotomy for Cancer: A Randomized Clinical Trial ABSTRACT.BACKGROUND: Pain management after abdominal surgery is a critical issue in cancer patients undergoing laparotomy. Opioid analgesics commonly used postoperatively have significant side effects and can postpone restoring normal life. Administration of analgesics before the surgery by inhibiting pain cascades may be an effective method for more efficient pain control. ABSTRACT.OBJECTIVES: This study aimed to investigate the effect of the preemptive use of oral pregabalin-acetaminophen-naproxen on pain control and morphine consumptions in cancer patients undergoing laparotomy. ABSTRACT.PATIENTS AND METHODS: A total of 40 cancer patients scheduled for open abdominal surgery were randomized into the two groups. one group received combination of pregabalin 150 mg, acetaminophen 1 g and naproxen 250 mg (the PAN group) an hour before laparotomy. Following the surgery, morphine was administered on a protocolized schedule based on patients' demand for pain control. Postoperative pain level was assessed using universal pain assessment tool (UPAT) at 0, 2, 4, 6, 12, 24 and 48 hours after the operation. The postoperative morphine dose and complications were noted. Data were analyzed using SPSS version 16. ABSTRACT.RESULTS: Patients in the PAN group had significantly lower UPAT scores at 0, 2, 4, 6, 12, 24 and 48 hours after the surgery than those in the control group (P = 0.008, 0.021, 0.008, 0.047, 0.004, 0.001, and 0.001). The mean dose of postoperative morphine consumption in the PAN group was 37% less than the control group (P = 0.001). The complications were not significantly different between the two groups. ABSTRACT.CONCLUSIONS: Preemptive use of pregabalin-acetaminophen-naproxen decreases intensity of pain and morphine consumption in the cancer patients after laparotomy without significant complications. BODY.1. BACKGROUND: After major surgeries, patients experience pain and a long convalescence period. Restoring baseline physiologic function after surgery has always been a goal for medical staff (1). Pain is one of the most common clinical problems especially in postoperative patients, which postpones achieving this aim. Reducing pain increases patient's satisfaction and decreases the hospitalization period (2). Moreover, relieving pain after laparotomy decreases ileus period and pulmonary complications such as mucous plaque, hypoxia, atelectasis and infection (3). Conventionally morphine is used for pain control after surgery, which has considerable side effects and exacerbates ileus and bowel dysfunction after abdominal surgeries (4, 5). Pain receptors in injured tissues send signals, inducing a cascade which causes increased peripheral and central neuronal response. These sensory processing alterations cause an extreme response to stimulus and pain. Preventing these signals would inhibit these cascades and reduce pain postoperatively (6). Preemptive analgesia is the use of analgesic agents before painful stimulus compared to conventional analgesia used after painful stimulus. This method was first introduced in animal studies for relieving pain after surgeries by Wall in 1988 (7) and Woolf in 1991 (8). Preemptive analgesia inhibits nocireceptors, and thus prevents the central alterations induced by afferent input after surgery. Preemptive analgesia also may reduce alterations in sensory central receptors responsible for central sensitization, neuropathic pain, hyperalgia and allodynia postoperatively (9, 10). In cancer patients, the effect of medication on the course of disease should be considered too. Preemptive analgesia may restore the interleukin-2 level to normal earlier, which has a key role in T cells' immune response in cancer (11). Also, immunosuppressive effects of opioids used for pain control after surgery may have some tumor promoting effects in cancer patients (12); so, decreased need for opioid analgesic after surgery can provide additional benefits for use of preemptive analgesia in cancer patients. Gabapentinoid medications are shown to be an effective analgesic in various surgeries for cancer and non-cancer patients even in eyelid surgery (13-15). Pregabalin inhibits the release of neurotransmitters such as glutamate, norepinephrine and substance P by binding to α2-δ1 protein of voltage-gated calcium channels. This change in neurotransmitters' concentration explains its various uses as anxiolytic, analgesic and anticonvulsant (16-18). Acetaminophen is a worldwide used analgesic and antipyretic. However, there is still debate about its exact mechanism of action. It primarily inhibits COX2 and then COX1. It may also affect additional inflammatory pathways by inhibiting other peroxidase enzymes such as myeloperoxidase (19, 20). Naproxen is an old introduced nonsteroidal anti-inflammatory and analgesic with a fully established profile of mechanism of action, adverse effects and dosing requirement. It has been shown in different studies that among nonsteroidal anti-inflammatory drugs (NSAIDs), naproxen is the one with the least effect on increasing cardiovascular risk (21-23). BODY.2. OBJECTIVES: This study aimed to investigate the effect of preemptive oral pregabalin-acetaminophen-naproxen on pain control and morphine consumption in cancer patients after laparotomy. This combination was chosen as a noble selection for preemptive analgesia in order to block different sites in pain pathway (from production to perception). BODY.3. PATIENTS AND METHODS: This double-blind study was conducted in Cancer institute. All patients have received informed consent for being a part of this study. An informed consent form was approved by ethics in research committee of Tehran University of Medical Sciences. Exclusion criteria included nonelective surgery, ASA physical status more than 3, history of drug sensitivity to preemptive analgesia agents, opioids addiction, non-cancerous peptic ulcers and coagulopathies (Figure 1). Figure 1.Flow Diagram of the Study Investigating the Effect of Three Agent Preemptive Analgesia, Pregabalin-Acetaminophen-Naproxen, in Laparotomy for Cancer A total of 40 cancer patients admitted for open abdominal surgery were recruited. We gave every patient a code according to a computer-generated list and then codes were randomized into two groups. One group received a combination of pregabalin 150 mg, acetaminophen 1 g, naproxen 250 mg (PAN) an hour before laparotomy. All patients were moved to operation hall and monitored for blood pressure, oxygen saturation, pulse rate, capnometry and ECG. All patients were premedicted by midazolam 2.5 mg and fentanyl 2 μg/kg. Anesthesia was induced by thiopental Na 3 - 5 mg/kg and atracurium 5 mg/kg. Anesthesia maintained by isoflurane 0.8% - 1.2% in 50% N2O - O2 mixture. Before skin incision 1 μg/kg fentanyl was injected. Atracurium and fentanyl were administered as needed during anesthesia. After skin closure, neostigmine (40 μg/kg) and atropine (20 μg/kg) were administered to antagonize remaining neuromuscular blockade. Then patients were moved to recovery room. After regaining consciousness completely, pain intensity was assessed using universal pain assessment tool (UPAT) (time 0). This assessment was repeated subsequently at 2, 4, 6, 8, 12, 24 and 48 hours thereafter. Morphine was administered on a protocolized schedule based on patients' demand. Time that patient demanded first analgesic medication, total dose of morphine and other complications in the postoperative period were recorded. We also measured surgeons' satisfaction with pain control in 24 and 48 hours after surgery with a 3-score scale ranging from completely satisfied to completely dissatisfied. Patients and staff responsible for administering drug preoperatively, and assessing the pain level and morphine dose postoperatively, were not aware of the specific codes and intervention mode of each patient. Surgeons were blinded to the study, too. Data were analyzed using SPSS version 16. Mean and standard deviation for all data were calculated and analyzed. We used unpaired t-test for comparing quantitative variables and chi-square or Fisher's exact test for qualitative ones. P value less than 0.05 was considered statistically significant for all tests. BODY.4. RESULTS: The mean age of the patients in the PAN group was 53.45 ± 10.76 and in the control group was 56.4 ± 8.60 years, which showed no statistically significant difference (P = 0.34). Male to female ratio was 0.66 and 1 in the PAN and control groups respectively, which was not significantly different (P = 0.75). Moreover, no significant difference was found in other characteristics including body mass index (BMI), ASA status, blood pressure, time gap between diagnosis and surgery duration between the two groups (Table 1). Table 1. Characteristics of Patients in the Pregabalin-Acetaminophen-Naproxen and Control Groups Variable PAN Group Control Group P Value Statistical Significance Age, y 53.45 ± 10.76 56.4 ± 8.6 0.34 NS Gender 0.75 NS Male 8 10 Female 12 10 BMI, Kg/m 2 22.85 ± 3.33 22.98 ± 2.26 0.88 NS ASA, I/II 10/10 12/8 0.15 NS Systolic blood pressure, mmHg 114.5 ± 13.91 116.5 ± 10.89 0.53 NS Diastolic blood pressure, mmHg 76.7 ± 4.9 79.15 ± 8.71 0.28 NS Cigarette smoking, No. (%) 4 (20) 8 (40) 0.15 NS Non-cancerous peptic ulcer, No. (%) 2 (10) 0 (0) 0.25 NS Gap between diagnosis and surgery, mon 54.2 ± 74.48 55.4 ± 47.94 0.94 NS Surgery duration, min 221.0 ± 83.65 244.0 ± 55.00 0.31 NS Abbreviations: BMI, body mass index; NS, Not significant; PAN, pregabalin-acetaminophen-naproxen. Fifty-five percent of all patients had gastrointestinal cancers and remaining 45% suffered from gynecologic cancers. In gastrointestinal cancer patients, affected areas were stomach, colon, pancreas and rectum/anus in 36%, 32%, 18% and 14% of the patients respectively. Also, 89% of gynecologic cancer patients suffered from ovary and adnexal malignancies and the rest 11% had uterus cancer. Most laparotomies included resection of the affected area with lymphatic resection. The overall mean pain intensity levels by UPAT in the PAN and control groups were 4.06 ± 0.77 and 5.3 ± 0.64, respectively which showed a statistically significant difference (P < 0.001). Pain intensity levels by UPAT at 0, 2, 4, 6, 12, 24 and 48 hours after surgery in the PAN group were 5.65 ± 1.04, 5.2 ± 0.76, 4.7 ± 0.80, 4.6 ± 0.94, 4 ± 1.02, 2.9 ± 0.96 and 1.4 ± 1.04, respectively. These values in the control group were 6.4 ± 0.59, 5.85 ± 0.93, 5.35 ± 0.67, 5.15 ± 0.75, 4.90 ± 0.79, 4.50 ± 1.10, and 3.80 ± 0.89, respectively. These scores difference was statistically significant in all time points. (P values 0.008, 0.021, 0.008, 0.047, 0.004, 0.001, and 0.001 respectively) (Figure 2). Figure 2.Pain Intensity at 0, 2, 4, 6, 12, 24 and 48 Hours After Surgery in the Pregabalin-Acetaminophen-Naproxen and Control Groups Patients in the control group needed first opioid analgesic 70 minutes earlier than patients in the PAN group (P = 0.003).Total opioid analgesic dose administered to the PAN group was 9.9 mg (37%) less than other group, which was statistically significant (P = 0.001, 17 ± 7.6 mg and 26.9 ± 6.8 mg in the PAN and control groups, respectively). Surgeons reported more satisfaction with pain control in the medication group in 24 and 48 hours after surgery (P = 0.001) (Table 2). Table 2. Pain Intensity, Time to Demand First Analgesic Dose, Total Morphine Dose and Surgeon Satisfaction With Pain Control in the PAN and Control Groups Variable PAN Group Control Group P Value Statistical Significance Pain intensity mean in all points (UPAT score) 4.06 ± 0.77 5.13 ± 0.64 0.001 S Time to demand first analgesic dose, min 124.29 ± 88.72 54 ± 35.89 0.003 S Total morphine dose administered, mg 17 ± 7.6 26.9 ± 6.8 0.001 S Surgeons satisfaction in 24 hours 2.3 ± 0.66 1.5 ± 0.51 0.001 S Surgeons satisfaction in 48 hours 2.9 ± 0.31 1.75 ± 0.64 0.001 S Abbreviations: PAN, pregabalin-acetaminophen-naproxen; S, significant; UPAT, universal pain assessment tool. In investigation of pain reduction in time periods after surgery, pain level reduction was more in time periods of 2 - 4 6 - 12, 12 - 24 and 24 - 48 hours after surgery in the PAN group compared to the control group. However, this effect was significant only in 12 - 24 and 24 - 48 hours postoperatively (Table 3). Table 3. Pain Reduction in Different Time Periods in the PAN and Control Groups Time Periods After Surgery PAN Group Control Group P Value Significance 0 - 2 0.45 ± 0.6 0.55 ± 0.68 0.62 NS 2 - 4 0.5 ± 0.688 0.05 ± 0.607 0.15 NS 4 - 6 0.1 ± 0.553 0.2 ± 0.69 0.618 NS 6 - 12 0.6 ± 0.68 0.25 ± 0.55 0.08 NS 12 - 24 1.1 ± 0.71 0.4 ± 0.681 0.003 S 24 - 48 1.5 ± 1.06 0.7 ± 0.59 0.001 S Abbreviations: NS, not significant; PAN, pregabalin-acetaminophen-naproxen; S, significant. We compared pain intensity at 2, 4, 6, 12, 24, 48 hours after surgery to time 0 in both groups. Pain reduction was more in 12, 24 and 48 hours periods after surgery in the PAN group compared to the control group. However, this was significant only in 24 hours and 48 hours after surgery (Table 4). Table 4. Pain Reduction in 2, 4, 6, 12, 24, 48 Hours After the Surgery Between the PAN and Control Groups Hours After Surgery PAN Group Control Group P Value Significance 0 - 2 0.45 ± 0.6 0.55 ± 0.68 0.62 NS 0 - 4 0.9 ± 0.75 1.05 ± 0.51 0.628 NS 0 - 6 1.05 ± 1.14 1.25 ± 0.55 0.486 NS 0 - 12 1.65 ± 1.80 1.5 ± 0.5 0.58 NS 0 - 24 2.75 ± 0.85 1.90 ± 0.85 0.003 S 0 - 48 4.25 ± 1.11 2.60 ± 0.59 0.001 S Abbreviations: NS, not significant; PAN, pregabalin-acetaminophen-naproxen; S, significant. Most common complication in first postoperative 24 hours in our study was nausea, followed by somnolence and dizziness. All these three complications in the PAN group were more than the control group, but this difference was not statistically significant (Table 5). Table 5. Postoperative Complications in the PAN and Control Groups a Complication PAN Group Control Group P Value Significance Nausea 75 65 0.37 NS Somnolence 70 50 0.17 NS Dizziness 60 40 0.38 NS Abbreviations: NS, not significant; PAN, pregabalin-acetaminophen-naproxen. a Values are expressed as percentage. BODY.5. DISCUSSION: In this clinical trial, preemptive use of PAN was associated with reduced pain intensity at 0, 2, 4, 6, 12, 24 and 48 hours after surgery significantly. The total administered morphine dose in the PAN group was 37% less than the control group. In terms of postoperative pain and reduced need for opioid by preemptive analgesia, similar findings are reported in different surgeries in other studies. Preemptive gabapentin on patients scheduled for laparoscopic cholecystectomy, lumbar discectomy, arthroscopic knee and dacryocystorhinostomy repair yielded significant opioid sparing and decreased pain score (24-27). In two other studies conducted on tonsillectomy and total abdominal hysterectomy, pain intensity difference was not significant between the gabapentin and placebo groups; however, the need for opioids reduced in both studies (28, 29). A study on patients undergoing abdominal hysterectomy due to benign diseases, has reported 36% less pain score but no significant opioid consumption difference (30). Arguably administering preemptive gabapentinoid drugs can decrease postoperative pain and need for opioid analgesics (31). We used pregabalin, acetaminophen and naproxen together in our study. This is the first study to investigate the use of this three-agent preemptive analgesia. Different medications are investigated for preemptive analgesia. Using combination of various agents and blocking more than one pathway has yielded more promising results. Combination of gabapentin and refecoxib is shown to be superior to use of single agent in total abdominal hysterectomy patients (32). Concurrent use of acetaminophen and naproxen is shown to increase analgesic efficacy in a systematic review analyzing 21 human studies (33). It's suggested that pregabalin with naproxen can have a synergic or at least additive anti hyperalgesia effect by experimental study on rats and nocireceptors thresholds (34). Riad et al. in his investigation on children scheduled for surgery due to inguinal hernia reported that preemptive use of acetaminophen and diclofenac suppository would reduce postoperative pain and need for analgesics more than patients receiving single agent (35). Another recent systematic review has shown opioid sparing effect for preemptive use of acetaminophen and NSAID in controlling pain after surgery (36). In most of these studies, researchers used visual analogue scale (VAS) for assessing pain intensity. However, now it is a debate whether some people might report intensity of stimulation instead of pain perception in visual analogue score. Other tools included faces scales such as Wong and Baker pain scale for children, verbal numerical rating scale, or a scale made by authors. To assess pain, we used UPAT, which is newly designed and a combination of visual analogue scale, faces scales and activity tolerance scale (37, 38). In our study pain intensity in all time points was significantly less than the control group, and most prominent pain reduction was seen in an interval of 12 - 48 hours postoperatively. Pain level reduction in intervals earlier than 12 hours after surgery was not significantly different between the two groups. Similar results were reported by other researchers. In a study on laparoscopic cholecystectomy, a group of preemptive gabapentin had lower pain scores in time intervals of 0 - 6, 6 - 12, 12 - 18, 18 - 24 compared to the tramadol and placebo groups (27). Eman et al. reported that preemptive pregabalin in total abdominal hysterectomy reduced pain significantly in a period of 4 - 24 hours after surgery. However, no significant difference was observed in the pain score at 1 hour after surgery (16). This insignificance in early hours after surgery may be explained by limited activity of patient in these hours. Possibly walking of patients in later hours increases intra-abdominal pressure, which stimulates pain receptors and thus highlights the difference between two groups. Time to demand first analgesic dose was significantly longer in PAN group in our study. Although in many studies, this item was not investigated, Similar studies yielded consistent results (16, 39, 40). However this effect was smaller in those studies and not statistically significant. This difference can be attributed to our multimodal preemptive analgesia, in these two other studies only preemptive pregabalin or pregabalin with a NSAID have been used in contrast to our three agent modality. Blocking of more pain pathways possibly delays the time to feel first signs of post-operative pain. In our study, most common complications were nausea, somnolence and dizziness which did not show a significant difference between the two groups. Pregabalin complications include nausea, somnolence, dizziness, ataxia, diplopia, and weight gain, which are mostly seen in chronic use (41). Preemptive pregabalin was associated with similar side effects in other studies. Sedation, nausea/vomiting, dizziness, gait disturbance have been reported in studies on single agent gabapentinoid drug (27, 29). We used 150 mg pregabalin in our study, which is the major drug implicated in more nausea/vomiting and somnolence in the case group. Various studies have been conducted on determining the optimum dose of pregabalin for preemptive analgesia. In a clinical trial by jokela, pregabalin 150 mg with 800 mg ibuprofen was superior to pregablin 75 mg with ibuprofen 800 mg in terms of controlling pain without significantly increased side effects (40). In another clinical trial by jokela, pregabalin 600 reduced postoperative analgesic need more than pregablin 300 mg, but caused considerable significant side effects of dizziness, headache and blurred vision (39). Pandey showed that increasing gabapentin dose more than 600 mg, does not affect postoperative pain in lumbar discectomy (26). It seems that specific type of surgery should be considered in settling the recommended preemptive pregabalin dose. However, in studies on preemptive NSAIDs especially in children, bleeding is a concern, (36) in our study no perioperative bleeding is noted in the PAN group. We should note that in decision to evaluate preemptive analgesia side effects, avoiding opioid noticeable side effects such as respiratory depression should be taken in account. Despite different investigations on preemptive analgesia, best choice of drugs, efficient dose and time to use them, possibility of multiple dosing or continuing medication after surgery are not completely determined. More investigations are needed for making guidelines about preemptive analgesia in specific surgeries and age groups.

TITLE: Effects of dexmedetomidine in combination with fentanyl-based intravenous patient-controlled analgesia on pain attenuation after open gastrectomy in comparison with conventional thoracic epidural and fentanyl-based intravenous patient-controlled analgesia ABSTRACT: Background: This study was investigated the effects of dexmedetomidine in combination with fentanyl-based intravenous patient-controlled analgesia (IV-PCA) on pain attenuation in patients undergoing open gastrectomy in comparison with conventional thoracic epidural patient-controlled analgesia (E-PCA) and IV-PCA. Methods: One hundred seventy-one patients who planned open gastrectomy were randomly distributed into one of the 3 groups: conventional thoracic E-PCA (E-PCA group, n = 57), dexmedetomidine in combination with fentanyl-based IV-PCA (dIV-PCA group, n = 57), or fentanyl-based IV-PCA only (IV-PCA group, n = 57). The primary outcome was the postoperative pain intensity (numerical rating scale) at 3 hours after surgery, and the secondary outcomes were the number of bolus deliveries and bolus attempts, and the number of patients who required additional rescue analgesics. Mean blood pressure, heart rate, and adverse effects were evaluated as well. Results: One hundred fifty-three patients were finally completed the study. The postoperative pain intensity was significantly lower in the dIV-PCA and E-PCA groups than in the IV-PCA group, but comparable between the dIV-PCA group and the E-PCA group. Patients in the dIV-PCA and E-PCA groups needed significantly fewer additional analgesic rescues between 6 and 24 hours after surgery, and had a significantly lower number of bolus attempts and bolus deliveries during the first 24 hours after surgery than those in the IV-PCA group. Conclusions: Dexmedetomidine in combination with fentanyl-based IV-PCA significantly improved postoperative analgesia in patients undergoing open gastrectomy without hemodynamic instability, which was comparable to thoracic E-PCA. Furthermore, this approach could be clinically more meaningful owing to its noninvasive nature. BODY.BACKGROUND: Radical open gastrectomy is one of the major upper abdominal surgeries that have been reported to cause acute postoperative pain 1. Moreover, the severity of pain is higher especially in this upper-abdominal surgery, which can lead to the impairment of the respiratory effort due to the restriction of the movement of the thoracic cage and abdomen, as well as the decreased respiratory capacity 2, 3. Such changes have a negative impact on the course of postoperative recovery 4. Conventionally, pain after open gastrectomy has been controlled with thoracic epidural patient-controlled analgesia (E-PCA) or intravenous PCA (IV-PCA) 1, 4. Thoracic E-PCA has an excellent effect in controlling postoperative pain, when properly positioned 1, 5-7. However, as it is a relatively invasive technique, its application is limited by specific contraindications such as infection or bleeding tendency, and there is a possibility of malpositioning of the catheter in the spinal nerve roots leading to severe postoperative neurologic deficits due to ischemia of the sensory and motor nerves 5, 7-10. Therefore, despite its potential benefits, the clinical use of E-PCA may have even declined because of these types of complications 1, 11. In case of IV-PCA, higher doses of opioids are required to control postoperative pain effectively; however, this often leads to the discontinuation of IV-PCA because of persistent adverse effects such as nausea, vomiting, and pruritus 1, 12, 13. Dexmedetomidine is well recognized as an extremely preferential α2-receptor agonist that has sedative and analgesic effects without unfavorable respiratory suppression 14-16. Previous studies have reported that dexmedetomidine administration during surgery could reduce the amounts of opioids and analgesics used after surgery 17-20. Furthermore, current studies on the combination of dexmedetomidine with various opioid-based IV-PCA techniques have demonstrated that this combination treatment could help provide better analgesia and opioid-sparing effects without any remarkable unfavorable effects 21-24. Hence, in this prospective, randomized clinical trial, we investigated the effects of dexmedetomidine in combination with fentanyl-based IV-PCA on pain attenuation in patients undergoing open gastrectomy in comparison with conventional thoracic E-PCA and IV-PCA. BODY.MATERIALS AND METHODS.STUDY POPULATION: This investigation was approved from the Institutional Review Board and Hospital Research Ethics Committee of Severance Hospital (Yonsei University Health System in Seoul, Korea; IRB protocol No. 4-2014-0883), and consequently registered at http://clinicaltrials.gov (registration No. NCT02325882). After acquiring written informed consent from all patients, 171 patients with stomach cancer, of age 20 to 65 years and American Society of Anesthesiologists physical status I/II, who were planned to undergo elective conventional open gastrectomy, were enrolled between July, 2015 and March, 2016. The exclusion criteria were as follows: refusal of PCA application; histories of abdominal surgery; prior cardiac disease including unstable angina, congestive heart failure, uncontrolled hypertension; concomitant coagulopathy; presence of vertebral deformity or disease; concomitant pulmonary, renal, or hepatic disease; any contraindication to epidural catheterization; any allergy or hypersensitivity to fentanyl, α2-adrenergic agonists, or local anesthetics; use of any type of chronic pain killer or current opioid; cognitive, neurological, or psychiatric impairment; and incapability to report the pain intensity on the pain scale. All enrolled patients were educated on how to express the intensity of pain by using the numerical rating scale (NRS; 0, no pain, and 10, worst pain possible) 25, and on how to use the PCA machine in the preanesthetic room. BODY.MATERIALS AND METHODS.RANDOMIZATION AND PERIOPERATIVE PROTOCOL: The assignments of the patients were performed randomly into one of 3 groups (1:1:1) according to preset random numbers by using a computer-generated table (http://www.random.org) with no dividing blocks and stratification: conventional thoracic E-PCA (E-PCA group, n = 57), dexmedetomidine in combination with fentanyl-based IV-PCA (dIV-PCA group, n = 57), or fentanyl-based IV-PCA only (IV-PCA group, n = 57). In the E-PCA group, the procedure for epidural catheter insertion was completed before the induction of general anesthesia. After standard monitoring, a single investigator performed the epidural catheterization at the level of T7-8 or T8-9 by using a 17-gauge Tuohy needle, and a catheter was advanced 5 cm into the epidural space. Intravascular or subarachnoid placement of the epidural catheter was excluded by checking the absence of aspirated blood or cerebrospinal fluid. Furthermore, intrathecal delivery of the local anesthetic was ruled out by confirming that no rapid onset of neuroaxial block was developed after the administration of 3 mL of 1% lidocaine. Upon the initiation of peritoneal closure, the PCA machine (Accumate 1100®; Woo Young Medical Co., Ltd., Seoul, Korea) was started after 5 mL of 0.15% ropivacaine was administered via the epidural catheter. The PCA regimen was a mixture of 0.15% ropivacaine and 3 μg/mL of fentanyl in 0.9% normal saline solution with a total volume of 250 mL. All PCA machines for the 3 groups were programmed to deliver at the rate of 5 mL/h with a 0.5 mL per demand allowed every 15-minute lockout time. In the IV-PCA group, after 1 μg/kg of fentanyl was administered intravenously at the start of peritoneal closure, PCA machine was applied intravenously, which consisted 15 μg/kg of fentanyl and 0.3 mg of ramosetron (Nasea, Astellas, Tokuo, Japan), mixed with 0.9% normal saline solution to a total volume of 250 mL. Thus, in the IV-PCA group, fentanyl was infused basally at a rate of 0.3 μg/kg/ h with a bolus dose of 0.03 μg/kg and a lockout time of 15-min. In the dIV-PCA group, dexmedetomidine (100 μg/mL at 2 mL/vial; Hospira Worldwide, Seoul, Korea) was infused continuously at a rate of 0.1 μg/kg/ h from anesthetic induction until the start of peritoneal closure. Subsequently, the PCA, containing dexmedetomidine in addition to the fentanyl and ramosetron like in the IV-PCA group, was applied intravenously. Thus, in the dIV-PCA group, the background infusion rate of dexmedetomidine was 0.07μg/kg/ h with a bolus dose of 0.007 μg/kg, and that of fentanyl was 0.3μg/kg/ h with a bolus dose of 0.03 μg/kg allowed every 15-min lockout time. In all three groups, the agents for PCA and the study drug were prepared by an investigator who was not involved in the assessment of postoperative pain intensity. BODY.MATERIALS AND METHODS.ANESTHESIA: Anesthesia was accomplished along with the same standard protocol in all three groups. After the patient arrived in the operating room, premedication was done with 0.1 mg of glycopyrrolate administered intravenously. All patients were applied with noninvasive arterial blood pressure monitoring device for mean blood pressure (MBP) measurement, electrocardiogram (ECG) for heart rate (HR) monitoring, oxygen saturation (SpO2) measurement device, and bispectral index (BIS) monitor (Aspect A-2000®; Aspect Medical System Inc., Newton, MA, USA). Anesthesia was induced with 1.5 mg/kg of propofol, 0.5 μg/kg of remifentanil, and 1.2 mg/kg of rocuronium. Thereafter, mechanical ventilation was kept to maintain the end-tidal carbon dioxide at 30-40 mm Hg in 50% O2/air throughout the surgery. Anesthesia was maintained with 0.6-1.2 age-adjusted minimal alveolar concentration end-tidal sevoflurane and 0.02-0.2 μg/kg/min of remifentanil, which were adjusted according to stable hemodynamic variables, including MBP or HR maintained within 20% of the baseline and BIS scores between 40 and 60. Hypotension [MBP <60 mm Hg or systolic blood pressure (SBP) <90 mm Hg] was managed with fluid loading at 100 mL increments or intravenous ephedrine at 4 mg increments, and 0.25 mg intravenous atropine was used to manage bradycardia (HR <40 beats/min). For the prevention of postoperative nausea and vomiting (PONV), 0.3 mg of ramosetron was administered at the start of peritoneal closure, and naloxone and oxygen were prepared for the event of respiratory depression. In case of the development of persistent complications such as severe PONV, hypotension, bradycardia, and respiratory depression despite of appropriate treatment, applications with the PCA machine were discontinued. BODY.MATERIALS AND METHODS.DATA COLLECTION: When the patients were transferred to the postanesthesia care unit (PACU) after surgery, they were reinstructed about the use of the PCA machine. Thereafter, recovery nurses who were not involved in this study assessed the resting NRS scores at 0.5 h and encouraged the patients to push the bolus button whenever they feel pain at a resting NRS score of >3. For patients who showed poor response to the PCA, thus felt sustained pain at a resting NRS score of >4, additional rescue analgesics with pethidine at 12.5 mg increments were given. After PACU discharge, postoperative pain assessment was performed at 1, 2, 3, 6, 12, 18, 24, and 36 h after surgery by the attending nurses of the Postoperative PCA Management Services of our institution, who were not aware of the purpose of this study. Similarly, for patients who experienced sustained pain at a resting NRS score of >4 in the admission room, additional rescue analgesics of pethidine at 12.5 mg increments were also administered. After finishing the infusion of PCA, the machine was taken off and sent to the anesthesiology department for the evaluation of all records in relation to the deliveries and attempts with the bolus button. In addition to the records of the PCA machine, the number of patients who required additional rescue analgesics was also noted. MBP and HR data were collected at baseline; at PACU arrival; and at 0.5, 1, 2, 3, 6, 12, 18, 24, and 36 h after surgery. The level of sedation (assessed on a 5-point scale—0, fully awake; 1, drowsy/closed eyes; 2, asleep/easily aroused with light tactile stimulation or a simple verbal command; 3, asleep/arousable only with strong physical stimulation; and 4, unarousable) was assessed as well. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: On the basis of a preliminary study, the mean ± standard deviation (SD) of the resting NRS score at 3 h after surgery in the IV-PCA group was 5.35, and the corresponding value for the E-PCA group was 4.38. In order to detect an expected difference of 1 with a SD of 1.8 for the resting NRS score in the dIV-PCA, the obtained sample size in each group was 51 patients with α = 0.05 and β = 0.8. Assuming a possible dropout rate of 10%, 57 patients were determined to be required in each group. Statistical analyses were performed by using SAS software version 9.2 (SAS Inc., Cary, NC, USA) and IBM SPSS Statistics 20 (SPSS Inc., Chicago, IL, USA). All values were shown as mean ± SD, number of patients (proportion), or median (range). One-way analysis of variance was performed to analyze all parametric variables among the three groups, and nonparametric data were analyzed by using the Kruskal-Wallis test. For categorical data, the Chi-square test or Fisher's exact test was used in the analysis when applicable. A linear mixed model was used in the analysis for repeated-measure variables such as NRS, MBP, and HR. Post-hoc analyses with Bonferroni correction were applied when the interaction of group, time, and group by time showed statistical significance. A P value of <0.05 was taken to indicate statistical significance. BODY.RESULTS: Of 190 patients evaluated for eligibility, 171 patients were initially registered and assigned into the 3 groups. Ten patients in the E-PCA group were eliminated because PCA was discontinued owing to persistent hypotension. In the dIV-PCA group, 3 patients were excluded from the analysis for the following reasons: one patient did not receive the allocated intervention because of another surgery, one patient discontinued PCA because of persistent dizziness, and one patient had deleted PCA data due to a mechanical problem of the PCA machine. Five patients in the IV-PCA group were removed from the analysis for the following reasons: one patient did not receive the allocated intervention because of another surgery, three patients discontinued PCA because of persistent nausea, and one patient had deleted PCA data due to a mechanical problem of the PCA machine. The remaining 153 patients successfully completed the study without any complications (Figure 1). The demographic and intraoperative variables were shown (Table 1). Apart from the total administered dose of remifentanil and ephedrine, there were no significant differences among the 3 groups. The total administered dose of remifentanil was higher in the IV-PCA group than in the E-PCA and dIV-PCA groups (Bonferroni corrected P = 0.017 and P < 0.001, respectively). In addition, the patients in the E-PCA group required more ephedrine than those in the IV-PCA group (8.4 ± 9.1 vs. 4.0 ± 4.8 μg; Bonferroni corrected P = 0.013). The pain scores at rest were shown in Figure 2. Postoperative pain intensity was significantly lower in the dIV-PCA and E-PCA groups than in the IV-PCA group, however, it was comparable between the dIV-PCA group and the E-PCA group. After post-hoc analysis with Bonferroni corrections, the NRS scores for resting pain in the dIV-PCA group were lower than those in the IV-PCA group at all time points during the 36 h after surgery (P < 0.01, Bonferroni corrected), and the E-PCA group showed lower NSR scores than those in the IV-PCA group at 0.5, 2, 3, 6, 12, 18, 24, and 36 h after surgery (P < 0.01, Bonferroni corrected). Moreover, patients of the dIV-PCA group required significantly fewer additional analgesic rescues than did patients of the IV-PCA group between 2 and 24 h after surgery, and patients in the E-PCA group needed significantly fewer additional analgesic rescues than those in the IV-PCA group between 6 and 24 h after surgery (Table 2). Figure 3 showed the number of bolus attempts and the number of successful bolus deliveries during the first 36 h after surgery. Patients in the dIV-PCA and E-PCA groups had a significantly lower number of bolus attempts and bolus deliveries than those in the IV-PCA group during the first 24 h after surgery (both P < 0.05, Bonferroni corrected). Significant differences in MBP and HR were observed among groups in the linear mixed model analysis (P = 0.007 and P < 0.001, respectively) (Figure 4). MBP in the E-PCA group was lower than that in the IV-PCA group at 3, 12, and 18 h after surgery, although more ephedrine was administered in the E-PCA group than in the IV-PCA group (P = 0.023, 0.010, and 0.033, respectively; Bonferroni corrected). Furthermore, patients in the dIV-PCA group showed lower MBP than those in the IV-PCA group at 1, 3, 6, 12, 18, 24, and 36 h after surgery (P < 0.05, Bonferroni corrected). HR was lower in the dIV-PCA group than in the E-PCA group at 2, 3, and 6 h after surgery (P = 0.02, 0.01, and 0.02, respectively; Bonferroni corrected). However, no patient in either group required atropine administration. The other postoperative adverse effects were not significantly different among the 3 groups (P > 0.05; Table 3). In addition, there were no patients who exhibited respiratory depression. BODY.DISCUSSION: This prospective randomized study demonstrated that for patients undergoing open gastrectomy, dexmedetomidine in combination with fentanyl-based IV-PCA significantly improved postoperative analgesia than fentanyl-based IV-PCA, which was comparable to thoracic E-PCA. Furthermore, such improved effects could be achieved without hemodynamic instability by using this dexmedetomidine-fentanyl combination as a noninvasive treatment. It is generally recognized that intense pain occurring during the postoperative period may have a major impact on the postoperative clinical outcomes. Insufficient analgesia might cause psychological distress as well as physical impairment, several postoperative complications, and even progression to chronic pain 2, 26. Especially, pain after the major abdominal surgery such as open gastrectomy could lead to restriction of thoracic and abdominal respiration as well as attenuation of vital capacity and tidal volume breathing, which probably have adverse effects on the respiratory drive 27, 28. In addition, it may result in significant cardiovascular changes, cognitive impairment, delayed recovery of bowel motility, and neuroendocrine instability, which will most likely have a deleterious effect on the postoperative recovery process 4. Thus, postoperative pain management concomitant with maintenance of hemodynamic stability is very crucial. In the last few decades, thoracic E-PCA and IV-PCA have been generally used for postoperative analgesia in patients after open gastrectomy 1, 4. Several studies have reported that thoracic E-PCA is considered more effective than IV-PCA in relieving postoperative pain 4, 29, 30. Furthermore, current research indicates that thoracic E-PCA is considered the "golden" standard in the management of pain after the major upper abdominal surgery, owing to its excellent analgesic effects 1, 5-7. However, it is a relatively invasive technique and its application is limited by specific contraindications such as infection or bleeding tendency 7. In addition to these limitations, there is a possibility of several complications such as hematoma, or severe postoperative neurologic deficits resulting from malpositioning of the catheter in the spinal nerve roots 8, 9. Therefore, despite its potential benefits, the clinical use of E-PCA may have even declined because of these types of complications 1, 11. IV-PCA requires a higher dose of opioids in order to acquire satisfactory analgesic effects. This, in turn, produces adverse effects such as nausea, vomiting and pruritus, which causes patients to discontinue the use of intravenous PCA 1, 12, 13. Indeed, in the present study, 3 patients in the IV-PCA group chose to discontinue the use of PCA because of persistent PONV. For postoperative recovery, it is very crucial to amplify pain relief without increasing the adverse effects of analgesics. The multimodal analgesic approach, which involves using analgesics with different action mechanisms, might be a good strategy in the current setting 31, 32. Of the various available multimodal protocols, the combination of an opioid with one or more adjunctive drugs, such as nonsteroidal anti-inflammatory agents, pure opioid antagonists, and ketamine, has been considered the expedient option for IV-PCA in current postoperative pain management 33-35. Dexmedetomidine, an extremely selective α2-adrenergic agonist that has hypnotic, sedative, and analgesic actions and generates sympatholytic responses, does not cause unfavorable respiratory suppression 14-16. Currently, it has been suggested that combination treatment with dexmedetomidine and opioid-based IV-PCA could provide better analgesic and opioid-sparing effects without any remarkable detrimental influences 21-24. However, to the best of our knowledge, no prior studies have investigated the impact of dexmedetomidine in combination with fentanyl-based IV-PCA on the attenuation of postoperative pain intensity in comparison with thoracic E-PCA and IV-PCA. In the present study, we found significantly reduced resting NRS scores in the dIV-PCA group compared with those in the IV-PCA group during the first 36 h after surgery, although the number of bolus deliveries and attempts was significantly lower in the dIV-PCA group than in the IV-PCA group for the first 24 h after surgery; this finding was in accordance with those of previous reports 21-24. Moreover, patients in the dIV-PCA group required significantly fewer additional rescue analgesics during 2-6, 6-12, and 12-24 h after surgery than those in the IV-PCA group (*P = 0.004, P < 0.001, P < 0.001, respectively; Bonferroni corrected). In the dIV-PCA group in comparison with the E-PCA group, comparable analgesic effects were achieved. A tendency was shown that the number of bolus deliveries and attempts were lower in patients of the E-PCA group than those in patients of the dIV-PCA group; however, no statistical difference was observed after post-hoc analysis with Bonferroni correction. Epidural-induced hypotension is also very common, which is partly due to cardio-depressant activity and arteriovenous vasodilation 7, 36, 37. In the present study, persistent hypotension (SBP <90 mm Hg) developed in 10 patients of the E-PCA group. Consequently, these patients were excluded because of the discontinuation of use of the PCA machine (Figure 1). Except for the 10 patients who were dropped from the E-PCA group, none of the patients in all groups developed severe hemodynamic instability (SBP <90 mm Hg, MBP <60 mm Hg). Previous trials have been conducted with various dosages for an infusion rate of dexmedetomidine in PCA mixture from 0.02 to 0.6 μg/kg/ h within the range of the recommended dose by the manufacturer (0.2-0.7 μg/kg/ h) 21-24, 38. In the present study, we selected 0.07 μg/kg/ h as the infusion dose and 0.007μg/kg/ h as the bolus dose with a maximum limit of 0.1μg/kg/ h in order to acquire the postoperative analgesic effect concomitant with maintaining hemodynamic stability. MBP in the dIV-PCA group were significantly lower than those in the IV-PCA group at 1 and 3-36 h after surgery; however, at all time points, the MBP in the dIV-PCA group were >65 mm Hg. The patient who showed the lowest MBP was in the E-PCA group, which was 61 mmHg. Furthermore, 4 patients in the E-PCA group and 3 patients in the dIV-PCA group developed intermittent mild hypotension (SBP <100 mm Hg), with no statistical difference. Moreover, no bradycardia (HR <40 beats/min) that had to be treated with atropine occurred in all of the 3 groups. Thus, these study findings may have clinical implication, considering that low dose of dexmedetomidine-fentanyl combination significantly improved postoperative analgesia while maintaining stable hemodynamics; especially for those patients who have limitations in applying the E-PCA. In addition, no significant difference was detected in postoperative adverse effects among the 3 groups (P > 0.05). The incidence of PONV in our trials was not consistent with the findings of previous reports 21, 38. This discrepancy might be derived from the low doses of dexmedetomidine (infusion rate, 0.07 μg/kg/ h; bolus rate, 0.007 μg/kg/ h; maximum limit, 0.1 μg/kg/ h) used in this study. Moreover, it might also be attributed to the removal of 3 patients from the IV-PCA group because of persistent PONV. This study has several limitations. First, the patients received three different PCA regimens via different routes in accordance with the group allocation. However, we did not control this confounding factor because the objective of our study was to investigate the effect of dexmedetomidine in combination with IV-PCA on pain intensity compared with the standard methods and regimens of PCA. Second, it still needs to be clarified whether the effects of dexmedetomidine in combination with IV-PCA on pain attenuation, compared with those of E-PCA, are dose dependent. In addition, more long-term follow-up data are required to evaluate the effects of dexmedetomidine-opioid combination on postoperative outcomes, including chronic pain. Thus, further investigations are imperative. Third, we included patients with a wide age range (20 to 65 years), who underwent two types of surgeries (subtotal or total gastrectomy). Although the extent of postoperative pain intensity varies depending on the age, sex, and type of surgeries, the similar demographic variables among the 3 groups in the present study may have helped in preventing these variables from affecting the results of this study. Finally, it is uncertain whether the effects of dexmedetomidine on the attenuation of pain intensity were due to analgesic effect of itself or an indirect effect that decrease the remifentanil-induced hyperalgesia by reducing intraoperative remifentanil amounts. Therefore, more studies performed in regard to various setting would be needed. BODY.CONCLUSIONS: Dexmedetomidine in combination with fentanyl-based IV-PCA significantly improved postoperative analgesia in patients undergoing open gastrectomy than fentanyl-based IV-PCA alone, comparable to thoracic E-PCA. Such improved effects could be achieved without hemodynamic instability; furthermore, this approach could be clinically more meaningful owing to its noninvasive nature.

TITLE: Neural Habituation to Painful Stimuli Is Modulated by Dopamine: Evidence from a Pharmacological fMRI Study ABSTRACT: In constantly changing environments, it is crucial to adaptively respond to threatening events. In particular, painful stimuli are not only processed in terms of their absolute intensity, but also with respect to their context. While contextual pain processing can simply entail the repeated processing of information (i.e., habituation), it can, in a more complex form, be expressed through predictions of magnitude before the delivery of nociceptive information (i.e., adaptive coding). Here, we investigated the brain regions involved in the adaptation to nociceptive electrical stimulation as well as their link to dopaminergic neurotransmission (placebo/haloperidol). The main finding is that haloperidol changed the habituation to the absolute pain intensity over time. More precisely, in the placebo condition, activity in left postcentral gyrus and midcingulate cortex increased linearly with pain intensity only in the beginning of the experiment and subsequently habituated. In contrast, when the dopaminergic system was blocked by haloperidol, a linear increase with pain intensity was present throughout the entire experiment. Finally, there were no adaptive coding effects in any brain regions. Together, our findings provide novel insights into the nature of pain processing by suggesting that dopaminergic neurotransmission plays a specific role for the habituation to painful stimuli over time. BODY.INTRODUCTION: The accurate perception of nociceptive information, such as an electric shock, is crucial for survival and depends on the absolute intensity of the stimulus. For instance, increasing the magnitude of aversive stimulation leads to increased pain perception as well as increased neural activity in pain responsive brain regions, including somatosensory cortices, insular cortex and mid/anterior cingulate cortex (ACC; for a review see Iannetti and Mouraux, 2010). Although such absolute coding has frequently been reported, the processing of aversive information also depends on contextual factors. Specifically, neural responses to nociceptive stimuli decrease as a function of repetition. This is known as habituation (Glaser and Whittow, 1953). This habituation effect in response to electro-dermal stimulation was observed in the above-mentioned pain associated brain regions over consecutive experiment blocks, as shown with fMRI (Bingel et al., 2007; Mobascher et al., 2010) and simultaneous EEG-fMRI (Christmann et al., 2007). Similarly, in an fMRI study, Nickel et al. (2014) observed habituation to electrical stimuli in the secondary somatosensory cortex, insula, ACC, dorsolateral prefrontal cortex and inferior parietal lobule. In line with these neural effects, repeated exposure of the same painful stimulus, including heat, leads to decreased pain ratings (May et al., 2012). While neural habituation to pain appears to be a protective mechanism in healthy humans, dysfunctional habituation may play a role in the chronification of pain (e.g., Bingel et al., 2007; Rodriguez-Raecke et al., 2014). Indeed, studies in chronic pain patients, for instance with chronic low back pain, migraine or fibromyalgia, showed attenuated habituation to pain when compared to healthy controls (e.g., Peters et al., 1989; Schoenen et al., 1995; de Tommaso et al., 2011). Importantly, several chronic pain pathologies such as fibromyalgia or burning mouth syndrome have been associated with dopaminergic deficits (e.g., Brefel-Courbon et al., 2005; Wood et al., 2007; Potvin et al., 2009; de Tommaso et al., 2011; Jarcho et al., 2012) indicating a link between habituation to pain and dopaminergic neurotransmission. For instance, patients with Parkinson's disease, which is mainly characterized by a deficit of dopamine, showed attenuated habituation to nociceptive stimulation while they were off medication, while habituation was evident when dopamine was increased by levodopa treatment (dopamine precursor; Brefel-Courbon et al., 2005; Schestatsky et al., 2007). Moreover, formalin-induced nociception can be enhanced through the injection of D2 antagonists into the dorsolateral striatum (Magnusson and Fisher, 2000), and the dopaminergic substantia nigra/ventral tegmental area (SN/VTA) responds to painful stimuli as a function of probability (e.g., in animals: Matsumoto and Hikosaka, 2009; Bromberg-Martin et al., 2010; in humans: Bauch et al., 2014; Pauli et al., 2015) further suggesting a role of dopamine in pain processing. Apart from habituation, contextual predictions also modulate pain processing. For instance, in direct comparison a high intensity electrical stimulation is perceived as more painful than a low intensity electrical stimulation (i.e., absolute coding); however, when high intensity stimulation is expected but the low one is delivered, it may be perceived as less intense. At the neural level, this can be related to neural adaptation which is a general property of neurons (Ohzawa et al., 1982; Brenner et al., 2000; Fairhall et al., 2001; Brown et al., 2008), and, more specifically, to the prediction error mechanism, which quantifies the difference between expected and received outcomes. Importantly, prediction errors adaptively scale according to the expected range of momentarily possible outcomes, which allows neurons to maintain high sensitivity. This neural mechanisms is well established in the reward literature (see below) and known as "adaptive coding" (e.g., Tobler et al., 2005; Bunzeck et al., 2010; Kobayashi et al., 2010; Park et al., 2012; Diederen et al., 2017). For instance, in a single cell recording study in monkeys (Tobler et al., 2005), visual cues were followed with equal probability by either a small or medium reward (i.e., outcome), and another cue was associated with an upcoming large or medium reward. In both contexts, the larger of the two possible reward outcomes (i.e., medium and large) increased activity in the SN/VTA, while the relatively smaller reward led to activity decreases (i.e., scaled prediction errors). Similar forms of adaptive coding have been observed in the human ventral striatum, prefrontal cortex and medial temporal lobe (Tremblay and Schultz, 1999; Nieuwenhuis et al., 2005; Padoa-Schioppa and Assad, 2008; Padoa-Schioppa, 2009; Bunzeck et al., 2010). Moreover, there is evidence that dopamine modulates adaptive coding of appetitive information in the human midbrain and striatum (Diederen et al., 2017), which seem to be functionally connected (Park et al., 2012). With regard to pain processing, several imaging studies have shown that contextual predictions (similar to adaptive coding) modulate activity in higher order pain-associated brain regions, including the insula and ACC (Wiech and Tracey, 2009; Ploner et al., 2011; Leknes et al., 2013; for a different finding see Winston et al., 2014). In particular, Leknes et al. (2013) presented within a block-design moderately painful heat stimuli either in a context with intense pain (i.e., relief context) or in a context with non-painful warm stimulation (i.e., control context). Both contexts were predicted by a unique cue; as such the moderately painful heat stimulus could be the best outcome (i.e., relief context) or the worst outcome (i.e., control context). In line with the literature on adaptive reward processing, pleasantness ratings were dependent on the context (called "hedonic flip" by the authors), and skin conductance as well as hemodynamic responses in the insula and dorsal anterior cingulate were higher in the control context as compared to the relief context. Although this activity pattern provides initial evidence for adaptive coding, the link to dopaminergic neuromodulation has not been demonstrated. In sum, there is independent empirical evidence for: (a) absolute and (b) adaptive coding of painful stimuli, and for (c) neural habituation in pain responsive brain regions. However, the relationship to dopaminergic neurotransmission in healthy human subjects remains unclear. Therefore, we used a double-blind within-subject pharmacological fMRI study (placebo/haloperidol: dopamine antagonist) to investigated the role of dopamine in pain processing with a focus on absolute coding, adaptive coding and neural habituation. To this end, the experiment consisted of two absolute tasks (phase I and phase III), where subjects rated the absolute intensity of an electro-tactile stimulus (low, medium, high) received on the back of the hand. Between these two phases, we used a task in which contextual predictions to a nociceptive event were manipulated across trials (i.e., adaptive task, phase II). Here, volunteers were presented with three different visual cues (i.e., triangle, square, diamond) predicting: (1) a high or medium electro-tactile stimulus; (2) a medium or low electro-tactile stimulus; or (3) a high or medium electro-tactile stimulus. We hypothesized neural habituation as well as adaptive coding in pain-associated areas, including the insula and ACC (Leknes et al., 2013), prefrontal cortex and possibly mesolimbic brain regions (SN/VTA, ventral striatum; Bunzeck et al., 2010; Leknes et al., 2013; Winston et al., 2014; Diederen et al., 2016). Moreover, we expected that neural habituation to absolute pain magnitude and adaptive coding diminishes in dopaminergic and pain-associated brain regions, when blocking dopaminergic D2 receptors with haloperidol. BODY.MATERIALS AND METHODS.PARTICIPANTS: Twenty-six participants took part in the fMRI experiment, but only 20 were included in further analyses (mean age: 25 years; age range: 19–28 years; 13 women) due to technical problems with the scanner or digitimer (electrical stimulation). All participants were healthy, right-handed and had normal or corrected-to-normal vision, without a history of neurological, psychiatric, or medical disorders or any current medical problems. This study was carried out in accordance with the recommendations of "Ethikkomission der Ärtzekammer Hamburg" with written informed consent from all subjects. BODY.MATERIALS AND METHODS.TASK: In order to assess dopaminergic effects on pain processing, a randomized double-blind within-subject design (i.e., 1.5 mg haloperidol/placebo) was used. Haloperidol is an established substance to treat psychiatric disorders including schizophrenia. More specifically, haloperidol is a dopaminergic antagonist that blocks mainly D2 receptors (e.g., Kapur et al., 1997). In correspondence with previous research, our dose of haloperidol has been shown to be an acceptable compromise between sufficient D2 receptor inhibition (between 60% and 80%) and minimization of side effects (Kapur et al., 1997). The entire experiment took place in 3 days. During the first day, participants practiced a condensed version of the experiment to ensure high performance in all tasks, which will be explained in more detail below (see Figure 1). Furthermore, they received information regarding the MRI procedure and pharmacological manipulation. Participants were assigned to receive placebo on day 2 and haloperidol on day 3 or vice versa in a randomized and double-blinded fashion. The time interval between placebo-day and haloperidol-day was at least 7 days long (mean interval: 15 days) to allow a washout of haloperidol. Figure 1Experimental design. Participants underwent three phases. In the first and third phase (absolute task), electrical stimulation with three different magnitudes were presented randomly intermixed to the back of the hand. Subjects had to make low/medium/high judgments (see experimental procedures for details). In between both phases, participants took part in the adaptive task, where they learned the association between three contexts and three visual cues that predicted with 50% probability either: (1) a high or a medium stimulation; (2) a medium or a low stimulation; or (3) a high or a low stimulation. Participants made a relative low/high judgment (see experimental procedures for details). The experimental procedure was identical on both days of the pharmacological manipulation. Drug intake was scheduled 2.5 h before the start of the first phase in the MRI scanner, when haloperidol reaches significant plasma concentration (e.g., Andreou et al., 2014), and participants were asked to not eat 2 h before drug intake. Blood pressure was measured and a questionnaire about their subjective state and possible side effects was completed at three time points: before drug intake, 2.5 h after drug intake and after the experiment (i.e., approximately 4.5 h after drug intake). After placing the participant into the MRI scanner, an electrode was placed on the back of their right hand, which was followed by calibrating the magnitude for the highest possible electrical stimulation that was applied during the experiment. Three different stimulation magnitudes were used during the experiment: low, medium and high. The stimulation magnitude was adjusted by varying the number of trains of consecutive electrical pulses of 2 ms each. Electrical stimulation was applied for 500 ms in total (i.e., high stimulation: 10 trains of 2 ms electrical pulses separated by an interval of 50 ms; medium stimulation: six trains of 2 ms pulses separated by an interval of 83.3 ms; low stimulation: four trains of 2 ms pulses separated by an interval of 125 ms). The time window between the first pulse and offset of the last pulse was identical for all shock intensities. This procedure was based on several published studies using the same electro-tactile stimulation system (Haaker et al., 2013; Lonsdorf et al., 2014; Sjouwerman et al., 2015). During calibration, participants were asked to rate the intensity of the electrical stimulation with 10 trains of electrical pulses on a visual analog scale (VAS) ranging from 0 (i.e., electrical stimulation is not perceptible) to 10 (i.e., electrical stimulation is intolerable). An intensity of seven for the electrical stimulation was used as highest possible nociceptive stimulus throughout the experiment. The magnitude of the medium and low stimulation conditions were adjusted accordingly by reducing the number of trains of pulses to six or four, respectively. The experiment consisted of three consecutive phases: an absolute task (phase 1); an adaptive task (phase 2); and again an absolute task (phase 3; see Figure 1). The visual stimuli were presented via a mirror system attached to the head coil of the scanner. Throughout the first phase (absolute task), a fixation cross was presented in the center of a gray screen. A series of in total 60 consecutive electrical stimulations was applied to the back of the right hand. The three stimulation magnitudes were randomly intermixed (20 trials per condition) and each stimulation was separated by an inter-stimulus interval of 3000 ± 100 ms. Participants were asked to judge the magnitude of the electrical stimulation (i.e., low, medium, high) by pressing one of three buttons. The contingency between magnitude and button was randomly assigned across participants. Accuracy and speed were stressed. This phase took approximately 5 min in total. Ten practice trials were shown before the actual task to ensure that participants familiarized themselves with this task. The second phase included a modified version of an established paradigm used in reward studies investigating adaptive coding (Tobler et al., 2005; Bunzeck et al., 2010; Park et al., 2012; Diederen et al., 2017). During this second phase (i.e., adaptive task), three visual cues (60 triangles, 60 squares; 60 diamonds; see Figure 1) were randomly intermixed and presented in central vision for 1500 ms on a gray background. Each cue was followed by a fixation cross that was shown throughout the remaining trial. Three-thousand millisecond after the offset of the cue, an electrical stimulation was applied for 500 ms. The three types of cues were associated with different contexts and predicted the occurrence of an electrical stimulation with either: (1) a high or medium; (2) a medium or low; and (3) a high or low stimulation intensity, whereby both stimulation intensities had the same occurrence probability in each type of context (e.g., in context 2, 50% of the stimulations had a medium and 50% a low intensity). The fixation cross disappeared 4000 ± 100 ms after stimulation offset. Note, that this paradigm is an event-related design. On the first day (see above), all volunteers explicitly learned the association between the type of visual cue and stimulation magnitude in the behavioral lab outside of the scanner. The task was to judge whether the relatively low or high electrical stimulation was applied by pressing one of two buttons using the right index or middle finger, respectively. The response buttons were randomized across participants. Before the actual task in the scanner on day 2 and 3, participants were familiarized again with the task in a short practice consisting of 10 trials and accuracy and speed was stressed. The adaptive task was split into four blocks of 45 trials, and took approximately ~40 min including breaks. Approximately 5 min after phase two ended, participants continued with the absolute task (i.e., phase 3) consisting of a different trial randomization than in phase 1. Finally, structural scans were acquired for approximately 15 min (see below). Note that the relatively short jitters of 100 ms (Figure 1) might be longer in order to significantly improve design efficiency and should be adjusted in future experiments. BODY.MATERIALS AND METHODS.FMRI DATA ACQUISITION: The fMRI acquisition was performed on a 3-tesla system (Siemens Trio) with echo planar imaging (EPI). During functional imaging, 48 T2*-weighted images per volume (i.e., covering whole head) with BOLD contrast were obtained (matrix, 64 × 64; 48 oblique axial slices per volume angled at −30° along the anteroposterior axis; spatial resolution: 2 × 2 × 2 mm; TR = 2870 ms). For each subject, functional MRI data were acquired for both absolute tasks each consisting of 140 volumes and the adaptive task that was split into four scanning sessions each consisting of 148 volumes per session. Six additional volumes per scanning session were recorded at the beginning of each block to allow for steady-state magnetization; these were excluded from the analyses. At the end of the experiment, anatomical images of each subject's brain were collected using multi-echo three-dimensional fast low angle shot (FLASH) acquisition for mapping T1 (TR = 19 ms), and magnetization transfer (TR = 24 ms) at 1-mm3 resolution (Weiskopf and Helms, 2008; Steiger et al., 2016). BODY.MATERIALS AND METHODS.FMRI DATA ANALYSIS: All fMRI images were realigned to the first volume, unwarped, spatially normalized to the Montreal Neurology Institute space, and smoothed with a 4 mm Gaussian kernel using SPM12 (Ashburner et al., 2014). The fMRI time series data were high-pass filtered (cutoff = 128 s) and whitened using an AR(1) model. For each subject, we computed four first-level analyses by including each combination between pharmacological treatment and task (i.e., (1) placebo and adaptive task; (2) placebo and absolute task; (3) haloperidol and adaptive task; and (4) haloperidol and absolute task). First, we defined four regressors for the absolute task under placebo treatment: one regressor for each stimulation magnitude (i.e., low, medium and high), and one regressor for trials with incorrect responses (i.e., errors). Second, we computed a first-level analysis for the same task under haloperidol treatment, which included the same four types of regressors as in the former analysis. A third first-level analysis was computed for the adaptive task under placebo. Here, we defined 10 regressors: one regressor for each of the three cues (i.e., (1) high/medium; (2) medium/low; and (3) high/low); one regressor for each of the six possible stimulation outcome (i.e., (1) high stimulation in context 1; (2) medium stimulation in context 1; (3) medium stimulation in context 2; (4) low stimulation in context 2; (5) high stimulation in context 3; and (6) low stimulation in context 3), and one regressor for trials with incorrect responses (i.e., errors). Fourth, we computed a first-level analysis for the adaptive task under haloperidol including the same regressors as defined in the third analysis. Note that the order of the trials for the different stimulation magnitudes was fully randomized. To capture residual movement-related artifacts, six covariates were included (the three rigid-body translation and three rotations resulting from realignment) as regressors of no interest in all four models. Two separate second-level random-effects analyses (i.e., for absolute task and adaptive task) were computed on the contrast images resulting from the four first-level analyses. In the first second-level model, the hemodynamic effects of stimulation magnitude were entered into a 2 × 2 × 3 way analysis of variance (ANOVA) with the factor drug (placebo/haloperidol), time (phase 1/phase 3) and the factor stimulation magnitude (low/medium/high) to test for absolute pain effects and the influence of dopamine across time. Second, a 2 × 3 × 2 way ANOVA with the factor drug (placebo/haloperidol), stimulation context (context 1/context 2/context 3) and stimulation outcome (relatively low/relatively high) was computed to investigate adaptive pain effects and the impact of dopamine (see Figure 1). This enabled us to investigate main effects and their interactions. We were interested in brain regions associated with an adaptive coding effect irrespective of drug treatment, which was realized by contrast weights of "1" for all relatively high stimulations and "−1" for all relatively low stimulations (see "Results" section). All contrasts were initially thresholded at p < 0.001 (uncorrected). Since we hypothesized a priori regions for the adaptive effect (i.e., insula and ACC: Leknes et al., 2013), we corrected for multiple comparisons using small volume correction (SVC; p < 0.05, family-wise error (FWE)-correction, k > 5 voxels). The masks for the regions of interest were defined using the WFU-Pickatlas (Maldjian et al., 2003). Otherwise, FWE was used as implemented in SPM12. The sources of the effects were localized by overlaying the SPMs on a T1-weighted group image, which was generated by averaging all normalized T1-images, respectively (spatial resolution of 1 × 1 × 1 mm). BODY.RESULTS.BEHAVIORAL RESULTS: QUESTIONNAIRE REGARDING SIDE EFFECTS: To account for potential drug effects on subjective well-being (i.e., side effects: dry mouth, dry skin, blurred vision, lethargy, nausea, dizziness and headache), participants rated their subjective state on a 7-point Likert scale three times during the experiment (before drug administration, 2.5 h after drug intake and at the end of the experiment after ~4.5 h). Mean ratings across the seven symptoms for the three measurements and both drug treatments are displayed in Table 1. The 2 × 3 ANOVA on subjective well-being (mean rating across seven side effects) with drug (placebo/haloperidol) and time (before drug intake, after 2.5 h, at the end) revealed neither a main effect of drug nor an interaction between time and drug (all p's > 0.531). The analysis resulted in a significant main effect of time (F(1.82,43.67) = 5.64; p = 0.009). However, post hoc paired t-tests revealed no difference between the three time points when p values were Bonferroni corrected (all p's > 0.113). Table 1 Likert ratings for potential side effects (1 = no side effects; 7 = extreme side effects) before, during and after the fMRI experiment in the placebo and haloperidol condition ( n = 20). Treatment 1st assessment (before DI) 2nd assessment (2.5 h after DI) 3rd assessment (4.5 h after DI) Placebo 1.54 (0.46) 1.43 (0.30) 1.44 (0.39) Haloperidol 1.54 (0.44) 1.41 (0.39) 1.53 (0.42) Values represent mean ratings across-subject means (SD). DI, drug intake . During calibration, averaged intensity ratings for the highest electrical stimulation on the VAS from 0 (i.e., electrical stimulation is not perceptible) to 10 (i.e., electrical stimulation is intolerable) did not differ between placebo (M = 1.42 mA; SD = 0.45; range 1.42–2.3 mA) and haloperidol treatment (M = 1.45 mA; SD = 0.63; range 1.44–3.00 mA; p = 0.840). BODY.RESULTS.BEHAVIORAL RESULTS: ABSOLUTE TASK: Mean reaction times (RTs) are displayed in Table 2. A 2 × 3 × 2 way ANOVA with the factor drug (placebo/ haloperidol), stimulation magnitude (low/medium/high) and time (phase 1/phase 3) revealed a significant main effect of stimulation magnitude (F(2,1.48) = 31.37; p < 0.0001). Mean RTs for medium stimulation were slower than for high (t(19) = −3.76; Bonferroni corrected p = 0.009) and low stimulation (t(19) = 8.50; Bonferroni corrected p = 0.003); and participants responded faster to high stimulation than to low stimulation (t(19) = 4.17; Bonferroni corrected p = 0.009). Analyses showed no main effect of drug, time or an interaction between any of the factors (all p's > 0.191). Table 2 Reaction times (RTs) of hits in the absolute task for the first and third phase ( n = 20). Drug Phase Stimulation magnitude Low Medium High Placebo I 1115 (193) 1384 (192) 1259 (180) III 1119 (206) 1373 (293) 1191 (168) Haloperidol I 1111 (301) 1465 (227) 1241 (377) III 1135 (172) 1390 (210) 1268 (257) Values represent RTs of correct responses across-subject means in ms (SD) . The mean proportion of hits for each condition is displayed in Table 3. A 2 × 3 × 2 way ANOVA on the mean hit rate revealed no main effect of stimulation magnitude, drug treatment and there was no interaction between any factors (all p's > 0.081). Table 3 Proportion of hits in the absolute task for the first and third phase ( n = 20). Drug Phase Stimulation magnitude Low Medium High Placebo I 0.83 (0.11) 0.70 (0.14) 0.72 (0.21) III 0.76 (0.19) 0.74 (0.19) 0.77 (0.19) Haloperidol I 0.74 (0.24) 0.66 (0.15) 0.64 (0.22) III 0.76 (0.16) 0.75 (0.14) 0.68 (0.12) Values represent proportions of correct responses across-subject means (SD) . BODY.RESULTS.BEHAVIORAL RESULTS: ADAPTIVE TASK: The mean RTs are depicted in Table 4. A 2 × 3 × 2 ANOVA with the factor drug (placebo/haloperidol), context (1/2/3) and relative stimulation outcome (low/high) revealed a significant main effect of context (F(1.91,40.03) = 44.03; p < 0.0001), stimulation outcome (F(1,19) = 34.07; p < 0.0001) and a significant interaction between context and stimulation outcome (F(1.94,36,84) = 85.49; p < 0.0001), but there was no effect of drug (p's > 0.214). Mean RTs in response to the relatively lower stimulation were faster than to the high stimulation (see Table 4). Mean RTs in context 3 were significantly faster than RTs in context 1 and context 2, where the physical difference between the stimulation is relatively smaller. Mean RTs in context 1 and 2 did not differ. Mean RTs in response to the low stimulation magnitude in both contexts (i.e., context 2: t(19) = 10.53; p < 0.0001); context 3: (t(19) = 3.42; Bonferroni corrected p = 0.009; other p = 0.107) were faster in comparison to the response to the relatively high stimulation outcome. There was no difference in mean RTs between the medium and high stimulation (i.e., context 1). Table 4 RTs of hits in the adaptive task ( n = 20). Drug Context Relative stimulation magnitude Low High Placebo 1 (med/high) 1276 (243) 1272 (272) 2 (low/med) 1104 (145) 1447 (212) 3 (low/high) 1041 (161) 1119 (186) Haloperidol 1 (med/high) 1377 (243) 1289 (267) 2 (low/med) 1127 (147) 1489 (254) 3 (low/high) 1070 (219) 1158 (221) Values represent RTs of correct responses across-subject means in ms (SD) . The proportion of correct responses is summarized in Table 5. A 2 × 3 × 2 ANOVA revealed a significant main effect of context (F(1.79,34.07) = 75.65; p < 0.0001) and a significant interaction between context and stimulation outcome (F(1.34 25.54) = 4.09; p = 0.043), but no interaction with the factor drug (other p's > 0.089). Separate post hoc t-tests for each context revealed no difference between both stimulation outcomes (all p's > 0.093). Table 5 Proportion of hits in the adaptive task ( n = 20). Drug Context Relative stimulation magnitude Low High Placebo 1 (med/high) 0.90 (0.09) 0.87 (0.12) 2 (low/med) 0.86 (0.12) 0.78 (0.18) 3 (low/high) 0.96 (0.06) 0.96 (0.08) Haloperidol 1 (med/high) 0.86 (0.12) 0.87 (0.10) 2 (low/med) 0.87 (0.10) 0.75 (0.17) 3 (low/high) 0.93 (0.10) 0.93 (0.08) Values represent proportions of correct responses across-subject means (SD) . BODY.RESULTS.IMAGING RESULTS FOR ABSOLUTE TASK (PHASE 1 AND 3): Imaging data for the absolute task in phase 1 and 3 were analyzed in a 2 × 3 × 2 ANOVA with the factors drug (placebo/haloperidol), stimulation magnitude (low, medium, high) and time (phase 1/phase 3). In a first step, we aimed to pinpoint brain regions that were associated with the processing of pain irrespective of drug and stimulation magnitude across both phases and drug conditions (i.e., main effect of pain, Figure 2A and https://neurovault.org/images/57884/). Whole brain analyses revealed BOLD effects in left central operculum (−54 −18 22; 24981 voxels; FWE corrected p < 0.0001); right putamen (14 6 −10; 396 voxels; FWE corrected p < 0.0001); right angular gyrus (34 −62 50; 62 voxels; FWE corrected p = 0.014); and cerebellar vermal lobules I-V (4 −62 −12; 117 voxels; FWE corrected p < 0.0001). Although this activation pattern is widely distributed and rather unspecific in nature, it confirms previous studies on pain processing because it involves the expected brain regions (see "Introduction" section). Figure 2Main effect of electrical stimulation in the absolute task (A) and adaptive task (B). The highlighted voxels exhibited increased BOLD activity during noxious stimulation p < 0.001, family-wise error (FWE)-corrected at cluster level. Maps of activations are superimposed on a T1 group template. Note that both tasks require different cognitive demands; therefore, both main effects are not formally compared. In a second step, the analysis revealed BOLD responses that linearly coded absolute stimulation magnitude irrespective of drug treatment (i.e., main effect of absolute coding) in the left midcingulate cortex supplementary motor cortex (−2 −14 48; 206 voxels; FWE-corrected p < 0.0001) and left postcentral gyrus (−38 −24 48; 422 voxels; FWE-corrected p < 0.0001). Finally, a significant 2 × 3 × 2 interaction effect was observed in the left postcentral gyrus (−36 −24 46; 123 voxels; FWE-corrected p < 0.0001) and the left midcingulate cortex (−2 −14 48; 91 voxels; FWE-corrected p < 0.0001), contralateral to the stimulated region. This effect was driven by a linear activity increase of stimulation magnitude in phase 1 for both drug conditions (placebo and haloperidol), and, importantly, this linear effect disappeared in phase 3 in the placebo but not haloperidol condition (see Figure 3). Thus, a time-dependent neural habituation to painful events (placebo) was blocked by haloperidol. Figure 3fMRI results in the absolute task. Analyses revealed a significant interaction between stimulation magnitude, drug and time in left postcentral gyrus (A) and left midcingulate cortex (B). Under placebo in phase I, activity increased as a function of stimulation magnitude (absolute coding), and this effect was absent in phase III (i.e., habituation). Under haloperidol, however, there was no significant habituation from phase I to phase III. The significant interaction effects are highlighted by the asterisk. Maps of activations are superimposed on a T1 group template. Error-bars denote one standard error of the mean. BODY.RESULTS.IMAGING RESULTS FOR ADAPTIVE TASK: Imaging data for the adaptive task (phase 2) were analyzed in a 3 × 2 ANOVA with the factors drug (placebo/haloperidol), context (high/medium, medium/low, high/low). First, we were interested in pain-related activity independent of context and drug (i.e., main effect of pain, Figure 2B and https://neurovault.org/images/57885/). BOLD effects were evident in bilateral postcentral gyrus (−44 −22 56; 83447 voxels; FWE-corrected p < 0.0001; 64 −14 32; 1403 voxels; FWE-corrected p < 0.0001); left supplementary cortex/mid-cingulate cortex (−2 −6 50; 4075 voxels; FWE-corrected p < 0.0001); bilateral anterior insula (−42 −6 6; 582 voxels; FWE-corrected p < 0.0001; 44 −2 4; 587 voxels; FWE-corrected p < 0.0001). Similar to the main effect of pain in the absolute task, this activation pattern is widely distributed and rather unspecific in nature, but it confirms previous studies on pain processing (see "Introduction" section). In the next step, we identified brain regions with adaptively coded responses to stimulation outcome irrespective of drug treatment (i.e., main effect of adaptive coding). FWE-corrected whole brain analysis did not reveal any significant effects. Subsequently, a SVC was performed, using the insula and ACC as masks (see "Introduction" section: Ploner et al., 2011; Leknes et al., 2013); this analysis also did not reveal any significant main effects or interactions (p < 0.05, FWE-corrected, k > 5 voxel). BODY.DISCUSSION.SUMMARY OF MAIN FINDINGS: We investigated the neural mechanisms underlying pain processing with a focus on contextual effects and dopaminergic neurotransmission in a pharmacological fMRI experiment. Our data reveal that haloperidol decreased neural adaptation to electrical stimulation in pain-associated areas (Iannetti and Mouraux, 2010), including left postcentral gyrus and left midcingulate cortex. While this habituation effect was evident under placebo, it was absent after haloperidol intake (see Figure 3) suggesting a direct link to dopaminergic neurotransmission. BODY.DISCUSSION.FMRI FINDINGS FOR ABSOLUTE TASK: Absolute coding of pain magnitude in the left midcingulate cortex and left postcentral gyrus is consistent with previous fMRI studies reporting BOLD increases as a function of pain intensity in brain regions associated with pain or saliency processing. This includes primary and secondary somatosensory cortices, the insular cortex and mid/ACC (Coghill et al., 1999; Büchel et al., 2002; Iannetti and Mouraux, 2010). Specifically, the midcingulate cortex has been involved in cognitive control processes, preparation of defensive responses to threat (for review, see Shackman et al., 2011) and in discriminating between pain intensities (Büchel et al., 2002). The habituation effect in the left postcentral gyrus and mid-cingulate cortex in the placebo group corresponds to previous research showing that habituation to nociceptive stimuli can already be evident within a short period of time (e.g., Milne et al., 1991). For instance, electro-dermal stimulation is associated with a reduction of BOLD responses from the first to the second half of the experiment in primary and secondary somatosensory cortices, the insular and anterior/mid cingulate cortex (Mobascher et al., 2010; see also Ibinson et al., 2004; Bingel et al., 2007; Christmann et al., 2007). Similar findings on habituation have been reported by Rennefeld et al. (2010) and Nickel et al. (2014). Also in line with previous literature, our effects were evident contralateral to the stimulated hand (e.g., Peyron et al., 2000; Bingel et al., 2003). However, in comparison to former habituation studies, we used three different stimulation magnitudes, which all showed habituation over time. The absence of a graded reduction of BOLD response for the three different pain magnitudes in the third phase (i.e., absence of an absolute effect) indicates that habituation to pain is rather an all-or none phenomenon. Importantly, the interaction between drug and time provides evidence for the role of dopamine in pain processing. At the physiological level, haloperidol reduces dopamine availability by blocking dopamine D2 receptors (Kapur et al., 1997) and striatal dopamine D2 receptors are known to modulate pain processing (Hagelberg et al., 2004; Potvin et al., 2009). Thus, blocking dopaminergic neurotransmission may have prevented the habituation to electrical stimulation across time. Indeed, clinical studies suggest that habituation is reduced in chronic pain patients, such as fibromyalgia or migraine (Valeriani et al., 2003; Montoya et al., 2006; Smith et al., 2008), with a link to dopaminergic deficits (Potvin et al., 2009). For instance, Parkinson's patients showed reduced neural and behavioral habituation to pain stimuli in the absence of levodopa treatment (i.e., dopaminergic stimulation; Schestatsky et al., 2007; see also Martikainen et al., 2015). Moreover, animal models stressed that chronic pain is associated with decreased D2 receptor availability and excitatory functions of D2 neurons in the nucleus accumbens (Schwartz et al., 2014). Finally, intraventricular or striatal microinjections of haloperidol increased acute pain and apomorphine (dopamine agonist) reduced nociception (Lin et al., 1981; Magnusson and Fisher, 2000; Mansikka et al., 2005). Thus, our findings provide evidence for the role of dopamine in pain processing by showing reduced neural habituation following receptor blockage in pain-associated brain regions. BODY.DISCUSSION.NO FMRI FINDINGS FOR ADAPTIVE TASK: Contrary to our predictions, adaptive coding was not evident in the mesolimbic system, including the ventral striatum (e.g., Bunzeck et al., 2010; Park et al., 2012) and dopaminergic SN/VTA (Tobler et al., 2005; Matsumoto and Hikosaka, 2009; Bauch et al., 2014; Diederen et al., 2016). One possibility for this null finding is that the spatial resolution of fMRI could have been too low to dissociate between subsets of SN/VTA neurons showing adaptive coding and others responding in an absolute fashion. Alternatively, adaptive coding within the dopaminergic system might depend on task properties. In fact, hemodynamic responses within the human SN/VTA and ventral striatum were adaptively coded in a paradigm where reward distributions alternated in short blocks (rather than trial wise) and had to be learned throughout the experiment (Diederen et al., 2016). This indicates that adaptive coding to appetitive and aversive information may be more pronounced in implicit and blocked learning paradigms. Together with higher spatial resolution, both aspects should be regarded in future studies. A third possibility is that adaptive coding of nociceptive events does not depend on the dopaminergic mesolimbic system but is driven by other neuromodulators such as the opioid or norepinephrine system (e.g., Wager et al., 2007; Scott et al., 2008). For the insula and ACC, we also hypothesized adaptive coding effects on the basis of a study by Leknes et al. (2013), which could not be confirmed. Here, our null effects might be due to differences in the design (block vs. event related), or related to differences in sensory modality (heat vs. electrical stimuli). A final possibility is that adaptive coding is not as relevant to pain processing as it is for reward. Indeed, rewarding stimuli, such as monetary incentives, have a much wider range, which might require higher fidelity, while aversive stimuli on the other hand have a natural upper limit. BODY.DISCUSSION.LIMITATIONS: There was no behavioral effect of haloperidol in any task of the combined pharmacological fMRI study. Instead, participants were equally able to differentiate between stimulation intensities irrespective of time and drug. This suggests that dopamine does not influence the perceptual and discriminative processes of nociceptive information per se, but may indirectly modulate pain processing via higher cognitive functions, such as learning or valuation processes (see also Becker et al., 2013; Tiemann et al., 2014). Since we only sampled the discriminating performance between different pain magnitudes, it is an open question whether subjective pain ratings to the different electrical stimulation also habituate over time (e.g., Bingel et al., 2007; Mobascher et al., 2010) and vary as a function of haloperidol. Alternatively, the effect of haloperidol on neural processes but not behavior may be due to the relatively low single dosage. Indeed, similar reports (i.e., no effects of drug on behavior) have been published in the field of placebo research (Wrobel et al., 2014) and pain sensitivity (D2 antagonist sulpiride, Becker et al., 2013). As a final remark, we would like to point out that future research needs to include other non-nociceptive types of stimuli to investigate whether the dopaminergic effect on habituation is specific to pain processing. BODY.CONCLUSION: Haloperidol changed the habituation to painful events over time in left postcentral gyrus and left midcingulate cortex. As such, our results point towards a previously unreported mechanism linking dopaminergic neuromodulation and habituation to pain in healthy humans. BODY.AUTHOR CONTRIBUTIONS: EMB, VHR and NB designed research. EMB and CA performed fMRI data acquisition. EMB, NB, CA and VHR interpreted the data. EMB and NB drafted the article. CA and VHR revised the article critically for important intellectual content. EMB, CA, VHR and NB gave final approval for the article to be published; agreed to be accountable for all aspects of the work. BODY.CONFLICT OF INTEREST STATEMENT: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

TITLE: Pentoxifylline Plus Prednisolone versus Pentoxifylline Only for Severe Alcoholic Hepatitis: A Randomized Controlled Clinical Trial ABSTRACT.BACKGROUND:: Prednisolone and pentoxifylline (PTX) have been shown to be individually useful in severe alcoholic hepatitis with Maddrey discriminant function (MDF) score ≥32. Previous report suggests that PTX is probably superior to prednisolone alone. However the efficacy of PTX and prednisolone combination over PTX alone in the management of acute alcoholic hepatitis (MDF score ≥32) is yet unrevealed. ABSTRACT.AIM:: The present study was initiated to find out the efficacy of combined pentoxifylline and prednisolone versus PTX alone in acute alcoholic hepatitis in respect of short and intermediate term outcomes. ABSTRACT.SUBJECTS AND METHODS:: A total of 124 patients with severe alcoholic hepatitis (MDF score ≥ 32) initially were evaluated. 62 patients who fulfilled the inclusion and exclusion criteria were randomized and divided into 2 groups. Group 1 received PTX only, whereas Group 2 received PTX plus Prednisolone. The total duration of follow-up was 12 months. Student's t-test, Chi-square test, the Kaplan-Meier methods were used for statistical analysis. ABSTRACT.RESULTS:: A total of 60 patients, 30 in each group were available for final analysis. In Group-1, 6 patients expired at the end of 1 year (5 within 3 months and another after 3 months). In Group 2, 10 patients expired at the end of 1 year (9 within 3 months and another after 3 months). Though survival probability is higher among Group 1 patients but the difference is not statistically significant. ABSTRACT.CONCLUSION:: The combination of PTX plus Prednisolone yields no additional benefit in terms of mortality and morbidity from that of PTX monotherapy. BODY.INTRODUCTION: Alcohol abuse leading to alcoholic liver disease (ALD) is one of the most important causes of preventable morbidity and mortality in the world. Alcohol abuse is responsible for about 50% cases of liver cirrhosis.[12] As a consequence, alcoholic cirrhosis is one of the most common indications for liver transplantation in Europe and in North America.[34] ALD is a spectrum that ranges from fatty liver to alcoholic steatohepatitis (ASH) and eventually cirrhosis. Simple hepatic steatosis is the most common histological finding and occurs in 90% of heavy drinkers but is rapidly reversible with abstinence. Alcoholic hepatitis or ASH occurs in up to 35% of heavy drinkers and is usually a precursor of cirrhosis.[5] ASH is a clinico-pathological syndrome that denotes hepatocellular necrosis and inflammation. Pathologic characteristics include development of Mallory "alcoholic" hyaline, prominent intra-sinusoidal collagen deposition and infiltration with polymorphonuclear leukocytes of the hepatic parenchyma, usually at the peri-venular area. However histologically, the majority of patients with severe alcoholic hepatitis have either significant fibrosis or cirrhosis of the liver. Hence alcoholic hepatitis is often superimposed on chronic liver disease and is one of the most important causes of acute on chronic liver failure.[6] The pathogenesis of liver injury in acute alcoholic hepatitis remains elusive. Genetic factors and immune-mediated mechanisms have been postulated along with a direct hepatotoxic effect of alcohol.[7] Impaired immune response, endoplasmic reticulum stress, mitochondrial dysfunction and free-radical injury induced by alcohol and its acetaldehyde adduct metabolites, Kupffer cell activation and cytokine production, have important roles in accentuating the hepatocyte injury and disease precipitation.[89] Studies have shown a linear relationship between tumor necrosis factor-alpha (TNF-α) receptors and mortality from acute alcoholic hepatitis.[10] Glucocorticosteroids represent the most widely accepted but yet the most debatable therapy in patients with severe ALD. Corticosteroids have been shown to reduce cytokine production, suppress the formation of acetaldehyde adduct metabolites and neo-antigens and inhibit the production of collagen.[11] However down-regulation of steroid sensitivity, risk of infection and a rational therapeutic strategy of corticosteroids are all crucial aspects that need evaluation. Pentoxifylline (PTX), a non-specific phosphodiesterase inhibitor with anti-inflammatory (TNF-α inhibition) and anti-fibrogenic properties, has been found to be useful in patients with acute alcoholic hepatitis with maddrey discriminant function (MDF) score ≥32.[1213] Studies have shown that it also has a significant role in decreasing the risk of developing hepatorenal syndrome.[1415] The beneficial effects are believed to occur through down regulation of TNF-α, interleukin (IL)-1, IL-6, transforming factor-beta, interferon-gamma, stellate cell activation and procollagen I messenger ribonucleic acid (mRNA) expression.[16] Prednisolone and PTX have been shown to be individually useful in severe alcoholic hepatitis with DF score ≥32.[1417] A previous trial performed by our team had shown some evidence that PTX is probably superior to prednisolone alone.[15] However the efficacy of PTX and Prednisolone combination over PTX alone in the management of acute alcoholic hepatitis (MDF score ≥32) is yet unrevealed. The present study was initiated to probe into this issue and to measure the short and intermediate term outcomes of such therapeutic interventions. BODY.SUBJECTS AND METHODS: Totally 124 chronic alcoholic patients attending the liver clinic, outpatient department or emergency medical services of the Medical College and Hospital, Kolkata, were initially considered. The duration of the study was from January 2010 to August 2012. Patients were initially examined clinically, evaluated and subsequently were admitted for the duration of the study. The study protocol was approved by the institutional ethical committee. All the patients underwent investigations for liver chemistry (liver function test, prothrombin time), complete hemogram, random blood sugar, urea, creatinine, electrolytes, viral markers such as hepatitis B surface antigen, anti-hepatitis C virus antibody, hepatitis A virus immunoglobulin M (IgM), hepatitis E virus IgM, serum ceruloplasmin, 24-h urinary copper (as and when required), anti-human immunodeficiency virus (HIV) antibody, chest X-ray, antinuclear antibody, upper gastrointestinal (G.I.) endoscopy and Doppler abdominal ultrasound (as and when required). Patients who had a history of chronic alcohol intake of more than 50 g/day with clinical and biochemical features of severe alcoholic hepatitis (MDF score ≥32 and Aspartate aminotransferase [AST]: Alanine aminotransferase [ALT] >2:1 with absolute value of AST < 500 I.U/L and ALT < 200 IU/L) were included. Patients with any other potential etiology of liver injury (acute or chronic viral hepatitis, autoimmune liver disease, Wilson's disease) even in the background of chronic alcohol intake were excluded from the study. Furthermore patients who were positive for HIV antibodies or patients with a history of abstinence from alcohol in the last month were excluded. Patients with infection, sepsis or spontaneous bacterial peritonitis, acute pancreatitis, G.I. bleeding, hepatorenal syndrome or any other severe associated disease such as uncontrolled diabetes mellitus, systemic hypertension, heart failure, pulmonary disease or malignancy at the time of inclusion or in the previous 3 months were also excluded. Model for end-stage liver disease (MELD) score and Glasgow alcoholic hepatitis score (GAHS) and Child-Pugh score were calculated for all patients who were included in the study. Only those patients who gave a prior informed written consent for pharmacotherapy were considered for the final study. The recruited patients were then divided into 2 groups by a computer generated randomization table. Group 1: Patients receiving PTX only. Group 2: Patients receiving PTX plus prednisolone. Now the pharmacotherapy was started within a week of admission. Patients in Group 1 received PTX (Trental tablets, sanofi Aventis, Mumbai, India) at a dose of 400 mg thrice daily orally and a placebo tablet in the place of prednisolone for the first 4 weeks. Patients in Group 2 received prednisolone tablet (Wysolone, Wreath, Mumbai, India) at a dose of 40 mg once daily for 4 weeks and PTX tablets at a dose of 400 mg thrice daily for the same duration. During the study concomitant treatment with salicylates, non-steroidal anti-inflammatory drugs, budesonide, anti-TNF-α agents, Vitamin E, s-adenosyl methionine or ursodeoxycholic acid were not allowed. The investigator, who allocated the patients to the groups, administered the drugs and collected the clinical and laboratory data, as well as statisticians were all blinded regarding the nature of the pharmacotherapy. All the patients were admitted in the wards of the Department of Medicine, Medical College and Hospital, Kolkata for the initial period of 4 weeks. All investigations such as liver function tests, prothrombin time, electrolytes, renal profile and abdominal ultrasound were repeated after the initial 4 weeks of pharmacotherapy. After the initial 4 weeks, the study was opened and the patients allocated to different groups were revealed. Patients in Group 1 (PTX) who tolerated the drug well, continued to receive the medication at the same dose for the next 8 weeks and then stopped. After 4 weeks of initial therapy, the dose of prednisolone in Group 2 was tapered by 5 mg/week over a period of 7 weeks and then stopped and received PTX like Group 1 patients. Only those patients who were clinically stable at the end of 4 weeks were discharged and later followed-up in the liver clinic. All the patients were counseled for strict alcohol abstinence at the time of discharge from the hospital. The patients were reviewed at least once a month in the liver clinic. During follow-up, all the patients were examined clinically and asked about drug compliance, intake of alcohol or potential drug adverse effects. Liver function tests, prothrombin time, renal function test, electrolytes and abdominal ultrasounds were performed as and when required. MDF, MELD, GAHS and Child-Pugh scores were calculated for all the patients during follow-up. BODY.SUBJECTS AND METHODS.STATISTICAL ANALYSIS: Student's t-test was used for analysis of continuous variables. Chi-square test was used for categorical variables. All results of continuous variables were expressed as mean (SD). Survival curves were estimated according to the Kaplan-Meier method and were compared using the log-rank test. Results were considered statistically significant at P < 0.05. SPSS statistics version 17.0 (developer IBM) software was used for statistical analysis. BODY.RESULTS: A total number of 124 patients were initially evaluated. Out of them, 64 patients who fulfilled the inclusion criteria without any other potential etiology of liver injury or severe co-morbid states were considered. One patient refused to give consent for the study and another one refused admission. 62 patients who fulfilled the inclusion and exclusion criteria and who gave informed written consent were randomized and divided into 2 groups. Group 1 (PTX only) had 31 patients while Group 2 (PTX plus Prednisolone) had 31 patients. The total duration of follow-up was 12 months, with the patients being examined and evaluated in the liver clinic on a monthly basis. One patient in Group 1 developed severe vertigo within 7 days after starting PTX and one patient in Group 2 withdrew voluntarily from the study and hence they were excluded. Hence, a total of 60 patients, 30 in each group, were considered for the final analysis. The baseline parameters (clinical and biochemical) of the total 60 patients, 30 in each group were comparable and shown in Table 1. Table 1 Comparison of base line parameters of patients receiving PTX (Group 1) versus those receiving PTX plus prednisolone (Group 2) in the treatment of severe alcoholic hepatitis (mean [SD]) In Group – 1, 6 patients expired at the end of 1 year. Out of the 6 patients lost, three patients succumbed in the first 4 weeks; two expired between 4 weeks to 3 months of therapy, while one expired between 3 months to 1 year of therapy. PTX therapy was stopped prematurely (i.e., within 3 months) in 5 patients because of the development of life-threatening complications, all of them died. Out of 5 patients, 3 patients expired following massive (GI) bleeding, 1 patient was lost of progressive hepatic encephalopathy and 1 patient died of sepsis, not responding to conservative management including antibiotics, fluid therapy etc., 1 patient died of GI bleeding after 3 months of therapy. In Group – 2, 10 patients expired at the end of 1 year out of these 10 patients, 1 succumbed in the first 4 weeks, 8 more were lost between 4 weeks to 3 months of therapy while 1 more was lost between 3 months and 1 year of therapy. PTX plus prednisolone therapy was stopped prematurely (i.e., within 3 months) in 9 patients due to development of life-threatening complications. Out of these 9 patients, 3 patients developed sepsis and all of them died of septic shock despite of adequate antibiotics and hemodynamic support, 1 patient was lost of progressive hepatic encephalopathy, 2 patients had upper GI bleeding and succumbed to hemodynamic failure, 3 patients died of hepatorenal syndrome, not responding to conservative management. This was in sharp contrast to Group – 1, in which none of the patients developed hepatorenal syndrome. 1 patient died of GI bleeding after 3 months of therapy. Although, the survival probability was higher among patient receiving only PTX (83.3% [25/30] at the end of 3 months and 80% [24/30] at the end of 1 year) when compared to those receiving PTX plus prednisolone (70% [21/30] at the end of 3 months and 66.7% [20/30] at the end of 1 year), P values were not statistically significant as elaborated in Kaplan-Meier analysis shown in Figures 1 and 2 (P = 0.37 at 3 months and P = 0.32 at 1 year, Log rank test). Figure 1Survival curves (Kaplan-Meier life table analysis) of patients receiving pentoxifylline (PTX) (Group – 1) as compared to patients receiving PTX plus prednisolone (Group – 2), at the end of 3 months of therapy Figure 2Survival curves (Kaplan-Meier life table analysis) of patients receiving pentoxifylline (PTX) (Group – 1) as compared to patients receiving PTX plus prednisolone (Group – 2), at the end of 1 year of therapy Twenty seven patients in Group 1 and 29 patients in Group 2 were evaluated in the liver clinic at the end of 4 weeks. The study was opened at this point in time and the allotment of patients to the different groups was revealed. The investigation done at baseline were repeated and the patients were re-admitted if deemed necessary. The patients were followed-up on a monthly basis and the investigations were repeated at the end of 3 months, 6 months and 1 year. Although the patients did relatively well beyond 3 months of follow-up, only 1 more patient from each group succumbed to the disease. The morbidity/complication profiles among the two groups were comparable [Table 2]. Nausea followed by vomiting and dyspepsia were the most common adverse effects encountered in both groups. Patients receiving PTX complained of nausea and vomiting more frequently, whereas dyspepsia was more common among those receiving PTX plus prednisolone. Within a week after initiation of PTX, 2 patients from PTX group and 1 patient from PTX plus prednisolone group also developed mild vertigo which subsided spontaneously within few days. Oral thrush, impaired glucose tolerance, poor wound healing were some of the significant problems faced by the patients in the PTX plus prednisolone group. Table 2 Morbidity/complications profile of patients receiving PTX (Group 1) or PTX plus prednisolone (Group 2) in the treatment of severe alcoholic hepatitis Table 3 shows the baseline profile of patients who succumbed to various complications, as compared to those surviving at the end of the study (i.e., 1 year). It shows that baseline MDF, MELD, GAHS, Child-Pugh score and International Normalized Ratio (INR) were significantly higher among patients who succumbed to the disease as compared to those who survived [P < 0.001, Table 3]. It also shows that baseline albumin was significantly lower among patients who expired as compared to those who survived [P < 0.01, Table 3]. Table 3 Comparison of baseline parameters of patients succumbing to various complications to those surviving at the end of the study (mean [SD]) Table 4 shows the progression of MDF, MELD, Child-Pugh and GAHS score in patients over 12 months. MDF score and MELD score were observed to be significantly lower among patients receiving PTX at the end of 4 week, as compared to those receiving PTX plus prednisolone (P < 0.05 and < 0.01 respectively). But there were no significant differences in MELD, MDF, Child-Pugh and GAHS score between two groups at the end of 3 months, 6 months and 1 year. Table 4 Progression of scores evaluating the severity of liver disease of patients receiving PTX (Group 1) as compared to those receiving PTX plus prednisolone (Group 2) in the treatment of severe acute alcoholic hepatitis BODY.DISCUSSION: More than 17 controlled trials and at least 13 meta-analyses have reported the effects of corticosteroids in the treatment of ALD in the past 40 years with conflicting results. However current guidelines of the American College of Gastroenterology recommend the use of glucocorticosteroids in the treatment of patients with severe alcoholic hepatitis as defined by the Maddrey score (DF ≥ 32).[1819] PTX, a non-specific phosphodiesterase inhibitor, with combined anti-inflammatory and anti-fibrotic properties, has also emerged as an alternative therapeutic approach. The beneficial effects are believed to occur through various mechanisms such as inhibition of phosphodiesterases, increased cAMP level and down regulation of TNF-α, IL-1, IL-6, TGF-β, interferon-gamma, stellate cell activation and pro-collagen I mRNA expression.[16] It has been shown to block the activation of hepatic stellate cells in culture.[20] The use of (PTX) in the treatment of severe alcoholic hepatitis has been supported by Akriviadis et al.[14] in a double-blind placebo-controlled trial, which showed that 24% of PTX-treated patients and 46.1% of control patients died during hospitalization. The survival benefit of PTX was found to be related to a significant reduction in the development of hepatorenal syndrome. Although favorable results of (PTX) used in the treatment of severe alcoholic hepatitis patients with a MDF score ≥32 have been previously reported, it is not currently recommended as a first line treatment for alcoholic hepatitis owing to lack of evidence for its efficacy as compared to the standard treatment with corticosteroids. In a very recent issue of World Journal of Gastroenterology,[15] our team compared for the first time the two treatment modalities head to head in a randomized controlled study, demonstrating the advantage of PTX over corticosteroids in terms of patients' survival and risk-benefit profile. Prevention of Hepatorenal syndrome may account for the survival advantage of PTX over corticosteroids in patients with severe alcoholic hepatitis. In the present study, we compared the efficacy of combining PTX to prednisolone over PTX alone. The group receiving PTX and prednisolone had no additional improvement in survival. There was a trend toward reduced survival in the combination group (70% at the end of 3 months and 66.7% at the end of 1 year) when compared to those receiving only PTX (83.3% at the end of 3 months and 80% at the end of 1 year), although statistically non-significant. This increased mortality in PTX plus prednisolone group may be explained by the complications of steroids such as sepsis, GI. bleeding. MDF score in PTX group was significantly lower than PTX plus prednisolone group at the end of 28 days of therapy (34.89 [8.29] in Group 1 vs. 43.53 [20.19] in Group 2, P = 0.04). MELD score in the PTX group was also significantly lower than PTX plus prednisolone group at the end of 28 days of therapy (15.26 [2.22] in Group 1 vs. 18.76 [5.70] in Group 2, P = 0.004). So combination of PTX and prednisolone therapy was no way superior to PTX alone. One study suggested that patients with a MDF score >54 were at a higher mortality risk from use of steroids than from not being treated.[21] In our study, only 1 patient survived among 9 patients with MDF score >70, those who received PTX plus prednisolone. Three patients in the PTX plus prednisolone group died due to hepatorenal syndrome but none died of this complication in the PTX group. In spite of increased occurrence of nausea and to a lesser extent vomiting, among patients in the PTX group, they were not severe enough to warrant termination of therapy. Within a week after initiation of PTX, 2 patients from PTX group and 1 patient from PTX plus prednisolone group developed mild vertigo which subsided spontaneously within few days. Oral thrush, impaired glucose tolerance, poor wound healing were some of the significant problems faced by the patients in the PTX plus prednisolone group. Retrospectively, on analyzing different liver function scores at the time of inclusion, higher MDF, MELD, GASH, Child-Pugh score, INR and low albumin level were associated with the occurrence of increased mortality among patients with severe alcoholic hepatitis. Limitation of our study is inability to document histological changes in two groups of patients because of unavailability of transjugular liver biopsy in our hospital. So no therapeutic advantage or mortality benefit was observed by combining PTX with prednisolone. However, further studies with a larger cohort of patients are needed to confirm our findings. BODY.CONCLUSION: ALD is a major global health burden and newer horizons in the treatment will help better medical control of this disease entity. PTX being one such important molecule has emerged with a promising role. Our present study thus reveals a very important observation that combining these two modalities yields no additional benefit in terms of mortality and morbidity from that of PTX monotherapy.

TITLE: Vitamin A status and body pool size of infants before and after consuming fortified home-based complementary foods ABSTRACT.BACKGROUND: Home fortification using sachets of micronutrient powder (e.g. "Sprinkles") is a food-based approach offering an alternative to high dose vitamin A (VA) supplements for infants. The primary objective was to investigate the impact of VA-home fortification on infant VA pool size. The secondary objective was to compare VA status of infants assessed by the modified relative dose response (MRDR) test before and the 13C-retinol isotope dilution (13C-RID) test in the same infants after vitamin A supplementation. ABSTRACT.METHODS: A randomized-controlled trial was conducted in 7–9 month old infants in Ghana. Eligible children were randomly allocated to receive a daily sachet of "Sprinkles" with or without VA for 5 months added to complementary foods. The MRDR test indirectly determined VA liver reserves at baseline and the 13C-RID determined VA body pool at follow-up in the same cohort of children. ABSTRACT.RESULTS: At baseline, the MRDR values (95 % CI) for infants were comparable in the intervention and control groups: normal at 0·032 (SD 0·018) (0·025–0·038) and 0·031 (SD 0·018) (0·024–0·038), respectively. After intervention, total body stores (TBS) and liver retinol concentrations did not differ between intervention and control groups; TBS were 436 (SD 303) and 434 (SD 186) μmol, respectively, and estimated liver concentrations were 0·82 (SD 0·53) and 0·79 (SD 0·36) μmol/g liver, indicating adequate reserves in all children. ABSTRACT.CONCLUSIONS: Both the MRDR and 3C-RID tests confirmed that the infants had adequate VA status before and after home fortification of their complementary foods. These tests offered more information than serum retinol concentrations alone, which predicted VA deficiency using current suggested cutoffs not corrected for inflammation status. BODY.BACKGROUND: The growth rate of breast-fed infants in developing countries during the first 6 months of life is comparable to that of infants in developed countries. However, infants in developing countries deviate from this satisfactory growth pattern after this period [1]. This has been attributed to lack of nutrient-dense complementary foods and is further exacerbated by persistent micronutrient deficiencies [2] thus making children in developing countries vulnerable to diseases and death during the weaning period. One means of addressing this problem in poor communities where infants and young children consume monotonous cereal-based diets, is by feeding infants complementary foods containing micronutrients, such as vitamin A (VA), iron and zinc sprinkled on the food immediately before feeding [3–5]. Vitamin A deficiency is a public health problem in many countries and diminishes the ability of young infants to fight infections predisposing them to an increased risk of early death [6]. Infections occurring during the infant's life lead to increased risk of morbidity [7, 8], VA excretion in urine, and increased VA requirements [9]. High dose supplements are an effective way to ward off the deleterious effects of VA deficiency [10], and reduce mortality [11] and severe morbidity [12, 13] in children 1–5 y of age in less developed countries. Children are born with low VA stores and depend on their mother's milk for VA. Before an infant is introduced to complementary foods, the mother may not be able to provide enough VA to boost the child's liver stores if she herself has low VA stores [14] or does not consume rich sources of VA during lactation. Home fortification is a food-based approach offering an alternative to administration of high dose VA supplements directly to infants and young children [3]. A novel practical formulation of micronutrient powders in single dose sachets, commonly called "Sprinkles", was developed for home fortification of weaning foods to address the problem of micronutrient deficiency in young infants. "Sprinkles" can be added once daily to any complementary food immediately before serving. Sachets typically contain iron and zinc; vitamins A, C, and D; and folic acid [15]. Sprinkles can be used to meet the infants' high VA requirements for rapid growth after 6 months of age [15–17]. The success of home-based strategies needs to be evaluated by assessing VA status [18]. Serum retinol concentrations, which are homeostatically controlled yet depressed during times of infection due to the acute phase response [19, 20], are only useful when liver reserves are severely depleted but many children suffer from a marginal VA status [21]. The modified relative dose response (MRDR) test indirectly determines VA liver reserves. As liver VA reserves become depleted, apo-retinol-binding protein accumulates in the liver. A challenge dose of 3,4-didehydroretinyl acetate is administered and the response of 3,4-didehydroretinol (DR)-holo-retinol-binding protein complex is measured in the serum ~5 h after dosing [21–23]. The MRDR test is a categorical indicator of VA status and is typically positive at <0.1 μmol retinol/g liver [21]. The MRDR test distinguishes between moderately inadequate and adequate VA status, based on the ratio of DR to retinol (DR:R) in serum after dosing [24]. Stable isotopes are used to determine the VA body pool by using deuterium or 13C-retinol as a tracer [25, 26]. The tracer dilution technique is the only indirect measure that provides a quantitative estimate of total body VA pool size [27] and stable isotopes lack the potential deleterious effects of radioisotopes on human health [28]. The primary objective of this study was to investigate the impact of VA-home fortification on infant VA pool size using the 13C-retinol isotope dilution (13C-RID) test at follow-up among children who received "Sprinkles" with or without VA added to complementary foods. VA status of infants was determined at baseline with the MRDR test because it requires a smaller volume of blood and is less expensive to analyse than the 13C-RID test. Thus, although the two methods were not used concurrently at baseline and endline, the secondary objective was to use the MRDR test and the 13C-RID test in the same cohort of infants because this has not been done before. BODY.METHODS.STUDY SITE: The trial was carried out in 7 villages surrounding Kintampo located in the Brong Ahafo Region of Ghana. The district has a resident population of about 140,000, the majority of whom have a relatively poor socioeconomic status [29]. Anthropometric data also indicated a prevalence of stunting of 32 % and wasting of 4 % among children aged 12 months [30]. BODY.METHODS.PARTICIPANTS AND STUDY INTERVENTIONS: This community-based study included infants aged 7–9 months (n 93) and their ages were verified by inspecting their vaccination cards. Children of this age were selected to ensure that weaning had been established after they were identified by trained field workers. Eligible children were enrolled at home and randomly allocated to receive daily "Sprinkles" with or without VA using a computer generated random number table. Eligibility criteria included willingness of mothers to provide consent, to stay in the study area throughout the study duration and to feed the child with the contents of the micronutrient sachets. The child was also expected to eat complementary foods in addition to breast milk, and haemoglobin needed to be >70 g/l. Those in the VA group received a daily dose of a powdered fortificant (MNP-Sprinkles; Mumbai, India) containing 12 · 5 mg of elemental iron (as microencapsulated ferrous fumarate) plus ascorbic acid (30 mg), retinyl palmitate (400 μg RAE), and zinc (5 mg). The control group received a similar fortificant that did not contain VA. Blinding of the intervention was carried out by a neutral group of persons who packaged the supplements in identical packages with codes unknown to the investigators. Field workers delivered weekly supplements to mothers for use 7 d/week. At the conduct of this trial, VA supplementation was a national policy given to infants when they reached 6 months of age through national campaigns. Children enrolled in this study were excluded from taking the routine VA supplementation until the end of the 5-month study by marking their vaccination cards stating that they were enrolled in another study and should not be given routine VA supplementation at 6 months of age as is the practice in Ghana. This was effectively ensured by inspecting the identity cards of all children enrolled to determine if they were part of any other ongoing studies or programs that routinely administer VA supplementation. Infants were followed for 5 months. Mothers were instructed to mix a single sachet of "Sprinkles" with a small amount of food and to add water and sugar as needed to ensure that the child consumed the entire sachet. Breast feeding is universal in this area and mothers were not prevented from breast feeding their infants during the intervention. Where there were two eligible children in a household, only one was randomly selected. This was done to prevent contamination if it happened that the two children belonged to different groups and they happened to share food with each other. BODY.METHODS.SAMPLE SIZE: Sample size was based on previous stable isotope work done by Tondeur et al. [31] in Kintampo. We estimated that 15 infants per group would be sufficient to detect a 5 % difference in VA pool size with a 5 % SD on the basis of a type 1 error set at 0·05 and a 0·8 probability of detecting a true difference between the two groups. Incidentally, at the time this study was performed, fifteen children per group was considered adequate for the determination of the VA body pool size for supplements as stated by the Vitamin A Tracer Task Force [27]. The primary objective served as the basis for the sample size calculation but the sample size requirements for the MRDR test to be descriptive of the VA status as stated by the Vitamin A Tracer Task Force [27] was larger and hence the need for more children to be recruited. BODY.METHODS.STUDY PROCEDURES: A detailed explanation of the purpose, risks, and benefits were verbally explained and consent was sought for children's participation from their mothers. In the presence of a witness, mothers who were literate signed a consent form but fingerprints were obtained for those who could not sign. The child of each consenting mother was issued a study ID card containing identification information, which was used by trained staff to replenish their weekly supply of Sprinkles and for database management. Information on compliance was obtained by collecting the used empty sachets weekly from mothers and because the study was double-blind, compliance was expected to be similar in both groups; however, the allocation of the empty sachets were not verified to determine which group they belonged to in order not to disclose the groups to which the children had been assigned. All information collected was considered confidential and was de-identified. The Institutional Ethics Committee of the Kintampo Health Research Centre (Office for Human Research Protections Federal Wide Assurance Number 00011103 and IRB registration number 0004854) approved the study protocol. The study was registered with clinical trials.gov NCT 01751009. Information was collected on socioeconomic status (occupation), marital, and education status. Morbidity questionnaires were used to collect health data by fieldworkers for the 5 months through surveillance fortnightly, the health of the child was assessed and information was collected on whether the child had been taken to a health facility within the past two weeks. At the beginning and end of the 5 months study period, anthropometric, haemoglobin [32], CRP [33] (QuickRead, Orion Diagnostica, Finland), and ferritin (Spectro Ferritin, Ramco Laboratories USA) [34, 35] assessments were carried out. Haemoglobin concentrations were measured with the use of a portable HEMOCUE-Hemoglobin photometer (Hemocue Inc, Angelholm, Sweden). Haemoglobin was considered low if <100 g/l [36]. Depleted iron stores were defined as ferritin < 12 μg/l and elevated CRP was defined as >5 mg/L. BODY.METHODS.MODIFIED RELATIVE DOSE RESPONSE TEST: The MRDR test involved giving an oral dose of 5 · 3 μmol 3,4-didehydroretinyl acetate dissolved in 290 μl corn oil in the morning using a 0 · 3 ml insulin syringe. The children were dosed at their homes and 5 h later a heel prick blood sample (~500 μl) was taken. The samples were stored on ice away from light in a cooler until transported to the laboratory. Clotted blood samples were centrifuged at 600 X g for 10 min and the serum was stored at –20 °C until shipped. After completion of the trial, samples were shipped frozen to the Vitamin A Assessment Laboratory at University of Wisconsin-Madison. All samples arrived frozen and were immediately stored at -80 °C until analysis. The samples were analysed for DR and R using a standardised method developed specifically for small serum volumes [37]. MRDR values (DR:R) >0 · 06 were used to indicate VA deficiency. BODY.METHODS.EXTRACTION AND HIGH-PRESSURE LIQUID CHROMATOGRAPHY (HPLC) PROCEDURES: The standard HPLC method was followed as published for 200 μl serum [37] except three extractions were made with 300 μl hexanes instead of two [38]. BODY.METHODS.DESCRIPTION OF : Blood samples of 7 children from both groups were randomly taken in May 2010 after the last sachets were used to serve as a measure of natural abundance of 13C [39]. The remaining [33] children (14 VA group; 19 control) were given an oral 1 μmol (288 μg retinol equivalents) dose of 13C2-retinyl acetate followed by 14 d to allow for tracer mixing with the retinol pool in children [40, 41]. Blood (2 ml) was collected from 33 infants for assessment of the VA liver stores and results were obtained for 24 infants because insufficient serum was obtained from some infants. The optimal amount of serum required for the test is 1 · 5 ml, although we were able to get reliable readings on 0 · 5 ml. The samples were analysed using the method of Howe et al. [39] modified by Valentine et al. [42]. The gas chromatography/combustion/isotope ratio mass spectrometer was run as previously described by Howe et al. [39]. BODY.METHODS.CALCULATION OF TOTAL BODY VITAMIN A STORES: Total body VA was calculated using the following mass balance equation, substituting for c, and rearrangement:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \begin{array}{c}\hfill \left({\mathrm{F}}_{\mathrm{a}}\mathrm{x}\ \mathrm{a}\right) + \left({\mathrm{F}}_{\mathrm{b}}\mathrm{x}\ \mathrm{b}\right) = \left({\mathrm{F}}_{\mathrm{c}}\mathrm{x}\ \mathrm{c}\right)\hfill \\ {}\hfill \mathrm{c} = \mathrm{a} + \mathrm{b}\hfill \\ {}\hfill \mathrm{b} = \mathrm{a}\ \left({\mathrm{F}}_{\mathrm{a}} - {\mathrm{F}}_{\mathrm{c}}\right)\ /\ \left({\mathrm{F}}_{\mathrm{b}} - {\mathrm{F}}_{\mathrm{c}}\right)\hfill \end{array} $$\end{document}Faxa+Fbxb=Fcxcc=a+bb=aFa−Fc/Fb−Fc Where Fa = atom percent (%At) of the dose*0 · 01 = 0 · 1 (2 of 20 atoms labeled), Fb = %At at baseline*0 · 01 based on the mean of natural enrichment samples, and Fc = %At at day 14 after dosing*0 · 01 (%At for each of the individual results). Additionally, a = μmol VA absorbed from the dose, which is assumed to be 80 % in this group of infants who are susceptible to multiple infections [41, 43], b = uncorrected body pool at baseline (unknown), and c = μmol VA in body pool after dosing = a + b. It is then corrected for loss of tracer in the body over the 14 d by accounting for the half-life of retinol in young children [44], so the corrected body pool of VA = b x e^(-kt) where k = ln(2)/32 and t = time in days of the serum collection after the dose was administered. Finally, the total body stores (TBS) were corrected for the serum to liver ratio of 0 · 8 because the infants were not fed a low-VA containing diet during the equilibration period, which is supported by one human study [45]. Total liver reserves were assumed to be 80 % of TBS and liver weight was assumed to be 4 % of total body weight in these infants [43, 46]. Of the 14 completing children in the intervention group who were actually tested using stable isotopes, 10 were males and 4 were females, and for the 19 in the control group, 7 were males and 12 were females. BODY.METHODS.DATA MANAGEMENT AND STATISTICAL ANALYSES: Field supervisors checked all forms manually for completeness. Forms were double entered on computers, range and consistency checks performed, and discrepancies resolved with reference to the original form using Microsoft Visual Foxpro version 9 · 0 Data Management Software. Data were analysed using Stata version 11. Simple descriptive analysis of baseline measures (e.g. demographic, socioeconomic, biochemical) was performed across the treatment groups to confirm their comparability. Categorical demographic characteristics were summarised as proportions, while continuous variables were summarised as means. Differences in means of quantitative variables, such as ferritin, MRDR, and CRP, between intervention and controls at baseline and endline were evaluated using t-tests. Normality of residuals for isotopic data was assessed by the Shapiro-Wilk test. Non-parametric analysis was carried out on ranked data. Anthropometric indices of height-for-age (HA), weight-for-age (WA), and weight-for-height (WH) were expressed as z-scores using the WHO Anthro for personal computers, Version 3 · 1, 2010. P < 0 · 05 was considered statistically significant. BODY.RESULTS.ENROLMENT OF SUBJECTS: The studied children were enrolled from January through June 2010. There were 30/47 (63 · 9 %) males in the intervention group and 19/46 (41 · 3 %) in the control group. Characteristics of mothers of children in both groups were similar (Table 1). Seven infants were lost to follow-up before blood samples could be collected for VA analyses leaving 86 eligible children (Fig. 1). Ten mothers moved out of the study area during the farming season, a mother reported that her child was sick and three mothers refused to allow blood samples of their infants to be collected. Blood samples were collected from a total of 72 children for haemoglobin analyses.Table 1Comparison of age of children and baseline data of mothers in the intervention and control groupsa InterventionControl P n 47 n 46NumberPercentNumberPercentParity1–32655 · 32656 · 54–61429 · 81226 · 00 · 09>6714 · 9817 · 4EducationNone2961 · 73167 · 4Primary714 · 9510 · 90 · 80Middle817 · 0715 · 2Secondary36.424 · 3Unknown012 · 1Marital statusMarried3268 · 12963 · 0Living together1225 · 51226 · 00 · 71Widowed0012 · 2Divorced0012 · 2Single unmarried36 · 436 · 5OccupationFarmer38 · 080 · 834 · 073 · 9Trader5 · 010 · 65 · 010 · 90 · 59Professional4 · 08 · 57 · 015 · 2Age, monthsMeanRangeMeanRangeBaseline8 · 27 · 9–8 · 18 · 48 · 1–8 · 70 · 30Endline14 · 113 · 7–14 · 114 · 414 · 0–14 · 70 · 23 aNo significant difference was noted among treatment groups for all characteristics shownFig. 1Numbers of infants at each stage in the trial, and the reasons for any losses to follow up BODY.RESULTS.VITAMIN A STATUS IN INFANTS: Out of 93 children enrolled, 63 blood samples were finally collected for MRDR testing at baseline to determine VA status. Even though 72 children were eligible, nine infants were not presented by their mothers for the final blood draw for the MRDR test due to suspected concerns with blood draw. The MRDR test (n 30 and 33 in the intervention and control groups, respectively) showed that there was no difference in the VA status between the groups even when infants with high CRP were excluded (Table 2). At baseline, the mean ratio (95 % CI) of MRDR for infants in the intervention group represented sufficient vitamin A status 0 · 032 (0 · 025–0 · 038) (SD 0 · 018), values did not differ from those of the control group, i.e., 0 · 031 (0 · 024–0 · 038) (SD 0 · 018). In contrast, the mean serum retinol concentrations were 0 · 812 (SD 0 · 238) (95 % CI 0.73–0.90) and 0 · 781 (SD 0 · 266) (95 % CI 0.69–0.87) μmol/L for the intervention and control groups, respectively, with 34.9 % of the children having a serum retinol concentration below 0 · 7 μmol/L, which is used as a cutoff value for VA deficiency. The MRDR and serum retinol concentrations were not correlated (r = 0.167, P = 0.19). After 5 months supplementation, the vitamin A status was assessed as endline on a subgroup of infants in both groups using the 13C-RID test. Liver retinol concentrations did not differ between groups (P = 0 · 87) and all children had adequate status. The intervention and control groups had TBS of 436 (SD 303) and 434 (SD 186) μmol, respectively. The estimated liver reserves were 0·82 (SD 0·53) and 0·79 (SD 0·36) μmol/g of liver for the intervention and control groups, respectively. Even removing the results of TBS of three potential outliers (545 – 697 μmol) out of the total 24 infants did not demonstrate a significant difference in the liver stores and therefore they were left in the statistical analysis. In this group of infants, both the MRDR and isotope dilution tests indicated adequate liver reserves in all children.Table 2Infant vitamin A status, hematologic and anaemia indexes at baseline and end line in intervention and control groupsInterventionControlLow vitamin A statusy Number%Number%All infants2/296 · 92/345 · 90 · 87Excluding infants with high CRPd 2/229 · 12/267 · 70 · 86Adequate vitamin A by MRDRχ All infantsχ 27/2993 · 132/3488 · 20 · 86Excluding infants with high CRPd 20/2290 · 924/2692 · 30 · 86Baseline indexesMeanMeanHaemoglobin (g/l)103101Number%Number%Prevalence of anaemiab 18/4738 · 320/4642 · 50 · 61Prevalence of low ferritinc 0/320.01/342 · 90 · 33Prevalence of elevated CRPd 7/3221 · 98/3423 · 50 · 87Endline indexesMeanMeanHaemoglobin (g/l)111108Number%Number%Prevalence of anaemiab 8/3423 · 512/3831 · 60 · 44Prevalence of low ferritinc 0/160 · 00/170.0-Prevalence of elevated CRPd 8/1747 · 07/1741 · 20 · 73 aχ ± SD bDefined as haemoglobin concentration <10 g/dl cDepleted iron stores defined as ferritin < 12 μg/l dElevated CRP > 5 mg/L ylow vitamin A status MRDR ≥ 0 · 06 χAdequate vitamin A status MRDR < 0 · 06 BODY.RESULTS.ANTHROPOMETRY AND HAEMATOLOGICAL INDICES: Weight and height of enrolled children did not differ at baseline and endline. There was no change observed in other anthropometric indices between the intervention and the control groups Table 3. The two groups were not significantly different with respect to their anaemia and CRP status at baseline. At the end of the supplementation period, more infants tended to be anaemic in the control group as compared with the intervention group (23 · 5 % vs. 31 · 6 %) but this was not significant (Table 2).Table 3Infant anthropometric status (z-scores) at baseline and end line in intervention and control groupsInterventionControl p Baseline anthropometryMeanSDRangeMeanSDRangeChild weight (kg)7 · 61.037 · 20, 7 · 907 · 31.047 · 0, 7 · 600 · 19Child height (cm)68 · 23.1967.3, 69.268 · 43.0167 · 5, 69 · 30 · 79Height-for-agea -0 · 81 · 16-1 · 18, -0 · 50-0 · 71 · 28-1 · 09, -0 · 330 · 62Weight-for-agea -1 · 01 · 06-1 · 32, -0 · 69-1 · 21 · 27-1 · 60, -0 · 850 · 37Weight-for-heighta -0 · 61 · 07-0 · 95, -0 · 32-1 · 11 · 12-1 · 40, -0 · 720 · 06Endline anthropometryChild weight (kg)8 · 31.777 · 70, 8 · 908 · 31.747 · 70, 8 · 900 · 95Child height (cm)74.13.3072 · 9, 75 · 374 · 53.3173 · 4, 75 · 60 · 61Height-for-agea -1 · 41 · 17-1 · 79, -0 · 95-1.21 · 16-1 · 55, -0 · 770 · 46Weight-for-agea -1 · 30 · 89-1 · 64, -1 · 00-1.31 · 16-1.67, -0.900 · 9Weight-for-heighta -0 · 90 · 72-1 · 19, -0 · 67-1.01 · 21-1.43, -0.610 · 7 aχ ± SD BODY.RESULTS.MORBIDITY ASSESSMENT FOR INFANTS DURING BIWEEKLY VISITS: An assessment of clinical conditions was carried out every other week from the start of supplementation until the end of the 5 months period. There were no differences in any of the conditions assessed, but more infants sought treatment in the intervention group compared with the control group during weeks 12 and 14 (Table 4).Table 4Comparison of morbidity reports for infants at 2 week visits between intervention and control groupsa InterventionControl P InterventionControl P NumberPercentNumberPercentNumberPercentNumberPercent2 weeks2 weeks4 & 6 weeks4 & 6 weeksCough8/4020 · 06/3815 · 80 · 6311/8213 · 47/769 · 20 · 40Diarrhoea12/4030 · 011/3828 · 90 · 9219/8223 · 224/7631 · 60 · 23Refuse breast2/405 · 00/380 · 00 · 164/824 · 92/762 · 60 · 46Feverishness5/4012 · 54/3810 · 50 · 7813/8215 · 815/7619 · 70 · 52Sought treatment in past 2 weeks8/4020 · 04/3810 · 50 · 2520/8224 · 417/7622 · 40 · 768 & 10 weeks8 & 10 weeks12 & 14 weeks12 & 14 weeksCough6/807 · 56/767 · 90 · 928/849 · 52/822 · 40 · 06Diarrhoea23/8028 · 721/7627 · 60 · 8828/8433 · 319/8223 · 20 · 15Refuse breast1/801 · 23/763 · 90 · 280/840 · 01/8291 · 20 · 31Feverishness19/8023 · 726/7634 · 20 · 1514/8416 · 78/829 · 80 · 19Sought treatment in past 2 weeks17/8021.213/7617.10.5116/8419.06/827.30.0316 & 18 weeks16 & 18 weeks20 & 22 weeks20 & 22 weeksCough3/793 · 84/814 · 90 · 701/731 · 45/816 · 20 · 12Diarrhoea20/7925 · 319/8123 · 50 · 7817/7323 · 319/8123 · 50 · 98Refuse breast2/792 · 52/812 · 50 · 981/731 · 45/816 · 20 · 12Feverishness15/7918 · 916/8119 · 70 · 9012/7316 · 423/8128 · 40 · 08Sought treatment in past 2 weeks14/7917 · 712/8114 · 80 · 6215/7320 · 516/8119 · 70 · 90 a% of subjects with side effects as a percentage of the total number of visits for the periods indicatedThere were no reports of convulsions and bulging fontanelles BODY.DISCUSSION: This study explored the use of stable 13C-tracer methodology to assess the VA body pool size in infants who were followed for a 5-months period of micronutrient home fortification with and without VA. Stable isotopes were used because they lacked the potential harmful effects of radioisotopes on human health making them ideal for studying a broad range of metabolic conditions [47]. The baseline VA status of the children who participated was normal as assessed by the MRDR test and the VA status remained normal after the intervention. The mean TBS of VA also did not differ between groups after the intervention. Serum retinol concentrations were <0.7 μmol/L in 34.9 % of the children at baseline, but this may have been due to the fact that ~23 % of them had inflammation, which was assessed with CRP. The approach of using two different methods of assessing vitamin A status at different times meant that it was not possible to compare VA body pool size at any one point using the 13C-RID test. The only option was to compare the VA body pool size of children in the intervention and the control group. This may have limited our power to detect an intervention effect. For ethical reasons, our comparative group was not a true placebo but received other micronutrients like zinc and iron, which are also known to enhance VA status and could have led to the lack of an intervention effect. Zinc is a cofactor in the β-carotene cleavage enzyme potentially making the VA more bioavailable from plant sources [48]. Zinc also is involved in the synthesis of retinol-binding protein and thus may influence transport [49]. The VA status of young infants is known to be influenced by the liver retinol stores at birth, consumption of VA from breast milk and other foods, and losses from infections and parasites [50]. Many infants in developing countries remain VA deficient at 6 months of age after the weaning period and will require additional VA [51, 52]. The present study showed that infants in the two groups had comparable VA status as assessed by the MRDR at baseline and 13C-RID tests at endline. The fact that there was no difference in the vitamin A status of infants between groups suggests that in rural communities in Ghana, this method of home fortification did not significantly improve the VA stores of infants in the intervention group compared with infants in the control group over the study period. This is likely due to the adequate VA status observed in this study. TBS were twice as high as Thai children who had marginal to deficient liver reserves and had no access to fortified foods [40], and half as much as Zambian children who had adequate to hypervitaminotic stores of VA on the background of VA supplementation and fortification [41]. The mean values obtained in these Ghanaian infants (~0.8 μmol/g liver) are the same as the midpoint of two Ghanaian infants (0.77 μmol/g liver) who died from serious infections [53]. In 6 to 12 month old US children, the mean value of vitamin A 0.30 ± 0.21 μmol/g liver is lower than the assessed vitamin A values with Ghanaian infants [54]. Ghana had many VA interventions in place when this trial occurred including VA supplements at immunisation contacts and post-partum supplementation to lactating mothers, which may have been missed on the identity cards. Furthermore, green leafy vegetables are widely consumed [38] and vegetable oil and wheat flour are now VA-fortified [55]. Three main strategies have been implemented for improving the VA status among populations: supplementation, food fortification, and dietary diversification [56]. De Pee et al. emphasised the need for effective VA programs in poor countries to include a mix of supplementation, fortification, and dietary diversification [57]. Filteau and Tomkins have advocated that the choice of strategy is context specific and must take into account climate, the agricultural potential of the region, local infrastructure, food beliefs, and socioeconomic status of the population [56]. The current study reveals that sensitive VA assessment is also necessary to demonstrate whether supplementation or fortification is needed in target groups. Young infants from developing countries are often VA deficient and studies from Bangladesh and Brazil have shown that a quarter to 90 % of children studied had inadequate liver stores assessed by MRDR and autopsy samples, respectively [58]. However, studies in American infants reported no VA deficiency in livers of 6–12 month old infants at necropsy [59]. Assessment of morbidity during our trial did not reveal any difference between those who had been given Sprinkles with VA and those whose sachets did not contain VA. This observation is similar to that found in a trial in Ghana where children were followed weekly to ascertain the occurrence of morbidity. There were no significant differences between the two arms (vitamin A and placebo) with respect to diarrheal and respiratory conditions, but children who received VA had significantly fewer clinical visits and hospital admissions [12]. Villamor and Fawzi have suggested that the protective effect of VA was mediated by a reduction in severity rather than the incidence of infections [60] and this study seems to agree with those findings even though in our study seeking hospital care was used as a proxy for severity. In a trial in Tanzania by Idindili et al., a clinical surveillance system did not confer any clinically important absolute effect on morbidity [61] and this was seen in earlier trials in Ghana in Kintampo [29] but in that trial there were differences in all anthropometric indices between the vitamin A and the placebo groups. The earlier study in Ghana enrolled younger children and gave three doses of 25,000 IU VA at 6, 10 and 14 weeks of age with immunization compared with children in this present study who were between the ages 7–9 months at enrolment. The impact of improvements in VA status is also likely to be related to the extent of deficiency in the population [12, 21]. It should be noted that mothers in the study communities had low socioeconomic status and results of the MRDR tests conducted at baseline showed that all infants had adequate VA status. The mothers may have received high dose supplements post-partum and this may have benefited the infants but no evidence exists to confirm whether this actually happened. No evidence existed, but the infants enrolled in the study may have received VA at their earlier immunization contacts. However, breast-feeding is universal and likely contributed to the adequate liver stores in these children. Vitamin A can be obtained from the diet as preformed VA (retinol and its esterified form, retinyl ester) in dairy and organ meats or as provitamin A carotenoids from vegetable and fruits; although, it is unlikely that infants in the study consumed much of these foods. In developing countries, 70–90 % of VA is obtained from provitamin A carotenoids in plant foods and these are absorbed much less efficiently (20–50 %) depending on VA status and other non-dietary factors [48, 62]. Some of the main staple foods in the studied area in Ghana are millet, sorghum, and groundnuts, which do not contain significant amounts of carotenoids. However, in Europe and the United States, 75 % of dietary VA is from preformed VA and the fortification of foods, such as milk, breakfast cereals, and some snack foods [63]. This study was not able to do more sophisticated comparisons with the MRDR and stable isotope dilution, such as sensitivity and specificity, because there was a significant amount of time in between the MRDR and isotope dilution tests. Future comparisons of biomarkers should consider this. Developing countries, such as Ghana, have often used serum retinol in the assessment of VA status but stable isotope methodology can be used, even though it is more expensive [27], to quantitatively estimate TBS of VA [21, 43]. BODY.CONCLUSIONS: This is the first study to assess VA status using the MRDR test before and stable isotope dilution technique after the intervention in the same children. Both the MRDR and 3C-RID tests confirmed that the infants had adequate VA status before and after home fortification of their complementary foods. These tests offered more information than serum retinol concentrations alone, which indicated VA deficiency. In fact, 34·9 % of the children were diagnosed with VA deficiency using serum retinol at baseline, which WHO defines as a severe public health problem. This is one of the reasons that WHO recommends that serum retinol concentrations should not be used alone as they are homeostatically controlled and do not change unless VA status is deficient [64]. Due to this phenomenon, other assays, such as the MRDR and RID tests, have been developed. In current studies using only serum retinol concentrations to assess VA status, it is highly recommended that CRP and α1-acid glycoprotein be measured to correct serum retinol concentrations [65]. Further population-based research needs to be conducted to determine the feasibility of using stable isotopes to evaluate different VA interventions [66].

TITLE: Dexmedetomidine versus esmolol to attenuate the hemodynamic response to laryngoscopy and tracheal intubation: A randomized double-blind clinical study ABSTRACT.CONTEXT:: Sympathoadrenal response to laryngoscopy and tracheal intubation manifests as transient, but distinct tachycardia and hypertension. ABSTRACT.AIMS:: The objective of this study is to compare the clinical effects of dexmedetomidine with esmolol and control in attenuating the presser response during laryngoscopy. ABSTRACT.SETTINGS AND DESIGN:: A randomized, prospective, double-blind, controlled study. ABSTRACT.SUBJECTS AND METHODS:: We studied consented, 90 adult, American Society of Anesthesiologists physical status I and II patients of either sex, scheduled for non-cardiac surgery requiring intubation. The patients were randomly divided into three groups (n = 30). Group C received placebo, Group E received 2.0 mg/kg of esmolol and Group D received 1.0 μg/kg of dexmedetomidine, intravenously over 10 min and 3 min before induction of general anesthesia. All patients were uniformly pre-medicated, induced and intubated using thiopentone and succinylcholine as per standard protocol. Heart rate (HR), systemic arterial pressures were recorded at baseline, after study drug infusion, after induction, immediately and 3, 5, 7, 10 min after intubation. ABSTRACT.STATISTICAL ANALYSIS:: Analysis of variance and t-test as appropriate. ABSTRACT.RESULTS:: The mean arterial pressure was significantly increased in patients receiving placebo (P < 0.0001) and esmolol (P < 0.0001) after laryngoscopy and intubation compared with baseline value and Group D (P = 0.6294). The rise in HR (P = 0.08481) and rate pressure product (P = 0.0666) at the time of intubation were minimal and was statistically significant up to 15 min in Group D. ABSTRACT.CONCLUSIONS:: Both the drugs attenuated the pressure response. Of the two drugs administered, dexmedetomidine 1.0 μg/kg provides a consistent, reliable and effective attenuation of pressure responses when compared to esmolol 2.0 mg/kg. BODY.I: Laryngoscopy and tracheal intubation are noxious stimuli that evoke a transient but marked sympathetic response manifesting as increase in heart rate (HR), blood pressure these changes are maximum immediately after intubation and lasts for 5-10 min, which may be well-tolerated by normal fit American Society of Anesthesiologists (ASA) 1 patients. In patients with cardiovascular disease the hemodynamic changes may lead to life threatening complications including myocardial ischemia, acute heart failure and cerebrovascular accidents.[1] Treatment modalities include topical lignocaine sprays, deeper planes of anesthesia by inhalational/intravenous (IV) agents or narcotics, calcium channel blockers, vasodilators such as sodium-nitroprusside; nitroglycerine etc.[2] Although there are several methods, research is still going on for attenuation of pressor response to laryngoscopy and intubation.[3] Esmolol is an ultra-short-acting, beta-adrenergic receptor antagonist;[4] with proven efficacy to provide hemodynamic stability during laryngoscopy and tracheal intubation without severe side-effects.[5] In contrast to this, there have been a very few reports on the effects of dexmedetomidine. Dexmedetomidine is an imidazole derivative and highly selective alpha (α)-2-adrenergic receptor agonist.[6] α2 -agonists produce hyperpolarization of noradrenergic neurons and suppression of neuronal firing in the locus cerelous leads to decreased systemic noradrenalin release results in attenuation of sympathoadrenal responses and hemodynamic stability during laryngoscopy and tracheal intubation.[7] The topic of study was selected because tachycardia and hypertension may get further enhanced in response, to laryngoscope and tracheal intubation, proven dangerous to cardiovascular disease patients. In a randomized, prospective, double-blind, controlled study, we compare the safety and efficacy of single bolus IV dose of dexmedetomidine with single bolus IV dose of esmolol and placebo in attenuating hemodynamic response to laryngoscopy and tracheal intubation. BODY.S: After approval of the study protocol by the Institutional Ethical Committee, written informed consent was obtained from each patient. 90 normotensive, ASA physical status I and II patients of either sex, aged 40-60 years, who were scheduled for elective non-cardiac surgery under general anesthesia (GA) requiring endotracheal (ET) intubation, were included in this study. All patients were thoroughly examined and routine investigations were carried out. The patients who refuse to consent, patients whose physical characteristics suggested difficulties in intubation (Mallampati grades III and IV), who had hypertension or cardiovascular, respiratory, neurological, psychological, hepatic, endocrinal, renal disease and taking any medication (e.g. opioids or sedatives in the week prior to surgery), having history of alcohol abuse or drug allergies, pregnant and lactating patients were excluded from the study. Baseline (average of three readings) vital parameters of patients' including HR, systolic arterial pressure (SAP), diastolic arterial pressure (DAP); mean arterial pressure (MAP) and oxygen saturation were recorded in the pre-operative ward. After 1 h patients were taken to the operation theatre. In the operating room an IV line was secured with 18-G venous cannula and Ringer's lactate infusion (6 ml/kg) was started. Routine standard monitors such as pulse oxymetry, electrocardiography (ECG) and non-invasive blood pressure were applied and monitoring started. All the patients were uniformly pre-medicated with IV ondansetron 0.08 mg/kg and glycopyrrolate 0.004 mg/kg, 10 min before induction. The study drugs were premixed to a volume of 10 ml and were presented as coded syringes by an anesthesiologist who is not involved in the study. The patients were blinded to the treatment group and all recordings were performed by an anesthesiologist blinded to the group allocation, thus the study was made double-blinded. The patients were randomly divided into three equal groups of 30 each (Balanced randomization using a computer program, block size 10). The patients in control Group C received 10 ml of physiologic saline, Group E received esmolol 2.0 mg/kg and Group D received dexmedetomidine 1.0 μg/kg as slow IV infusion over a period of 10 min. The patient's HR and blood pressure were stabilized and pre-oxygenated for 3 min after study drug infusion. Then the induction of anesthesia was performed with thiopentone 5.0 mg/kg and then succinylcholine 2.0 mg/kg was administered IV as per standard protocol. The patient's lungs were ventilated manually with 100% oxygen. Laryngoscopy was attempted 90 s after the administration of succinylcholine with Macintosh curved blade number 4 by an anesthesiologist having more than 9 years of experience. The trachea was intubated with appropriate size-cuffed disposable ET tube. Laryngoscopy and intubation was limited to 15-20 s in all patients, failure to intubate within this period was excluded from this study. After confirming the position and fixing the ET tube with adhesive plaster anesthesia was maintained with, 66% N2O in 33% oxygen and 1% sevoflurane in 6 l of fresh gas flow. Bolus IV dose of 0.08 mg/kg followed by intermittent dose of 0.02 mg/kg vecuronium was used for muscle relaxation. At the end of the surgery all patients were reversed with neostigmine 0.05 mg/kg and glycopyrrolate 0.008 mg/kg IV. Patients were extubated after adequate recovery and then shifted to anesthesia recovery room and monitored for complications such as pain, respiratory depression; hypertension, hypotension, bradycardia, drowsiness, rigidity, nausea or vomiting and attended appropriately, rescue treatment was also noted during anesthesia and recovery. Vital parameters such as HR, SAP, DAP and MAP were recorded, at baseline, after study drug infusion, after induction, immediately and 3, 5, 7 and 10 min after intubation and every 5 min there after using multipara monitor. No surgical intervention was allowed throughout the study period of 10 min. The hemodynamic alterations like a decrease in MAP greater than 20% below the baseline value or SAP less than 90 mm of Hg was treated with primarily by increasing the IV fluid infusion rate and then reducing sevoflurane concentration or incremental doses of ephedrine 4 mg bolus IV. Decrease in HR (<50 beats/min) was treated with atropine 0.5 mg IV. BODY.S.STATISTICAL ANALYSIS: After the initial pilot observations, it was decided that a 20% of difference should be the minimum detectable difference of means in all groups. The standard deviation (SD) of residual was also kept same (20% of average difference between the groups). The α value was 0.05 and the power (1-a) of the study was 0.80. Thus, the calculated sample size for each group was 23 patients. Preserving the designing effect it was decided to include 30 patients in each group. Groups were compared for demographic data (age, weight) and hemodynamic parameters (HR, blood pressure) by one way analysis of variance and paired t-test was used for comparison among the groups, while for comparison within the groups unpaired t-test was used. Probability was considered to be significant if less than 0.05. Data are represented as mean and SD. BODY.R: All Cases were selected from general surgery only; all the 90 patients completed the study. The demographic profile of the patients in terms of age, body weight, male:female ratio, ASA status, Mallampati Class were comparable and there were no significant differences among the three groups (P > 0.05) [Table 1]. Table 1 Patient's characteristics The increase in mean HR after intubation was seen in all the three groups. But the mean increase was minimal 5.83% in Group D (4 beats, P = 0.0848), when compared with Group E 14% (9.81beats; P = 0.0152) and Group C 30% (24.9 beats; P < 0.0001), which was highly significant (P < 0.0001). Also, only in the Group D, there was no significant rise of HR at any time interval [Figure 1]. Figure 1Mean heart rate of patients in Groups C-D The mean SAP levels in Group D were significantly lower than Groups C and E immediately after intubation (P < 0.001, P > 0.001 respectively) and until the end of surgery. Esmolol does not prevented the raise in SAP following intubation, but the raise was significantly less (P = 0.0269) when compared with the patients who does not received any drug [Table 2]. Table 2 Comparison of SAP (mm of Hg) in the three groups The DAP levels in Group D were significantly lower than Groups C and E at all times after intubation. In esmolol group, there is a transient raise 21.4% (16.63 mm Hg) in DAP following intubation (P < 0.0001) at other times it remained below the baseline level [Table 3]. Table 3 Comparison of diastolic arterial pressure (mm of Hg) in the three groups The MAP was comparable in all the three groups at baseline level. The MAP decreased following induction, which was significant in Group C (P = 0.024) but not significant in Group E (P = 0.088) and Group D (P = 0.3145). The MAP rose by 30% (28.04 mm Hg) in Group C, 26% (24.00 mm Hg) in Group E and only 2% (1.67 mm Hg) in Group D at intubation. The rise in MAP was highly significant after intubation in Group C (P < 0.0001) and significant in Group E (P < 0.05) which was not significant in Group D (P > 0.05) [Table 4]. Table 4 Comparison mean arterial pressure (mm Hg) level in three groups The rate pressure product (RPP) was calculated as the product of HR and SAP (RPP = HR × SAP). In our study the RPP during intubation revealed a highly significant increase in Group C (76.5%, P < 0.0001) and significant increase in Group E (49%, P < 0.001), whereas the increase was insignificant in Group D (16%, P > 0.0666). These changes were highly significant up to 15 min post-intubation. Although comparing Group E to Group D the increase in RPP in Group E at the time of intubation (P < 0.001) was statistically significant. The rise in mean RPP was least in Group D and highest in Group C [Figure 2]. Figure 2The comprehensive changes in rate pressure product of patients in Groups C-D BODY.D: In this study infusion of dexmedetomidine 1.0 μg/kg prior to induction of anesthesia suppressed the hemodynamic response to tracheal intubation in normotensive patients. This suppression in cardiovascular responses was found to be greater with dexmedetomidine than that resulted from infusion of esmolol 2.0 mg/kg. Tachycardia and hypertension are more common following laryngoscope and ET intubation. Prophylaxis include topical lignocaine sprays, deeper planes of anesthesia by inhalational agents; narcotics, calcium channel blockers, vasodilators such as sodium-nitroprusside; nitroglycerine etc.,[2] but they have got side-effects such as sedation, respiratory depression, hypotension and bradycardia. Dexmedetomidine has sedative, anxiolytic, analgesic and sympatholytic, effects may blunt the cardiovascular responses in the peri-operative period without causing significant respiratory depression. Among the β-adrenergic blocking drugs, esmolol seems to be an appropriate selection for attenuating the hemodynamic response to laryngoscopy and tracheal intubation, because of its cardioselectivity, rapid onset of action and short elimination half-life.[8] There have been several reports discussing the effects of esmolol on both HR and arterial blood pressure during laryngoscopy and ET intubation compared with placebo. Miller et al.[9] in their study have reported that 100 mg of single bolus dose of esmolol was effective for controlling the hemodynamic response to tracheal intubation in a Canadian multicenter trial. Liu et al. who used esmolol infusion to control hemodynamic responses associated with intubation, found significant decreases in HR and SAP prior to induction and post-intubation, the increase was 50% less in the esmolol-treated patients compared to the placebo group.[10] Esmolol decreases the force of contraction and HR by blocking beta-adrenergic receptors of the sympathetic nervous system which are found in the heart, blood vessels and other organs of the body. Esmolol prevents the action of two naturally occurring neurotransmitters epinephrine and nor-epinephrine, there by attenuates the tachycardia and hypertensive responses to laryngoscopy and tracheal intubation. Although esmolol is considered to have significant effect on both tachycardia and hypertensive response following ET intubation, Oxorn et al.[11] concluded that esmolol in bolus doses of 100 mg and 200 mg affects solely the chronotropic response in a significant manner. Kindler et al. found that esmolol administration before laryngoscopy was sufficient to control HR after intubation but it did not affect SAP.[12] Similarly, in this study, esmolol was not as effective on attenuating the hypertensive response as it was on attenuating the chronotropic response to tracheal intubation. In fact, a significant increase in SAP and a transient raise in DAP was observed after intubation compared to the baseline values and when compared with dexmedetomidine the increase in SAP was greater and more significant in this study. Direct acting α2 -adrenoceptor agonists represent clinically significant effects on the anesthetic requirements and on the sympathoadrenal and hemodynamic responses induced by anesthesia including tracheal intubation and surgery. Scheinin et al.[13] reported that 0.6 μg/kg dexmedetomidine decreased, but not totally suppressed, the hemodynamic response to tracheal intubation in healthy individuals. Keniya et al. stated that the pre-treatment with dexmedetomidine 1.0 μg/kg attenuated, but not totally obtunded the cardiovascular response to tracheal intubation after induction of anesthesia.[14] In this study, we did not observed any significant differences in HR and arterial blood pressure values between the baseline and post-intubation values in the dexmedetomidine group. Similar to the two studies mentioned above, the mean percentage variation analysis at the stated moments revealed an absence of any increase in HR, SAP and DAP in dexmedetomidine group suggesting dexmedetomidine as an effective agent for blunting the hemodynamic response to laryngoscopy and tracheal intubation. Bradycardia and hypotension have been reported in studies pertaining to the effect of dexmedetomidine administration on peri-operative hemodynamics.[151617] In contrast to the previously mentioned studies,[161819] we did not detected any excessive reduction in HR or systemic blood pressure values in the dexmedetomidine group compared with other groups. Moreover, in this study neither bradycardia nor hypotension was observed in the patients. Dexmedetomidine has been used IV in doses ranging from 0.1 to 10 μg/kg/h but higher doses have been associated with a significant increase in incidence of bradycardia and hypotension. Rapid administration of dexmedetomidine might produce tachycardia, bradycardia and hypertension followed by hypotension. We administered dexmedetomidine, 1.0 μg/kg slowly, over 10 min in our study hence no bradycardia or hypotension was found in our study. The α-adrenoceptors are involved in regulating the autonomic nervous system and cardiovascular systems. α2 -adrenoceptors are located on blood vessels, where they mediate vasoconstriction and on sympathetic presynaptic terminals where they inhibit epinephrine and nor-epinephrine release.[20] α2 -adrenoceptors are also located within the central nervous system and their activation leads to sedation, a reduction of tonic levels of sympathetic outflow and an augmentation of Vagal activity. This can result in a decrease in HR and cardiac output. The use of α2 -agonists in the peri-operative period has been associated with reduced anesthetic requirements and attenuated HR and blood pressure responses to stressful events.[2122] Monitoring of HR and ECG has shown no evidence of myocardial insult in any of the patients in any group in our study. It is advisable and safe to use dexmedetomidine in patients to attenuate the hemodynamic responses of cardiovascular system during laryngoscopy and ET intubation. BODY.C: Evaluation of baseline and immediately after intubation values, revealed a greater percentage variation in MAP in the esmolol and control groups as compared to the dexmedetomidine group. Therefore, within the constraints of this study we demonstrated that administration of a single dose of dexmedetomidine before GA induction was an effective method for attenuating the hemodynamic response to tracheal intubation.

TITLE: Educational interventions to improve the effectiveness in clinical competence of general practitioners: problem-based versus critical reading-based learning ABSTRACT.BACKGROUND: Evidence suggests that continuing medical education improves the clinical competence of general practitioners and the quality of health care services. Thus, we evaluated the relative impact of two educational strategies, critical reading (CR) and problem based learning (PBL), on the clinical competence of general practitioners in a healthcare system characterized by excessive workload and fragmentation into small primary healthcare centers. ABSTRACT.METHODS: Clinical competence was evaluated in general practitioners assigned to three groups based on the educational interventions used: 1) critical reading intervention; 2) problem based learning intervention; and 3) no intervention (control group, which continued clinical practice as normal). The effect on the clinical competence of general practitioners was evaluated in three dimensions: the cognitive dimension, via a self-administered questionnaire; the habitual behavioral dimension, via information from patient's medical records; and the affective dimension, through interviews with patients. A paired Student ́s t-test was used to evaluate the changes in the mean clinical competence scores before and after the intervention, and a 3 x 2 ANOVA was used to analyze groups, times and their interaction. ABSTRACT.RESULTS: Nine general practitioners participated in the critical reading workshop, nine in the problem-based learning workshop, and ten were assigned to the control group. The participants exhibited no significant differences in clinical competence measures at baseline, or in socio-demographic or job characteristics (p > 0.05). Significant improvements in all three dimensions (cognitive, 45.67 vs 54.89; habitual behavioral, 53.78 vs 82.33; affective, 4.16 vs 4.76) were only observed in the problem-based learning group after the intervention (p > 0.017). ABSTRACT.CONCLUSIONS: While no differences in post-intervention scores were observed between groups, we conclude that problem-based learning can be effective, particularly in a small-group context. Indeed, problem-based learning was the only strategy to induce a significant difference between pre– and post- intervention scores for all three CC dimensions. BODY.BACKGROUND: The Basic Package of Health Services (BPHS) is a set of strategies offered to all citizens by the government through federal and state participation, and it is government policy to provide universal access and coverage in response to priority health needs. This program offers a wide range of health services and involves low cost actions with high impact. The BPHS comprises 12 strategies that include 57 activities, 9 of which are performed by General Practitioners' (GPs) at primary healthcare centers. In order to complete these activities with quality care, GPs must maintain an adequate level of clinical competence. Clinical Competence (CC) reflects the ability of a physician to perform their activities in a healthcare setting when defining and managing a patient's health problems, and it involves problem solving skills (e.g., critical thinking and the application of clinical reasoning) and the ability to work as a team member and to communicate effectively [1]. CC involves three dimensions, the affective, cognitive and habitual behavior domains [2], although some authors refer to the affective dimension as "attitudes" [3]. CC has been defined as a pyramid with four parts: cognitive (what is known), competence (how it is known), performance (how it is proven) and actions (how it is done) [4]. Evaluating a physician's CC provides them with feedback regarding their achievements and their limitations in detecting and resolving clinical problems within their sphere of influence. In this way, they can better target their educational activities to improve any deficiencies detected [1,5,6]. Different strategies have been used in Continuing Medical Education (CME) to influence and improve a physicians ́ CC. One such strategy is collaborative active education (CAE) [7] or collaborative learning [8], which includes Problem-Based Learning (PBL), although the use of this technique in CME is limited [9,10]. PBL is considered a learning technique with great potential and it may be a key to improving medical practice. This type of learning involves an action-reflection-action process in which GPs identify problems from "clinical cases" or "diagnostic, therapeutic or malpractice controversies", they search for new information to attain a better understanding of a problem, and they finally formulate a hypothesis to explain and ultimately solve the issue at hand. PBL helps GPs to resolve problems by stimulating discussion, dialogue, reflection and participation in the educational strategy [11]. This is the most comprehensive CME strategy as it takes in all the variables involved in a group learning process, and it provides GPs with a self-learning opportunity. Another CME strategy is critical reading (CR). This is an active educational strategy that aims to improve in the ability of physicians to become aware of their position on a specific topic "almost automatically". In CR, implied assumptions and core ideas are identified through the debate between physicians and the author, allowing the physicians to identify the strong and weak points of the main arguments in the text. In this way, the physician can propose alternative arguments that may improve upon the authors' viewpoint, leading them to reaffirm or modify their own position. This strategy promotes an analysis of the text through a process of individual learning [12,13], and short learning courses have even been used previously as CME strategies [14]. Each of these strategies has produced mixed results and no single approach is clearly superior to others. The effectiveness of PBL interventions in CME have been systematically reviewed [10]. While an analysis of 6 relevant studies (two randomized clinical trials and four clinical trials) provided limited evidence that PBL improves the knowledge and performance of GPs, or patient health, only 3 of these studies evaluated knowledge, performance and participant satisfaction, of which 2 revealed positive results in all three areas [11,15]. Moreover, in the third study the only positive effect was evident on participant satisfaction [16]. Contradictory results were obtained in two of these studies [11,16] that used different lecture-based interventions, neither of which clearly defined the learning process in the reading group. We have yet to identify a study that compared the effects of PBL with those of CR intervention. PBL involves learning by guided discovery, while CR takes a repetition-based learning approach and significant learning improvements have been described for both strategies [17]. Thus, we sought to determine which of these educational strategies (CR or PBL) is more effective in improving physician CC in primary health services offered by the HISA in the city of Aguascalientes, México. BODY.METHODS.DESIGN AND SUBJECTS: An intervention study was carried out using 3 groups of physicians, 2 of which were subjected to educational intervention strategies (CR or PBL), and a third that acted as the control group. CC was evaluated and compared both within and between groups. BODY.METHODS.SETTINGS: The Health Institute of the State of Aguascalientes (HISA) provides healthcare to a population of nearly 389,000 residents of the city of Aguascalientes, Mexico, which is not covered by the Social Security services. It consists of 12 healthcare clinics employing 68 general physicians. A mean of 5.6 physicians per clinic perform an average of 11.2 medical consultations per day. BODY.METHODS.STUDY POPULATION, GROUP FORMATION, SAMPLE SIZE AND POWER ANALYSIS: As the HISA has no substitute physicians to cover permanent staff and since the health clinics cannot close, a random number list was generated using the EpiInfo program and used to select 38 of the 68 physicians working in the HISA. The physicians not selected covered the hours of the study participants in their clinics. Of the physicians selected for the study, 8 were not included because they were assigned to carry out different activities, or they were on vacation/leave of absence. The remaining 30 physicians were randomly assigned to 3 study groups using an electronic randomization list. A post-hoc power analysis was performed using the G Power 3.1.3 statistical program, as described previously [18]. A two-tailed Student ́s t-test was used to determine whether 10 physicians per study group was a sufficiently large sample to detect statistical differences within (difference of two dependent means) and between (difference of two independent means) groups, with an alpha level set at 0.017 and the effect size (dz) calculating group parameters (mean and standard deviation of each group). BODY.METHODS.CME INTERVENTIONS: The learning objectives were the same for both intervention groups: to improve the effectiveness of primary health care physicians in all three dimensions of CC in 9 priority health care programs (Hypertension, Diabetes Mellitus, Diarrheas and Acute Respiratory Infections, Prenatal Health, Nutrition, Family planning, Women's Health, Tuberculosis and Vaccines). Both interventions ran for 4 days, lasting 8 hours per day. In both interventions a session for each of the priority healthcare programs was planned, with the content focusing on preventive, diagnostic and therapeutic processes, as well as patient-doctor interactions. All the content was considered that were compliant with the quality of clinical healthcare guidelines established by the continuing healthcare quality program of the Mexican Health Ministry. Both interventions were carried out in the education department at the central office of the HISA; this department had classrooms and a library with access to electronic libraries, which were used by the participants after the sessions to gather new information. The first evaluation was carried out 3 weeks prior to the interventions the second took place four weeks post-intervention. The same instruments and data collection techniques were used to evaluate both groups. BODY.METHODS.CR EDUCATIONAL STRATEGY: An educational professional with expertise in critical reading was appointed as the group tutor, and the CR intervention was developed in three phases: 1. Planning. An expert in each of the priority health programs gathered reading material on the issues most relevant to the competence of a general physician in a primary healthcare setting. Up-to-date information was included (original articles, reviews and healthcare programs), and the tutor acted as a consultant and supervisor. 2. Implementation. The sessions were overseen by the tutor with help from a program expert (Chair). Participating physicians received the reading material 8 days before the intervention, which they were asked to review and analyze. At the first session, each of the physicians presented their initial opinion of the subject matter and the core ideas they had extracted from the first reading, and they discussed how these conformed with or differed from their original point of view (based on their experience and prior knowledge). Guided by the tutor, the physicians read the material again in order to identify the key concepts and core ideas. The tutor urged the physicians to concentrate on comparing their prior knowledge and theoretical positions with the arguments presented in the reading material, assessing individually whether it reaffirmed or changed their point of view. The physicians were required to support their opinions at the end of the session and to explain their position to the group, sharing information and describing their individual analyses. The purpose of this intervention was to improve the physician's ability to analyze reading material, systematically debate the associated issues and to enhance their capacity for statement development [19]. 3. Conclusion. At the end of the intervention, a group report was requested by the tutor to gather the opinions of the participants regarding the lectures, the dynamics of the workshop and their tasks. The second and third phases were adapted from Insfran-Sanchez [20]. Figure 1 presents each of the steps in the educational process and the role played by each participant. Figure 1 Educational process and the role of the participants in the Critical Reading Intervention. BODY.METHODS.PBL EDUCATIONAL STRATEGIES: An educational professional with expertise in PBL was appointed as the group tutor, and the intervention was designed in two phases, as described previously [21]. 1. Preparation. An expert on each of the priority health programs designed a clinical case study, using information from real and simulated patients, in which physicians could identify and solve clinical problems. The case studies also incorporated the most important content that a general physician should understand to be competent in a primary healthcare setting. All physicians received the clinical case studies 8 days before the intervention, which they were asked to review and analyze. 2. Tutorial. The strategy ran for 4 days, 8 hours per day. The aim of this intervention was to learn to identify problems, pose questions and debate the best solutions to the clinical problems at hand, a debate that was chaired by the tutor. Each session was divided into two meetings, as described previously [22]. A session for each of the health program lasted approximately 3 hours and a half; 1 hour and a half for the discussion meeting 1 hour for individual study, and 1 hour for the concluding meeting. The strategy was carried out under the direction of the tutor with help from a program expert (Chair). At the discussion meeting, the first 5 steps proposed by Schmidt were completed [23] and the physicians analyzed the problems posed in each clinical case study. After this meeting and prior to the following meeting, the physicians had time for individual study (step 6). In the concluding meeting (step 7), a physician from the group drafted a report on the intervention, describing the participants ́ experience of the presentation and dynamics of the workshop, and the resolution of the tasks involved. Figure 2 presents each of the steps in the educational process and the role played by each participant. Figure 2 Educational process and the role of the participants in the Problem-Based intervention. BODY.METHODS.CONTROL GROUP: This group received no educational intervention, nor any information or documentation relating to priority health programs, although they were evaluated in the same manner as the intervention groups. BODY.METHODS.DATA COLLECTION: Information on the physician's age, sex, seniority and time since medical graduation was collected using a structured questionnaire. The Cognitive Dimension was evaluated by a self-administered questionnaire completed by each physician in their office. The theoretical content of the questionnaires was reviewed by six clinical experts and the structure was reviewed by three researchers. For each priority health program clinical cases were presented, generating 70 items with one best answer Multiple Choice Questionnaires (MCQs). An example of clinical cases with questions is presented in Appendix A. Information from patient files was used to evaluate Habitual Behavior using a data compilation form with a check list. This form was designed in accordance with the quality of healthcare clinical guidelines established by the continuing healthcare quality improvement program of the Federal Ministry of Health for 4 of their priority health programs (Hypertension, Diabetes Mellitus, Diarrheas and Acute Respiratory Infections, Prenatal Health). This served as the basis to compile the forms for the Nutrition, Family planning, Women's Health, Tuberculosis and Vaccines programs. These forms were revised by 6 clinical experts and 3 researchers, and a total of 69 items from the 9 programs were generated, generating a register of the performance of a specific action with dichotomous answers. From the daily patient register of the previous days 2 patients per physician were identified or selected for each priority health program, a total of 18 patients per physician. Thus, each physician was evaluated on 138 items. The Affective Dimension was evaluated by interviewing patients using a validated questionnaire developed by Smith and Falvo, which measures patient perceptions of the patient-doctor interaction [24]. This approach has demonstrated adequate internal consistency (Cronbach's Alpha = 0.80 [25,26]), and was adapted and translated into Spanish. The Spanish questionnaire also demonstrated adequate internal consistency (Cronbach's Alpha = 0.90). Nineteen items were rated on a five-point Likert scale of agreement (1 to 5) that ranged from "strongly agree" to "strongly disagree". The questionnaire was completed in the waiting room of the healthcare clinics directly after patient consultation. Five surveys were administered per physician. Each instrument was applied by the principal investigator 3 weeks before and 4 weeks after the intervention. The quality of the data was evaluated immediately after collection and the EpiInfo Ver. 6.04 program was used to create a database. BODY.METHODS.STATISTICAL ANALYSIS: For each physician the Cognitive dimension was scored as the number of correct answers for the 70 items. From the 18 patient clinical files per physician, the Habitual Behavior dimension was scored as the mean of items for which there was a register in the patient's clinical files of the performance for each of the 138 actions. The scores for the Affective dimension were calculated for each patient as the mean of the 5 point Likert value for the 19 items. From the 5 patients, a mean score was then calculated per physician. For quantitative variables, measures of the central tendency and the dispersion were used to summarize the data. Variables with categorical scales were analyzed using percentages and absolute frequencies. To identify the differences between the three groups at the beginning of the study, qualitative variables (sex) were assessed using a chi-squared test, and quantitative variables (age, seniority and time since medical graduation) with a 1-way ANOVA. Similarly, a 1-way ANOVA was also used to compare pre-intervention scores within each of the 3 dimensions per group. In both cases, a p value of > 0.05 was considered statistically significant. A paired t test was used to identify differences between the means before and after the intervention for each group, and for each dimension. As each dimension represents a dependent variable, the alpha level was set at 0.017 (0.05/3 = 0.017). A 3 (groups) x 2 (times) ANOVA was performed for each dimension, with groups as between and times as within variables, and of their interaction, to compare the pre- and post-intervention scores for each group. Data processing and analysis was performed using the Statgraphics Centurion XV statistical package. BODY.RESULTS: Of the 30 GPs included in this study, 2 were removed from the analysis as they did not adequately complete the course (i.e.: they did not attend at least 90% of the sessions). Thus, 28 GPs completed the course, 9 in each intervention group and 10 in the control group, of which 46.4% were women. The mean age of the GPs was 44.01 (+/−5.94) years and their seniority in the HISA ranged from 1 to 28 years. Time since graduation from medical school ranged from 14 to 29 years. The majority of GPs were over 40 years of age and the average time spent working at the HISA following graduation was 4 years. No significant differences in any of these variables were detected between groups (Table 1). Table 1 Group characteristics   Critical Reading Problem-Based Learning Controls Total Age* 44.55 45.44 44.70 44.01   (± 5.98) (± 4.39) (± 6.88) (± 5.94) Seniority* 15.55 14.11 10.80 13.06   (± 5.65) (± 3.06) (± 7.37) (± 5.72) Time Since Graduation (yrs)* 17.88 17.88 18.40 17.9   (± 5.62) (± 2.71) (± 5.29) (± 5.21) * F test (p >0.05). BODY.RESULTS.INITIAL EVALUATION: Low scores were obtained in the cognitive dimension for all three groups, with the mean ranging from 43.11 in the CR group to 45.66 in the PBL group (maximum score = 70). In the habitual behavior dimension, the mean scores ranged from 52.3 in the control group to 57.44 in the CR group (maximum score = 138), whereas in the affective dimension, the CR group had the highest median scores (4.39). The negative score for skewness in the affective dimension of the PBL group indicates a shift of the peak to the right of the normal distribution, the negative kurtosis indicates that there are more scores in the tails that in the peak, 55.6% of the 9 physicians obtained the best possible score (5 points in the Likert scale). There were no significant differences between these groups in any of the three CC dimensions (p >0.05; Table 2). Table 2 Pre-intervention scores for each of the three dimensions of clinical competence in the three study groups   Mean Standard deviation Skewness Kurtosis F-test p-value Cognitive Dimension  Critical Reading 43.11 8.49 0.58 1.08      Problem-Based Learning 45.66 6.94 - 0.47 0.17 0.25 0.78  Controls 43.90 8.13 1.27 0.48     Habitual Behavioral Dimension  Critical Reading 57.44 13.62 - 0.17 - 0.16      Problem-Based Learning 53.77 13.80 0.25 0.42 0.30 0.74  Controls 52.30 16.66 - 0.95 0.84       Median Lower-upper quartile Skewness Kurtosis Kruskal-Wallis P-value Affective Dimension  Critical Reading 4.39 4.23-4.58 - 0.97 - 0.34      Problem-Based Learning 4.26 4.08-4.68 −2.87 - 3.69 0.53 0.76  Controls 4.26 3.8-4.54 - 0.92 −0.13     BODY.RESULTS.PRE VERSUS POST-INTERVENTION SCORES FOR EACH GROUP: Within the cognitive dimension, significant differences between pre and post-intervention scores were only detected in the PBL group (p > 0.017), in which a 9.22 point increase was observed. A post-hoc power analysis for this PBL group revealed that a sample of 9 physicians provided a power of 98% in detecting statistical differences for the effect size given by the mean scores within this group (Table 3). Table 3 Pre- and post-intervention scores for each study group in the three dimensions of clinical competence     n Mean 95% C.I. S.D. t-test p-value Power Effect size dz         Inf Sup           Cognitive Dimension  Critical Reading Pre 9 43.11 39.28 46.94 8.49 −2.97 0.017 0.51 0.98   Post 9 51.55 47.81 55.29 8.11          Problem-Based Learning Pre 9 45.67 41.83 49.49 6.95 - 5.77 0.000 0.98 1.93   Post 9 54.89 51.15 58.63 5.49          Controls Pre 10 43.90 40.26 47.54 8.13 −2.24 0.051 0.31 0.71   Post 10 49.90 46.35 53.45 8.92         Habitual Behavioral Dimension  Critical Reading Pre 9 57.44 50.24 64.65 13.62 −7.85 0.000 0.99 2.61   Post 9 85.00 79.71 90.28 5.31          Problem-Based Learning Pre 9 53.78 46.57 60.98 13.81 −6.60 0.000 0.99 2.21   Post 9 82.33 77.05 87.62 6.20          Controls Pre 10 52.30 45.46 59.14 16.67 2.51 0.037 078 1.19   Post 10 74.20 69.18 79.21 16.43         Affective Dimension  Critical Reading Pre 9 4.37 4.11 4.64 0.26 5.20 0.000 0.96 0.48   Post 9 4.77 4.66 4.87 0.14          Problem-Based Learning Pre 9 4.16 3.90 4.42 0.58 2.66 0.007 0.82 1.18   Post 9 4.76 4.65 4.87 0.25          Controls Pre 10 4.17 3.92 4.42 0.47 3.14 0.011 0.81 0.14   Post 10 4.79 4.63 4.83 0.25         The control group was the only group in which no significant differences were observed in the habitual behavioral dimension (p > 0.017). Indeed, post-hoc power analysis revealed that the sample size for the Critical Reading and the PBL group (9 physicians per group) had a statistical power of 99% to detect statistical differences for the effect size given by the mean scores within each group (Table 3). Significant differences in the pre- versus post-intervention scores for the affective dimension were detected for all three groups. The post-hoc power analysis revealed the sample size of each group to be adequate, with a statistical power of over 80% (Table 3). BODY.RESULTS.EVALUATION OF PRE VERSUS POST-INTERVENTION SCORES BETWEEN 3 GROUPS: In all three dimensions there were significant differences within the groups in the pre- versus post-intervention scores, yet no significant differences were observed between groups. No interaction between pre- and post-intervention differences was detected in any of the groups for any dimension (Table 4). A post-hoc power analysis revealed that the sample size was insufficient to detect statistical differences in each of the groups for the effect size given by the mean scores between each group after the interventions. Table 4 3 x 2 ANOVA of groups, times and their interaction   F-Ratio Df P-Value Cognitive Dimension       Between Groups 1.03 2 0.3662       Within Times 14.29 1 0.0004       Interaction 0.22 2 0.8007       Groups * Times       Habitual Behavioral Dimension       Between Groups 1.81 2 0.1735       Within Times 55.72 1 0.0000       Interaction 0.37 2 0.6954       Groups * Times       Affective Dimension       Between Groups 0.49 2 0.6172       Within Times 21.83 1 0.0000       Interaction 0.31 2 0.7349       Groups * Times       BODY.DISCUSSION: In this study, we have evaluated the CR and PBL based improvement in CC among groups of general physicians in Mexico. The low CC observed in the initial evaluation for the cognitive and habitual behavior dimensions may be due to physicians not staying up to date on the latest advances in their field, given their heavy workload [27] and working conditions [5]. Indeed, the isolated nature of a general physician ́s work provides little opportunity for regular discussion of clinical matters [28]. Other personal factors, such as a physician's motivation and attitudes, may also be reflected in their CC [29]. In relation to the priority health programs, a low CC in the cognitive dimension has been reported previously among family physicians in Mexico City using similar methods [5,27]. Moreover, similar results were also obtained in New Zealand [28], and low CC in the habitual behavior dimension was identified in standardized patients and audits of the clinical histories of primary care physicians in Spain [6]. The high CC scores observed in the affective dimension may be due to GPs assuming a paternalistic doctor-patient relationship, acting as the "good guy" in the predominant relation model proposed by Epstein [30]. In contrast to our results, deficiencies in the doctor-patient relationship have been reported in other studies [31,32], conflicting findings that may be explained by differences in population expectations not identified by the methods used here. We detected no significant improvement in the cognitive dimension after the CR intervention. By contrast, previous studies of medical students [13] and junior resident doctors [33] reported cognitive improvements following a CR intervention, a discrepancy that may reflect the type of knowledge evaluation used in each study. Here, we used questions relating to clinical case studies that tested the participant ́s application of knowledge, as opposed to the comprehension or interpretation of concepts and principles tested elsewhere. Moreover, the sample size in our group did not had the sufficient power to detect a significant median size difference in pre- versus post-intervention scores, which may require a larger sample in the CR group. The CR group exhibited a significant increase in the habitual behavior score. Although the CR intervention focuses on the acquisition of knowledge, the improvements in habitual behavior were not unexpected. In one of the stages of the strategy, the tutor and participants discuss the incorporation of knowledge in their working environment, mainly focusing on documents relating to healthcare programs. This strategy provides a better understanding of the actions to be recorded in patients' clinical files (habitual behavior dimension). As CR does not modify the affective dimension, this parameter was not evaluated in other studies. Nonetheless, we observed a significant increase in the affective dimension that may be explained by contamination from the PBL group (which does have an emotive focus), since both groups share a small clinical working area. After the intervention in the PBL group, we observed an increase in CC in all three dimensions. PBL is a strategy that promotes learning through guided discovery, increasing medical knowledge and reinforcing procedural skills, thereby increasing the probability that the GP will adequately complete the clinical files of their patients. PBL ultimately aims to provide a "Habitual Behavior" to improve therapeutic diagnostic procedures and skills [17]. A meta-analysis has demonstrated the positive effect of PBL strategies on knowledge application [34]. Indeed, our results for the PBL group matched those previously reported for a group of physicians that improved their knowledge and their therapeutic diagnostic skills for the treatment of headache patients [11]. Increased motivation has also been reported in groups subjected to PBL strategies [35], as well as improved communication and interpersonal skills [36], which may help identify affective problems in the physician-patient relationship. Taken together, these effects contribute to an improvement in the affective dimension in the post-intervention evaluation. After the intervention, the control group exhibited differences in the affective dimension, yet no improvement in knowledge or habitual behavior since they had not been exposed to any educational intervention. The increase in the affective dimension may have been due to a Hawthorne effect [37] whereby physicians who are aware that they are being observed pay special attention to their clinical activities, mainly by improving their relationships with patients. Since there were no significant differences between groups, it is not clear which of the two educational strategies is more efficient in improving the CC of primary healthcare physicians. Indeed, none of the groups were sufficiently large as to detect differences in effect (i.e.: in the differences in the mean scores between groups after the intervention). Based on the mean and S.D. post-intervention values for the CR and PBL groups in the thee CC dimensions, the sample size required to detect a significant difference would be 77 physicians per group, or a total of 154 physicians. In the city of Aguascalientes, the HISA employs only 68 physicians and thus, it will not be feasible to conduct such a study in this context. The use of small groups is common in comparative studies of educational methods [10]. Indeed, small groups may be preferable to plenary sessions to perform CR [20], and small group discussions in PBL have a positive effect on the intrinsic interest of those involved, producing better cognitive and motivational content [8]. Nevertheless, restrictions on sample size in these studies are common. In our study, we used small groups but with a broad evaluation in terms of quantity and content, which allows more consistent CC evaluation in a priority program. However, as in most studies with small groups it would be better to increase the number of groups and participants. A randomized controlled trial has been conducted to investigate the utility and efficacy of guideline dissemination in asthma management, using PBL with a small-group (23 family physicians) and a didactic lecture session (29 physicians), testing knowledge (nine items), skills (seven items) and attitudes (nine items) [38]. In agreement with the present findings study, both groups exhibited significant improvements in the knowledge, skills and attitudes dimensions. Performance varied over time, as evident through the significant main effect for time, although no differences were detected between PBL and more didactic learning sessions in terms of facilitating knowledge gain, knowledge retention, or changes in attitude regarding asthma management. A randomized controlled trial of 118 trainee occupational health physicians compared the effectiveness of PBL to lecture-based learning [39], as in our study, performance scores increased significantly in both groups, although no significant differences were observed between groups. It should be noted that these studies [38,39] used lecture-based formats in the control groups, whereas in this study, both PBL and CR were based on a collaborative active educational approach, which should also be considered when discussing the failure to detect significant differences. Different methods are used to evaluate improvements in CC as a result of continuing medical education. Thus, evaluating the Integral CC alone may fail to identify improvements in specific dimensions, as described in previous studies of medical students and resident doctors [13,33,40]. BODY.DISCUSSION.LIMITATIONS: The present study did not have sufficient power in terms of sample size to determine which of the two interventions was more effective in improving CC. However, the largest and most significant improvement in all three dimensions of CC was detected in the PBL group. In the present study, we used different methods for each of the CC dimensions, each with their own limitations. For example, the Habitual Behavior dimension was evaluated using information from the patient's clinical files, which only analyzes clinical skills within a limited range (omitting examination or patient management skills). Moreover, this information generally only demonstrates that the physician has registered a specific action, without indicating whether it was performed or understood correctly. This particular approach was used as this is the strategy recommended by the ministry of health in México to evaluate a physician's performance. The measure used to evaluate the affective dimension determines the patient's relative satisfaction. As in most measures of satisfaction, a ceiling effect is observed in the PBL group, reflecting an overestimation of this dimension. The values for skewness (−2.87) and kurtosis (−3.69) in this dimension for the PBL group indicates that most of the scores are shifted to the upper value of the Likert scale, 55.5% of the 9 physicians obtained the best possible score (5 points in the Likert scale) showing an important ceiling effect. Neither the CR nor the Control group showed a floor or a ceiling effect. Increased motivation and improvement of communication and interpersonal skills had been reported in groups subjected to PBL [35,36] therefore we cannot rule out that the effect observed is a true effect of the intervention. Nevertheless in future studies of the affective dimension of CC using this questionnaire, a seven point Likert scale, as well as an increment in the sample size may give results with higher variability. Some contamination occurred between the intervention groups and the control group, due to their sharing of common work spaces in small clinics. In future studies, this effect may be prevented by randomizing the clinics participating, whereby all physicians receive the same educational intervention rather than randomizing physicians into specific the study groups. Potentially confounding characteristics of the educational process were not analyzed, including the time physicians dedicated to their tasks outside of the sessions, the quality of the educational resources used in the strategies, the intrinsic motivation of the physicians, and their satisfaction with the learning experience. However, the consistency of our results with those of other studies in which these variables were analyzed [38,39] suggests that any effect of these variables was equivalent in all groups. BODY.CONCLUSIONS: While we found no significant differences between study groups, we can conclude that the PBL strategy led to the greatest increases in post-intervention scores and that it was the only strategy that produced a significant difference pre- versus post-intervention in our group of physicians for all three CC dimensions. These findings are in agreement with a previous study that reported no improvement in CC scores using PBL when compared with other conventional methods such as CR [41]. However, a study of graduate physicians reported that PBL was more effective than a lecture format in improving scores in the cognitive dimension [11]. PBL is an educational strategy that can be applied according to the competence of the study participants, taking into account differences in educational training, continuous education and health institutions. BODY.APPENDIX A: Sample of cases and questions for one of the priority health programs (Diabetes Mellitus). BODY.APPENDIX A.PREVENTION: Case 1: A 26-year-old man, with family history of a father with DM2 under medical treatment. The patient works as a clerk in a government office. He does not practice any physical activity after work. He has been a smoker since he was 15 years old smoking up to 7 cigarettes daily. He takes between 5 to 7 alcoholic drinks twice a week. He assists to a regular medical checkup in his health center. PE: BMI 32.7, BP 118/82, HR 81 bpm. Acanthosis nigricans in neck grade 1. 1. The General Practitioner informs about the presence of the following risk factor for DM2. a) Age, sedentary and smoking habit. b) Age, sex and family history of diabetes. c) Sex, Acanthosis nigricans, smoking habit. d) Family history of diabetes, obesity and sedentary. e) Family history of diabetes, smoking habit and age. 2. As a General Practitioner, you would recommend the following screening strategy. a) This patient does not require screening test because he is less than 45 years old. b) Inform the patient to return in 3 years for a capillary blood glucose test. c) Request a HbA1c test. d) Request a fasting capillary blood glucose test. e) Request an oral glucose tolerance test. 3. The result of the screening test was: 90 mg/dL: What is the course of action to follow? a) Repeat the capillary blood glucose test. b) Repeat the capillary blood glucose test in 3 years. c) Request a fasting plasma glucose test. d) Request an oral glucose tolerance test. e) c) Request aHbA1c test. BODY.APPENDIX A.DIAGNOSIS: Case 2: A 35-year-old woman, with family history of paternal grandfather with DM2. The patient smokes 6 cigarettes daily, takes alcoholic drinks every 15 days (approximately 4–8 cups) and has a sedentary life style. Her General Practitioner asks for laboratory tests having the following results: glucose 116 mg/dL, Total Cholesterol 286 mg/dL, LDL 160 mg/dL, HDL 34 mg/dL, Triglycerides 295 mg/dL. PE: BMI 33.5, BP 130/90, HR 78 bpm. 1. What would it be the diagnosis based on previous information? a) Obesity, high blood pressure, and type 2 Diabetes Mellitus. b) Obesity, glucose intolerance, and mixed dyslipidemia c) Obesity, high blood pressure, and glucose intolerance d) Obesity, mixed dyslipidemia, and altered fasting glucose level e) Overweight and type 2 Diabetes Mellitus. 2.-What others laboratory test would you request to complete your diagnosis? a) Repeat a fasting plasma glucose test. b) Request an oral glucose tolerance test. c) Request a HbA1c test. d) Request a General Urine test. e) Request a fasting plasma glucose test. 3. The patient returns with the results of laboratory tests having fasting plasma glucose of 109 mg/dl, and 2-hour glucose of 190 mg/dL. What is the diagnosis? a) Type 2 Diabetes Mellitus. b) Type 1 Diabetes Mellitus. c) Results are normal. d) Altered fasting glucose. e) Impaired glucose intolerance. BODY.APPENDIX A.TREATMENT: Case 3: Male 46 years old. Patient's father was diabetic since 55 years old with history of ischemic heart disease. Patient's mother has essential hypertension. The patient has been smoking since 20 years old, a pack daily for the last 2 years. His life style is sedentary. He attends public health services with a report of fasting plasma glucose test of 110 mg/dL. PE; BMI 37.4. BP 142/80. You request an oral glucose tolerance test reporting a fasting plasma glucose of 104 mg/dl, and 2-hour glucose of 138 mg/dL. 1. Is the patient candidate to start medical treatment as prevention for DM2?. a) Yes, he has pre-diabetes. b) Yes, he is less than 60 years old and has pre-diabetes. c) No, he is older than 40 years. d) No, the initial preventive management is changing his lifestyle. e) No, he has normal fasting glucose. 2. Does this patient need any other management? a) Dilated eye exam to screen for diabetic retinopathy. b) Cardiac stress test. c) Stop smoking. d) Chest radiography e) Vitamins B supplements. BODY.ABBREVIATIONS: CR: Critical Reading; PBL: Problem Based Learning; BPHS: Basic Package of Health Services; GP: General Practitioner; CC: Clinical Competence; CME: Continuing Medical Education; CAE: Collaborative Active Education; MCQs: Multiple Choice Questionnaire; HISA: Health Institute of the State of Aguascalientes. BODY.COMPETING INTERESTS: The authors have no competing interests to declare. BODY.AUTHORS’ CONTRIBUTIONS: JGO and CAPA participated in the study design, data collection, statistical analysis, data interpretation, and the drafting of the manuscript. YSB collaborated in data collection, statistical analysis and the drafting of the manuscript. JVM and MGA participated in the implementation of the educational strategies, data collection and the 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: http://www.biomedcentral.com/1472-6920/12/53/prepub

TITLE: Parent–child interaction therapy for preschool children with disruptive behaviour problems in the Netherlands ABSTRACT.BACKGROUND: Persistent high levels of aggressive, oppositional and impulsive behaviours, in the early lives of children, are significant risk factors for adolescent and adult antisocial behaviour and criminal activity. If the disruptive behavioural problems of young children could be prevented or significantly reduced at an early age, the trajectory of these behavioural problems leading to adolescent delinquency and adult antisocial behaviour could be corrected. Parent–Child Interaction Therapy (PCIT) is a short-term, evidence-based, training intervention for parents dealing with preschool children, who exhibit behavioural problems. Recently, PCIT was implemented in a Dutch community mental health setting. This present study aims to examine the short-term effects of PCIT on reducing the frequency of disruptive behaviour in young children. ABSTRACT.METHODS: This study is based on the data of 37 referred families. Whereby the results of which are derived from an analysis of parent reports of the Eyberg Child Behavior Inventory (ECBI), obtained during each therapeutic session. Furthermore, demographic information, extracted from client files, was also utilized. However, it must be noted that eleven families (27.5%) dropped out of treatment before the treatment protocol was completed. To investigate the development of disruptive behaviour, a non-clinical comparison group was recruited from primary schools (N = 59). ABSTRACT.RESULTS: The results of this study indicate that PCIT significantly reduces disruptive behaviour in children. Large effect sizes were found for both fathers and mothers reported problems (d = 1.88, d = 1.99, respectively), which is similar to American outcome studies. At post treatment, no differences were found concerning the frequency of behavioural problems of children who completed treatment and those who participated in the non-clinical comparison group. ABSTRACT.CONCLUSION: The findings of this study suggest that PCIT is potentially an effective intervention strategy for young children and their parents in the Dutch population. However, further research into the evaluation of PCIT using a randomised controlled trial is recommendable. BODY.BACKGROUND: Child disruptive behaviour disorders (DBDs), namely, conduct disorder (CD), and oppositional defiant disorder (ODD) as described by DSM-IV [1], are among the most common reasons for referring children and adolescents to mental health services [2]. Often, DBDs co-occur with attention deficit hyperactivity disorder (ADHD) [3]. Children with persistent high levels of aggressive, oppositional, and impulsive behaviours early in life are at a higher risk of serious adolescent and adult antisocial behaviour and criminal activity [4,5]. Although the prevalence rates of DBDs in the Dutch population has only been studied to a certain degree, one study concerning the prevalence of child psychiatric diagnoses of children between the ages of 6 and 8, using a structured diagnostic interview, revealed a mean prevalence rate of 12.8% for DBDs; 9.3% for girls and 15.2% for boys [6]. Within the last twenty years, several predictors and origins of DBDs have been identified. Most often, disruptive behaviour problems start in early childhood. Important risk factors relating to the development of chronic child disruptive behaviour problems can manifest during pregnancy and are often related to the history of a mother's social adjustment and lifestyle during pregnancy [7]. Moreover, the transition from preschool to elementary school years is a critical period for the further development of aggressive behaviour, which may persist over time if not treated [8-10]. The development of DBDs in young children and their consistency can be explained by an interplay of genetic and environmental risk factors [11]. Given the early development of disruptive behaviour problems and their stability, as well as long term negative outcomes, prevention and intervention at an early stage is important and more likely to be (cost)effective [12,13]. It can be expected that interventions which target young children who are at a high risk of chronic disruptive behaviour problems at an early age, will have a more significant impact, compared to interventions which are carried out five to ten years later, when behavioural problems may have become persistent [9,13]. If disruptive behaviour problems of young children could be prevented or significantly reduced early in life, the trajectory of early disruptive behaviour problems leading to adolescent delinquency and adult antisocial behaviour could also be prevented. Unfortunately, therapeutic approaches targeting children with disruptive behaviours struggle with two main issues. First, the majority of them lack empirical evidence [14], and second, most target older children, such as pre-adolescents or adolescents, thereby missing a crucial age group in which prevention and intervention is of utmost importance [7,13]. Currently, parent training programs, which use parents as the primary agent of change, are the most effective method in reducing disruptive behaviours in young children [15]. A review of the effects of early parent training programs aimed at preventing antisocial behaviour and delinquency, shows that parent training is an effective intervention strategy in reducing child disruptive behaviour, with a mean effect size of 0.35. However, this effect size still indicates a small to moderate effect [16]. Although parent training programs are an effective treatment for children with behavioural problems, further research is required [17]. BODY.BACKGROUND.PARENT–CHILD INTERACTION THERAPY: Parent–Child Interaction Therapy (PCIT) [18] is a short-term, evidence-based parent training intervention which is used widely as a treatment for young children with disruptive behaviour problems. This treatment is based upon social learning [19], as well as attachment theory [20] and its primary aim is to change dysfunctional parent–child interactions into those that can be characterized as authoritative parenting [21,22]. The treatment is designed to help parents build a warm and responsive relationship with their child and to manage their child's behaviour more effectively [23]. Several studies, mainly in the United States, have provided empirical evidence which indicated the effectiveness of PCIT, namely the improvement of parenting skills and the way parents interact with their children, as well as parental well-being, and the reduction of child disruptive behaviour with medium to large effect sizes [24]. Thereby, a body of evidence is growing on the effectiveness of PCIT to prevent child maltreatment [25]. PCIT has also proven to be robust across various groups and diagnoses. For instance, PCIT has been successfully adapted to meet the needs of several different cultural and language groups, including Puerto Rican [26], Mexican American [27], and Chinese [28]. Beside the cross cultural implementation of PCIT, PCIT has also explored new research directions including studies which work with several adaptations of the treatment which can in turn be used for different target groups. For example, PCIT has been tailored for physically abusive parents [29], prematurely born children [30], children with separation anxiety [31], and children with mental retardation [32]. In the past decade, the implementation of PCIT has expanded to several countries. However, evidence which illustrates the effectiveness of PCIT among children from other cultural backgrounds remains limited [33]. Although PCIT has been implemented in a number of European countries (e.g. the United Kingdom, Germany, Norway and Russia) [34], no evaluation studies are available in Europe. In the Netherlands, PCIT has been implemented in a community mental health setting in child and adolescent psychiatry since 2007. Most treatment outcome studies have been conducted at university clinics. Currently, the transferability of PCIT to community and other clinical settings is an important issue in evidence-based clinical practice. Delivering treatment in community mental health settings is often more challenging, and high rates of premature dropouts can limit its effectiveness. More research on PCIT is needed to examine the effectiveness of PCIT in real world clinics [35,36]. BODY.BACKGROUND.AIM OF THE STUDY: The present study describes the results of a preliminary evaluation of the short-term effectiveness of Parent–Child Interaction Therapy in the Netherlands which aims to reduce the disruptive behaviour of children. In a retrospective design, child disruptive behaviour was measured with the Eyberg Child Behavior Inventory (ECBI) [37]. We hypothesized that PCIT will have positive effects on the disruptive behaviour of young children. BODY.METHODS.PARTICIPANTS: Since the implementation of PCIT in a Dutch mental health setting, between January 2007 and July 2009, forty families were referred on the grounds of child disruptive behaviour. All of the families were contacted to provide permission for using their reports of the Eyberg Child Behavior Inventory (ECBI) [37] in this study. Because three families did not give their consent, data from 37 families were used in statistical analyses. Although the families who did not give their consent were composed of two-parent families, no significant differences were found in regard to other important demographic characteristics and scores on the ECBI at pre and post assessment between these three families and the participating families. A total of 37 families formed the clinical group (Table 1). All of the participating families lived in or nearby Amsterdam, The Netherlands. In addition, as determined by a child psychiatrist, 17 children (45.9%) met the diagnostic criteria according to the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) [1]. Only four children met the criteria for ODD only, six children for ADHD and only two children met the criteria for ASD (Autism Spectrum Disorder). Five children had co-morbid diagnoses. Two children met the criteria for both ADHD and ODD, two children met the criteria for ADHD, ODD and ASD, and one child met the criteria for ADHD and ASD. In all cases, a female caregiver/mother was involved in the treatment. In regards to fathers, 19 (51.4%) were involved in treatment sessions. Twenty-one children (56.8%) lived in two-parent families with their biological parents, and two children (5.4%) in this group were co-parented, meaning that the child lived with either divorced or separated parents, but in different homes. Thirteen children (35.1%) lived in single-mother families and three children (8.1%) had foster parents. The racial/ethnic composition of mothers was as followed; 62% Caucasian, 11% Surinamese, 8% Moroccan, 3% Turkish, and 16% from other, mainly African, countries. Table 1 Desriptive statistics of the Treatment and Non-Clinical Comparison Groups   Mean ( SD ) or Percent   TT ( n  = 37) NC ( n  = 58) Child age (years) 4.7 (1.5) 5.2 (0.8) Child sex (% male) 75.5 50.8 Mother age (years) 34.9 (6.7) 36.3 (4.1) Family status (% single parent) 35.1 1.7 Mother racial composition (% Caucasian) 62.0 96.6 Note. TT Total Treatment Group, NC Non-Clinical Comparison Group. In order to investigate the development of disruptive behaviour over a period of six months, a non-clinical comparison group was recruited which consisted of children from the same age category as those from the clinical group. These families were recruited by students on primary schools. The mothers in this group filled out the ECBI twice over a six month period (N = 59), and this group was composed of 30 boys and 29 girls (Table 1). No significant differences (p < .05) were found between the ages of the mothers and children in the non-clinical group and the clinical group. Although there was a significant difference in gender composition between the clinical and non-clinical group, there were no gender differences on the mean ECBI scores on all presented scales. BODY.METHODS.MEASURES.EYBERG CHILD BEHAVIOR INVENTORY (ECBI): The ECBI [37] is a 36-item parent report, which measures the degree of behavioural problems of children between the ages of 2 to 16. The ECBI assesses the behaviour on two different scales, the Intensity scale and the Problem scale. The ECBI Intensity scale measures the frequency of disruptive behaviour along a 7-point scale (1 = never to 7 = always), and the ECBI Problem scale measures whether or not parents view those behaviours as problematic (1 = yes, 0 = no). Several studies have demonstrated that both scales of the ECBI demonstrate a high level of reliability and validity in terms of measuring the disruptive behaviour of children [38,39]. Our study used a Dutch version translated by Raaijmakers, Posthumus, and Matthys (University of Utrecht, The Netherlands). The norms for a clinical range were used from the professional manual [37]. Scores above 132 on the intensity scale and above 15 on the problem scale were considered clinically significant. Both parents completed the ECBI if the father was involved in the treatment sessions. Therefore, for the pre and post assessment data, ECBI reports of the first session (orientation) and last treatment session (graduation) were used. BODY.METHODS.PROCEDURE: All participating families received PCIT delivered in the Dutch language by one of the eight therapists who were trained in two workshops by the program developers. They attended the first workshop at the University of Florida and the second at the University of Oklahoma. The original treatment manual [40] was translated into Dutch. Each therapist had a Bachelor's or Master's degree in mental health related fields and had experience in clinical work. Therapists started their cases right after the training workshop. Throughout the training and during follow-up consultations, a strong emphasis was put on treatment fidelity. For supervision purposes, all therapy sessions were videotaped. Although treatment adherence was not formally assessed, additional supervision sessions were provided. Due to the fast implementation process and organizational limitations, this study was retrospective. After the termination of PCIT, all parents were asked for their permission to use their reports of the ECBI [37] conducted during treatment, and some demographic information from the client-files for scientific research. BODY.METHODS.TREATMENT: Parent–Child Interaction Therapy (PCIT) is an intervention which focuses on children with disruptive behaviour problems and their caregivers [41]. PCIT consists of two phases of treatment, Child-Directed Interaction (CDI) and Parent-Directed Interaction (PDI). The first phase focuses on enhancing the parent–child relationship and the second on improving child compliance. Both treatment phases begin with a didactic parental teaching session followed by weekly sessions whereby the parent is coached by the therapist during play sessions with their child. The therapist provides the parent with feedback on their skills from an observation room behind a one-way mirror, via a bug-in-the-ear. Parents practice specific communication skills and behaviour management with their children. PCIT is customized per case and although it is often a short-term intervention, PCIT is not time-limited. In each session parent–child interactions are coded at the beginning to determine the family's progress toward pre-established mastery criteria. Parents have to master the CDI criteria before starting with the PDI phase of treatment. The PDI phase continues until parents reach the mastery criteria for the PDI skills and rate their child's behaviour well within a normal range. A consequence of this approach is that the number of sessions may vary among families. Nevertheless, each family receives the number of sessions necessary to master CDI and PDI skills in order to demote their child's behaviour below clinical levels [34]. BODY.METHODS.STATISTICAL ANALYSIS: The effectiveness analyses were performed on a sample of participants who completed the treatment. Paired samples t-tests were conducted on the mean scores of both parent's ECBI from pre and post assessments. If a score of a parent on the ECBI was missing on a pre or post assessment, the information of that parent was removed from the analyses for the particular scale. Effect sizes (Cohen's d) were calculated by dividing the pre and post test mean by the pooled standard deviation, whereby 0.2 indicated a small effect, 0.5 a medium effect, and 0.8 and higher a large effect size [42]. In all of the analyses, a two-tailed test was used and all p values < .05 were considered to be statistically significant. To determine whether the changes in disruptive behaviour in children were clinically significant, reliable change indices (RCI) [43] for each child were calculated by dividing the magnitude of change on the ECBI scales between pre and post assessment by the standard error of the difference score. Published norms for the ECBI clinical cut-off were used [38]. BODY.RESULTS.DESCRIPTIVE STATISTICS: Out of the 40 participating families who started with PCIT, 11 families (27.5%) dropped out before treatment was completed, and seven families (63.6%) dropped out within the first ten sessions of treatment. There were several reasons that caused families to terminate treatment prematurely. Four families required other, more intensive treatment (36.4%), and two families (18.2%) disagreed with the treatment approach, particularly the time-out procedure in the Parent-Directed Interaction phase. Another two families (18.2%) simply stopped showing up for treatment, another family (9.1%) was too busy to participate, one family (9.1%) had to stop treatment due to severe parental relational problems and for one family (9.1%), the child's behaviour improved enough to terminate treatment before meeting all skill levels by the parents. Those families who did complete treatment (n = 26), went through a number of treatment sessions ranging from 10 to 38 sessions per family (M = 17.4, SD = 6.9). Most families (80.8%) finished PCIT within 10 to 20 treatment sessions. The mean duration of the Child-Directed Interaction phase was 10 sessions (SD = 5.2) and for the Parent-Directed Interaction phase the mean duration was 7 sessions (SD = 2.6). The mean duration of PCIT measured in time was 6.6 months (SD = 2.7), ranging from 3 to 12 months, per family. BODY.RESULTS.OUTCOMES OF DISRUPTIVE BEHAVIOUR: Paired samples t-tests of pre and post measures revealed a significant reduction of the frequency of disruptive behaviour in children after treatment completion. Table 2 illustrates that at the end of the Child-Directed Interaction phase a significant decrease on both ECBI scales was already visible for both mothers and fathers. Overall, effect sizes between 1.48 and 1.99 at post-assessment were found for PCIT on child behavioural problems. Table 2 Changes on the Eyberg Child Behavior Inventory (ECBI)   n Pre Post t Effect size n Pre Post t Effect size Intensity Intensity (d) Problem Problem (d) M SD M SD M SD M SD Mothers                             CDI 25 156.4 32.0 128.2 28.9 6.2 *** .92 23 20.4 8.3 17.3 8.0 2.5 ** .38 PDI 24 127.3 28.3 102.8 23.7 4.5 *** .94 22 16.5 7.5 7.9 6.7 4.8 *** 1.21 Total treatment 23 154.0 32.2 100.2 20.5 8.4 *** 1.99 21 20.0 8.5 7.8 6.9 5.6 *** 1.56 Fathers                             CDI 14 151.9 31.8 128.9 34.8 2.6 ** .69 12 21.4 6.8 15.8 10.1 2.2 * .65 PDI 16 126.4 31.9 101.9 31.2 3.3 * .78 15 15.5 9.3 7.9 8.9 3.8 ** .83 Total treatment 15 153.3 30.9 101.0 24.3 6.7 *** 1.88 12 19.8 7.2 8.0 8.9 5.9 *** 1.48 Non-clinical group 1 59 80.5 20.4 80.8 22.8 -.2 -.02 56 3.3 5.3 2.3 4.2 1.8 .21 Note. ECBI Eyberg Child Behavior Inventory, CDI Child-Directed Interaction phase, PDI Parent-Directed Interaction phase. * p  < .10, ** p  < .05, *** p  < .001. 1 Post in the Non-Clinical Comparison Group corresponds to a six months follow-up; This Non-Clinical Group only represents mothers. In the non-clinical comparison group, no behavioural changes were reported at the six-month follow-up assessment. When the clinical group mothers were compared with the non-clinical group mothers on the ECBI Intensity scale at post treatment, no significant differences were found between the groups. However, mothers in the clinical group continued to view their child's behaviour as significantly more problematic (ECBI Problem Scale; t (81) = 2.21, p < .05) than mothers in the non-clinical comparison group. Figure 1 illustrates the mean scores of the ECBI Intensity scales for mothers in the different groups. This figure also includes the means of the total treatment group including the dropouts (n = 34) and the families who dropped out of treatment (n = 11) separately. Even when the dropouts are included, the means on the ECBI Intensity scale significantly improved from pre treatment to post treatment (Total Treatment Group; t (33) = 6.81, p < .001), and large effect sizes where obtained (d =1.15). Although Figure 1 shows a decrease in means between pre and post assessment for the families who dropped out of treatment prematurely, no significant differences were found in this group. Figure 1Mean scores on the Intensity scale on the Eyberg Child Behavior Inventory (ECBI) for mothers in groups. *Post = Post-treatment or six months follow up for the non-clinical comparison group. TC = Treatment Completers Group (n = 23); TT = Total Treatment Group (dropouts included) (n = 34); TD = Treatment Dropout Group (n = 11); NC = Non-Clinical Comparison Group (n = 59) BODY.RESULTS.CLINICAL SIGNIFICANCE: In order to measure individual change, the reliable change index [43] was calculated (Table 3). Participants of both the completer and dropout groups were classified according to the criteria of Jacobson et al. [44], and were presented in the same way as in Thomas and Zimmer-Gembeck [25]. In addition, based on the U.S. norms of the ECBI presented in the professional manual [37], 81.4% of the mothers of the total treatment group rated their child's behaviour at pre assessment in the clinical range on one or both of the ECBI scales. After terminating PCIT, 29.7% of the mothers of this total group (dropouts included) still rated their child's behaviour within the clinical range. Table 3 Frequencies and percentages of Treatment Completers and Dropouts in Reliable Change Index (RCI) Categories   Recovered Improved Unchanged Deteriorated False Positive   Completer Dropout Completer Dropout Completer Dropout Completer Dropout Completer Dropout Mothers ECBI Intensity 17 (73.9) 2 (18.2) 0 (0.0) 0 (0.0) 4 (17.4) 7 (63.6) 0 (0.0) 1 (9.1) 2 (8.7) 1 (9.1) ECBI Problem 15 (71.4) 2 (25.0) 0 (0.0) 0 (0.0) 6 (28.6) 5 (62.5) 0 (0.0) 0 (0.0) 0 (0.0) 1 (12.5) ECBI Intensity 10 (71.4) - 1 (7.1) - 3 (21.4) - 0 (0.0) - 0 (0.0) - ECBI Problem 8 (66.7) - 1 (8.3) - 3 (25.0) - 0 (0.0) - 0 (0.0) - Note. ECBI Eyberg Child Behavior Inventory; Scores > 132 on the Intensity scale and > 15 on the Problem scale were considered as clinically significant Recovered Passed RCI and clinical significance; Improved Passed RCI but no clinical significance, Unchanged Unchanged RCI and unchanged or deteriorated clinical significance, Deteriorated Deteriorated in both RCI and clinical significance, False Positive improved clinical significance but unchanged RCI; RCI > 1.96 = Reliable Change Index improved and recovered categories. 1 The only father in the dropout group had missing values on the pre-assessment. Using the reliable change index, most mothers (73.9%) reported a change in the frequency of their child's disruptive behaviour, whereby their child's behaviour was rated within the range of normal functioning. Nevertheless, 17.4% of the mothers who completed treatment still did not report a reliable change in their child's behaviour. Although eleven families dropped out of treatment before completing treatment protocol, two families (18.2%) in this group were still classified as recovered. However, most families who dropped out of treatment reported insufficient or even a negative change in their child's behaviour. BODY.DISCUSSION: Our study supports our hypothesis that Parent–Child Interaction Therapy (PCIT) has positive effects on the disruptive behaviour of Dutch preschoolers. The study indicates that behavioural problems declined significantly during treatment. After the implementation, 40 families were treated with PCIT and 37 of those were included in this present study. The majority of families (72.5%) finished treatment protocol, however 27.5% dropped out after having participated in at least one session. After treatment completion, most of the parents reported a significant reduction in the behaviour problems of their child. The effect sizes of the reduction of their child's disruptive behaviour problems were large, varying between 1.48 and 1.99 and were comparable with the effect sizes as reported in a meta-analysis on PCIT where they varied between 1.21 and 1.57 on the two ECBI scales [24]. Therefore, at post treatment almost all parents reported their child's behaviour in the range of normal functioning, and which did not differ from the non-clinical comparison group. In regards to the ECBI Intensity scale mean ratings of the non-clinical group, it is worth mentioning that these means indicate that Dutch ECBI norms differ from those mentioned in U.S. samples. However, these current findings are similar to other European ECBI standardization studies, which also found lower means on the ECBI [45,46]. Although it would be recommendable to study the Dutch ECBI norms in a larger sample, the differences between norms, as compared to the U.S. samples, may also lead to a reconsideration of the ECBI norms of normal functioning in the Dutch PCIT manual. In over 50% of the total cases, father involvement was achieved. Father reports of child disruptive behaviours at pre and post treatment were similar to those of the mothers. Even though father ratings are not often reported in treatment outcome studies [47], this finding suggests that fathers could profit from their involvement in treatment the same way that mothers do. The present findings are similar to the results of Schuhmann et al. [23] who also included fathers and analysed these results separately. The results of individual changes show that even for families who dropped out before treatment protocol was completed, PCIT can be a sufficient intervention strategy for reducing child behavioural problems. However, the results also conveyed that after completing PCIT, a small group of parents still reported the behaviour of their child to be within the clinical range. These results indicate that although some parents had reached the mastery skills of the PDI phase, PCIT was terminated before their child's behaviour was ranked within the normal range of functioning, which was also part of the PCIT termination procedure. This suggests that therapists need to obtain additional training in order to follow up on the PCIT protocol accurately. In this current study adherence to the treatment manual was not formally assessed. Future research should address this issue. BODY.DISCUSSION.STRENGTHS AND LIMITATIONS: Our study examined the service delivery of an evidence-based treatment in a mental health community setting. This contributes to bridging the gap between research-based approaches and routine practice. It thereby also contributes to the literature on evidence-based treatments for children with disruptive behaviour problems. Given the diversity of the sample, whereby 38% was categorized as non-western, this current study also contributes to the knowledge of the effectiveness of PCIT for immigrant families and families of non-western origin. It would be recommendable to study this specific group more extensively in further research. However, there are also a number of limitations inherent to this study. Although the non-clinical comparison group provided valuable information about the stability and the frequency of behaviour problems in this non-clinical group, no clinical control group was available and long-term effectiveness of treatment was not measured. Due to the absence of a clinical control group, improvements due to maturational or other factors could not be ruled out. However, disruptive behaviour problems of young children have a high degree of stability over time if not treated [8,9]. Regarding the large effect sizes on the decrease of reported child behaviour problems and the high stability of the behaviour of children in the non-clinical comparison group in this study, it seems unlikely that the improvements were simply spontaneous. Second, due to the retrospective design of this current study only parent-reports (ECBI) were available for the measurement of treatment outcome effects. As mentioned earlier, the lack of Dutch norms for the ECBI have consequences for the interpretations of the results in the Dutch context. Thereby, the normal range of functioning of a child on the ECBI is a part of the mastery criteria to terminate PCIT. Hence, more information on parent personality characteristics, parenting stress and child behaviour would provide a wider range of information for the treatment outcomes. This information is highly recommended for future research to address questions concerning the effectiveness of PCIT on other parent and child functioning areas. Furthermore, observational measures using the Dyadic Parent–child Interaction Coding System (DPICS) [48] are recommended for providing more information about the behaviours, as well as the quality of parent–child interactions. The inclusion of a diagnostic interview for concerning child behavioural problems and the use of more independent sources (e.g. teachers) could have also improved the study. The attrition rate (27.5%) in the current study was similar or slightly lower than other U.S. PCIT studies carried out in community mental health settings [35,49]. However, the attrition rate is still high and research is needed to identify the characteristics of specific families that are at risk of treatment drop out. Thus, more support from therapists and other professionals is needed to help high-risk families stay engaged and complete the treatment program. Nevertheless, the results do indicate that a premature termination of PCIT does not have to lead to negative outcomes on child behaviour in all cases. The limitations of this study can be associated with the preliminary nature of the research and can also be identified as a consequence of a fast implementation process. BODY.CONCLUSIONS: Despite the limitations of this study, it does provide significant evidence of short-term effectiveness of PCIT in the Netherlands. Nonetheless, future research is required to address the shortcomings of the present study. A randomised controlled trial is recommended for a further evaluation of PCIT, which can compare the results with a clinical control group and assess long-term effectiveness. Furthermore, studies in community mental health settings are necessary for obtaining knowledge about treatment effectiveness in a challenging population. Determining effective strategies for reducing treatment attrition is also important in these settings. Given the limited knowledge at this time, our findings are a step forward in the evaluation of PCIT as a promising intervention strategy in reducing child disruptive behaviour problems in the Netherlands. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS’ CONTRIBUTIONS: MA was involved in the data collection, performed the statistical analysis and drafted the manuscript. FC and LC participated in the design and data collection of the study. MJ and RL participated in the planning, supervision and co-ordination of the study as well as the critical revision of the draft of the manuscript. FB also critically revised the draft of the manuscript. All of the authors have read and given their approval to the final manuscript.

TITLE: Evaluation of the alignment efficiency of nickel-titanium and copper-nickel-titanium archwires in patients undergoing orthodontic treatment over a 12-week period: A single-center, randomized controlled clinical trial ABSTRACT.OBJECTIVE: The aim of this trial was to compare the alignment efficiency and intermaxillary arch dimension changes of nickel-titanium (NiTi) or copper-nickel-titanium (CuNiTi) round archwires with increasing diameters applied sequentially to the mandibular arch. ABSTRACT.METHODS: The initial alignment phase of fixed orthodontic treatment with NiTi or CuNiTi round archwires was studied in a randomly allocated sample of 66 patients. The NiTi group comprised 26 women, 10 men, and the CuNiTi (27°C) group comprised 20 women, 10 men. The eligibility criteria were as follows: anterior mandibular crowding of minimum 6 mm according to Little's Irregularity Index (LII), treatment requiring no extraction of premolars, 12 to 18 years of age, permanent dentition, skeletal and dental Class I malocclusion. The main outcome measure was the alignment of the mandibular anterior dentition; the secondary outcome measure was the change in mandibular dental arch dimensions during 12 weeks. Simple randomization (allocation ratio 1:1) was used in this single-blind study. LII and mandibular arch dimensions were measured on three-dimensional digital dental models at 2-week intervals. ABSTRACT.RESULTS: No statistically significant difference was observed between NiTi and CuNiTi according to LII (p > 0.05). Intercanine and intermolar arch perimeters increased in the CuNiTi group (p < 0.001). Inter-first premolar width showed a statistically significant interaction in week × diameter × application (p < 0.05). ABSTRACT.CONCLUSIONS: The effects of NiTi and CuNiTi round archwires were similar in terms of their alignment efficiency. However, the intercanine and intermolar arch perimeters, and the inter-first premolar width changes differed between groups. BODY.INTRODUCTION: By changing the mechanical properties of a material and, hence, the content of an alloy, we could possibly obtain ideal archwire characteristics required at each stage of fixed orthodontic treatment.1 Nickel-titanium (NiTi) wires are preferred by clinicians because compared to stainless steel wires, they have a wider working range and higher springback properties.2 Nitinol, which is categorized in the Mstab group, contains 55% nickel and 45% titanium, and is also known as "M-NiTi".3 This material has high springback values, despite having neither superelasticity nor a shape-memory effect.4 Copper-nickel-titanium (CuNiTi) wires, which fall into the Mact group, have a true shape-memory effect and show the mechanical properties of austenite above a transition temperature range. CuNiTi archwires can be ligatured easily at room temperature (approximately 25°C, below the transition temperature range) in the martensitic phase (soft and flexible). After intraoral application, this wire shows mechanical properties of the austenite phase under oral conditions, i.e., its hardness increases, becomes inflexible, and shows function while taking its true shape (form).56 In CuNiTi wires, the addition of copper into the alloy reduces hysteresis and helps control the transition temperature range.7 The copper content of CuNiTi archwires also enables these wires to exert more homogeneous forces from one side of the wire to the other, thereby providing faster and more efficient tooth movement.89 Several studies have evaluated NiTi archwires with different alloy compositions and, therefore, variable mechanical characteristics in vitro, 910 clinical performance in vivo,1112 and different effects on pain levels.13 However, the results obtained using in vitro910 vs. in vivo111214 conditions are controversial. Therefore, more in vitro studies are needed which are designed to evaluate the efficiency of NiTi wires at the initial stage of treatment with respect to the alleviation of crowding.15 A wide variety of archwire sequences have been used to compare the clinical efficiency of orthodontic archwires.161718 Identifying an archwire sequence that reflects the clinical routine would increase the usefulness of the study results in daily orthodontic practice.19 However, insufficient data are available to determine the archwire sequence that would alleviate crowding most rapidly and efficiently at the leveling stage of fixed orthodontic treatment.20 In the literature, several studies have compared the clinical efficiency of Mstab NiTi and Mact NiTi wires.121416 However, to the best of our knowledge, no in vitro randomized controlled study has evaluated the clinical performance of round archwires at the initial stage of orthodontic treatment when these are used sequentially with respect to the clinical routine. The aim of this study was to evaluate the efficiency of round NiTi and CuNiTi archwires used at the initial stage of orthodontic treatment in a sequence that reflects the clinical routine. The first hypothesis of this study is, "There is no difference between round NiTi and CuNiTi archwires in terms of the alleviation of crowding in the anterior segment of the mandible at the leveling stage." The second hypothesis is, "There is no difference between round NiTi and CuNiTi archwires in terms of the evaluation of arch perimeters and interdental widths at the leveling stage." BODY.MATERIALS AND METHODS: This study was a randomized, parallel-group, single-blinded, active-controlled trial with a 1:1 allocation ratio. The study was approved by the Suleyman Demirel University, Faculty of Medicine, Clinical Research Ethics committee (04.06.2014/96). The inclusion criteria for participants were as follows: (1) mandibular anterior dental crowding (Little's Irregularity Index [LII] > 6 mm); (2) 12 to 18 years of age; (3) permanent dentition; (4) treatment requiring no extraction of premolars or any other teeth; (5) skeletal and dental Class I relationships; (6) normal overjet and overbite; and (7) systemically and periodontally healthy. The exclusion criteria were as follows: (1) unwilling to be assigned to any of the treatment options; (2) caries and impacted or missing teeth except for third molars; (3) orthodontic treatment history; (4) posterior crossbite; (5) craniofacial syndrome or skeletal asymmetry; and (6) periodic non-steroidal anti-inflammatory drug use. Written informed consent was obtained from all study participants and their parents before the study was carried out. BODY.MATERIALS AND METHODS.INTERVENTIONS: The patients were divided into the NiTi group and CuNiTi group. Roth prescription brackets with a 0.018-inch (in) slot (Mini Sprint-Prescription: Roth 0.018 in; Forestadent, Pforzheim, Germany) were used in both groups. The orthodontic light-cured adhesive system used for bonding all brackets was Transbond XT (3M Unitek, Monrovia, CA, USA) (Figure 1). In the NiTi group, a 0.014-in round NiTi wire (natural arch form, Nickel-Titanium Archwire; Ortho Organizers, Carlsbad, CA, USA) was applied as the initial archwire immediately after the bonding procedure. At the next appointment, which was scheduled 6 weeks later, the patients received a 0.016-in round NiTi wire (natural arch form, Nickel-Titanium Archwire). The next appointment was scheduled 6 weeks later, at which time the study was terminated. The total study duration was 12 weeks. Both archwires were tightly and fully ligated to each wing of the brackets by using ligature wires (Figure 1). In the CuNiTi group, a 0.014-in round Tru-Arch CuNiTi 27°C (mandibular–small; Ormco Corp., Glendora, CA, USA) was applied as the initial archwire immediately after the bonding procedure. The next appointment was scheduled 6 weeks later, at which time the patients received a 0.016-in round Tru-Arch CuNiTi 27°C (mandibular–small). The next appointment was scheduled 6 weeks later, when the study terminated. The total study duration was 12 weeks. Both archwires were tightly and fully ligated to each wing of the brackets by using ligature wires. To avoid occlusal interferences that may occur during the study, a thermoplastic retainer with an acrylic bite block (2 mm) in the premolar-molar region was applied to the upper jaw from the beginning to the end of the study in each patient (Figure 1). Patients were asked to wear these retainers at all times except mealtimes. All clinical procedures were carried out by the same clinician (BA). All patients underwent scanning at the beginning of the study (T0) and at the 2nd (T1), 4th (T2), 6th (T3), 8th (T4), 10th (T5), and 12th (T6) weeks of treatment by using a three-dimensional (3D) intraoral scanner (TRIOS; 3Shape, Copenhagen, Denmark). The parameters measured in this study were evaluated using 3D digital casts obtained at 2-week intervals by using a 3D software program (OrthoAnalyzer; 3Shape). The amount of alignment achieved during the study using each different archwire was measured according to LII. In the assessment of LII, each 3D dental cast was measured three times, and the mean value of the measurements was recorded (Figure 2A). The length of the arch perimeter between the mandibular canines and mandibular first molars was measured to evaluate the changes in the anterior arch perimeter and total arch perimeter resulting from the treatment (Figure 2B). Interdental and interalveolar measurements were performed using the abovementioned 3D software program to evaluate the transversal arch width changes resulting from the treatment using different archwires (Figure 2C). BODY.MATERIALS AND METHODS.OUTCOMES AND CHANGES AFTER TRIAL COMMENCEMENT: The main outcome measure of this study was the alignment of the mandibular anterior dentition, achieved using sequentially applied round archwires over 12 weeks. The secondary outcome measure was the change in mandibular dental arch dimensions. The first initial archwire applied was a 0.014-in round archwire; after 6 weeks, the archwire was changed to a 0.016-in round archwire in both groups. During the 12-week study period, the changes were recorded using a 3D digital intraoral scanner at 2-week intervals. Using computer software, the irregularity index and mandibular arch dimensions were measured for each of the 3D digital dental models obtained at 2-week intervals for 12 weeks. The archwires used in the NiTi and CuNiTi groups were (Mstab) NiTi and (Mact) CuNiTi, respectively. All patients were clinically examined every 2 weeks, and the mandibular dental arch of each patient was scanned using an intraoral 3D scanner. Their complaints were also addressed where necessary. Patients were recalled after 1 to 3 days in case of broken brackets (Table 1). New brackets were bonded to teeth by using the same bonding materials and techniques. No changes in outcome were observed after study commencement. BODY.MATERIALS AND METHODS.SAMPLE SIZE CALCULATION: In this study, a preliminary calculation was performed to calculate the sample size by using G*Power software version 3.0.10 (Franz Faul Universität, Kiel, Germany). To achieve 95% power, the study included 36 patients per group (for alveolar inter-first molar width feature; mean, 50 mm; standard deviation, 0.35; alpha level, 0.05). BODY.MATERIALS AND METHODS.RANDOMIZATION: This study used the volunteer sampling method. The two study groups were designated using simple randomization (coin method), with an allocation ratio of 1:1 according to the type of NiTi archwire. The NiTi group, comprising 26 women and 10 men, was treated using NiTi archwires, while the CuNiTi group, comprising 20 women and 10 men, was treated using CuNiTi (27°C) archwires. BODY.MATERIALS AND METHODS.BLINDING: During the single-blind study, the allocation of wires was concealed from the participants, but the clinician had this information. No other wire was used, and no other treatment was performed on the maxilla or mandible throughout the study. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Data were processed using IBM SPSS Statistics ver. 23.0 (IBM Corp., Armonk, NY, USA). The normal distribution of data was assessed using the Kolmogorov-Smirnov Z-test, and a normal distribution was found for all features. A Levene test of homogeneity was used to determine whether the distribution was similar in the groups. The data were analyzed using repeated measures of a variance analysis technique by using a factorial system (rANOVA). Repeated measures were performed in levels of wire diameter and week. In this study, the application factor had two levels (NiTi and CuNiTi); the archwire diameter factor had two levels (0.014 in and 0.016 in); and the week factor had three levels (2, 4, and 6). In order to determine the exact differences between the NiTi and CuNiTi groups in these time intervals, the covariant adjustment was calculated for the initial value of each measurement. The starting points of the means for each group were also equalized. Turkey's method was used for post hoc comparisons, and values of p < 0.05 were considered significant. After a 2-week interval, 20 study models were randomly selected and re-measured for reproducibility of the measurements (r: Cronbach's alpha, 0.871–0.963). BODY.RESULTS: Volunteer inclusion began in October 2014 and ended in August 2015. When the volunteer groups were composed, 80 patients fulfilling the criteria were assessed for eligibility. After the randomization of 76 subjects to the NiTi and CuNiTi groups for the study with a 1:1 allocation ratio, the treatment protocol was carried out. However, at the end of the study, the data of only 66 subjects (46 females and 20 males) were analyzed (Figure 3). Since differences were found between the groups according to baseline irregularity index values, a covariant adjustment was calculated for the initial value of each measurement. The distributions of the chronologic ages of the study participants and treatment durations for the groups are given in Table 2. No statistically significant three-way interaction was observed between week × diameter × application types in terms of LII (p > 0.05). The length of the arch perimeter between the canines and the length of the arch perimeter between the first molars were significantly higher in the CuNiTi group than in the NiTi group in terms of the main effect (application factor) (p < 0.001). A statistically significant three-way interaction was observed between week × diameter × application types in terms of the inter-first premolar width change. When this interaction was evaluated (a) in terms of weeks (capitals, right-hand side; Table 3), the mean values obtained using the 0.014-in NiTi archwire in the second and fourth weeks of application (p > 0.05) were lower than those obtained in the sixth week (p < 0.05). For the 0.016-in NiTi archwire, the mean values obtained in the second week were lower than those obtained in the fourth and sixth weeks. The mean values obtained using the 0.014-in CuNiTi archwire in the second week were significantly lower than those obtained in the fourth and sixth weeks (p < 0.01). (b) In terms of diameter (lower case, right-hand side; Table 3), for the NiTi group, the mean values obtained using the 0.014-in archwires were significantly lower than those obtained using the 0.016-in archwires in the second, fourth, and sixth weeks (p < 0.05). For the CuNiTi group, the mean values obtained using the 0.014-in archwires were lower than those obtained using the 0.016-in archwires in the second, fourth, and sixth weeks (p < 0.05). (c) In terms of application (lower case, left-hand side; Table 3), the mean values obtained for the 0.014-in archwire application were statistically higher in the NiTi group than in the CuNiTi group in the second week (p < 0.05); however, these were statistically lower in the fourth week. In the sixth week, the mean values obtained in the NiTi group were statistically higher than those obtained in the CuNiTi group (p < 0.05). The mean values obtained using 0.016-in archwire application were significantly lower in the NiTi group than in the CuNiTi group in the second week (p < 0.05); however, these were significantly higher in the fourth week. In the sixth week, the mean values obtained in the NiTi group were significantly lower than those obtained in the CuNiTi group (p < 0.05). BODY.DISCUSSION: In this study, NiTi and CuNiTi archwires on the mandibular arch were compared in vivo to determine the efficiency of alignment and the changes in intermaxillary arch dimension by randomizing 66 patients between two different NiTi groups. Initial crowding evaluated using LII was 10.24 ± 2.10 mm and 10.60 ± 2.43 mm, respectively, for the NiTi and CuNiTi groups. The total amount of alleviation of crowding with the sequential (6 weeks for each archwire) application of 0.014-in and 0.016-in archwires was 4.07 mm in the NiTi group and 3.58 mm in the CuNiTi group. In this study, the NiTi and CuNiTi archwires showed statistically non-significant results in terms of the alleviation of crowding. These results are compatible with those of other studies.111214 Our study groups included patients with severe crowding. However, our result is similar to that of studies on groups with severe and/or moderate crowding.111214 Previous studies have demonstrated that during the alleviation of crowding under both moderate and severe crowding conditions, NiTi wires provide successful treatment results with preadjusted edgewise appliances.21 The arch perimeter, measured parallel to the occlusal plane, was affected by the protrusion of the incisors. The ligation of malposed teeth to the archwire without space-gaining methods could cause forced (obligatory) labial/buccal tipping, which could result in protrusion or dental expansion. This protrusion may be more evident in the anterior region, since crowding often occurs in this region. Moreover, the weaker roots of the anterior teeth predispose them to dental tipping. Insufficient occlusal contacts, which can limit dental tipping unlike in the posterior teeth, could be the reason for the shift of this protrusion through the anterior region.22 In our study, the mean values for the intercanine arch perimeter were higher in the CuNiTi group than in the NiTi group (p < 0.01) from the second week. The covariates were adjusted for initial values of all examined parameters. This difference in intercanine arch perimeter appeared at the end of the second week and remained until the end of the study. The application of CuNiTi archwires involves stronger forces than does the application of NiTi wires, and they exhibit rapid hardening in the oral environment because of their alloy structure.23 This difference in intercanine arch perimeter may be attributed to the anterior teeth in the CuNiTi group protruding faster as a result of the archwire alloy structure. Hence, both NiTi and CuNiTi archwires have similar effects on the alleviation of crowding and expansion of the intercanine arch width. The significantly increased changes in the CuNiTi group than in the NiTi group in terms of the length of the intercanine arch perimeter show that this group may have a greater tendency towards protrusion, which is a risk factor for stability.24 Some studies have suggested an increase in arch perimeter after nonextraction orthodontic treatment in patients with Class I malocclusion.2224 For the intermolar arch perimeter, the mean values obtained using CuNiTi archwires were significantly higher than those obtained using NiTi archwires (p < 0.01). Moreover, this measurement showed higher values in the CuNiTi group throughout the treatment protocol. This result was also compatible with the increase in intercanine arch perimeter. The shape of the mandibular arch is in structural and functional harmony.25 During orthodontic treatment, the prevention of intercanine arch width changes could increase the long-term success in terms of retention.26 In this study, no statistically significant difference was observed between the groups in intercanine arch width (p > 0.05). The increases in intercanine width were 1.44 mm in the NiTi group and 1.63 mm in the CuNiTi group. Previous studies have reported increases in intercanine widths of between 0.54 and 1.96 mm.222425 However, studies have also reported that this increase in width was lost after the retention period.25 This suggests that the risk of relapse, which could occur after treatment in both the groups, should be evaluated in further studies. A statistically significant three-way interaction (week × diameter × application) was noted between the groups in terms of the inter-first premolar width (p < 0.05). The increase in inter-first premolar width for the 0.014-in NiTi archwire in the sixth week was more significant than that in the second and fourth weeks (p < 0.05). For the 0.016-in NiTi archwire, the increases in arch width in the fourth and sixth weeks were similar and significantly more evident than those in the second week (p < 0.05). The interdental width obtained at the end of the fourth and sixth weeks with the 0.014-in CuNiTi archwire was more statistically significant than that obtained at the end of the second week (p < 0.05). For the 0.016-in CuNiTi archwire, the increase obtained at the end of the second, fourth, and sixth weeks was not statistically significant (p > 0.05) (capitals, right-hand side; Table 3). As the study progressed, an increase in arch width was obtained for both types of archwires. Studies evaluating the changes in intercanine, interpremolar, and intermolar widths after fixed orthodontic treatment have reported that the most notable difference was observed in the premolar region.2226 The first reason for this could lie in the arch form (Figure 4) of the archwires used in the study. It has been revealed that during the leveling and aligning phases of treatment, the Tru-Arch archwires, which were used in this study, could produce statistically significant increases in the transverse dentoalveolar width and the perimeter of the maxillary arch.27 As the forms of arches used in this study were similar to each other (Figure 4), the obtained changes in arch widths were an expected conclusion. The second reason could be the eruption path of the first premolar teeth in the lingual direction.22 The application of orthodontic forces to the lingually erupted premolars from the buccal side may have caused more dramatic changes in this region. In this study, the inter-first premolar width measurements were statistically higher in the NiTi group than in the CuNiTi group at the end of the second week; these were reversed in the next period, and this pattern repeated until the end of the study. This could be explained by the increase in the length of the intercanine arch perimeter in the lower anterior region at the end of the second week in the CuNiTi group. In the first week of application of the CuNiTi archwires, the rapid hardening of the wire in conjunction with the influence of its mechanical properties may have caused protrusions in the anterior region, where the roots were weaker than in the posterior teeth, rather than contributing to the increase in transversal arch width. The reversal of this pattern in the fourth week, where the CuNiTi archwire caused transversally greater expansion in the premolar region, suggests that CuNiTi had started expanding the arch after a sudden protrusion in the first week. In the sixth week, the pattern again favored the NiTi group, and this was maintained in the following weeks. Studies evaluating the changes in inter-first premolar width in fixed orthodontic treatment have shown that the results obtained vary between 1.46 and 3.22 mm because of the differences in materials and methodology.2428 In contrast to the results of our study, Fleming et al.24 found no statistically significant increase in inter-first and inter-second premolar width changes before and after fixed orthodontic treatment at the leveling stage. The reason for this difference could be the difference in the design materials and methods. The changes in interalveolar widths evaluated in this study were statistically non-significant (p > 0.05). Lundsträm29 has shown that the apical base is not affected by orthodontic tooth movement and mastication. Additionally, alveolar buccal changes do not follow dental expansion.28 Since neither group showed changes in alveolar widths, this is considered a favorable factor for retention.30 In this single-center study, the treatments were performed by a single clinician. Thus, the generalizability of the results of this study may be limited. BODY.CONCLUSION: The effects of sequentially applied 0.014-in and 0.016-in NiTi and CuNiTi round archwires were similar in terms of their alignment efficiency in the mandibular anterior region.In the second week of use of the 0.014-in archwire, the intercanine arch perimeter and total arch perimeter increased in the CuNiTi group than in the NiTi group. This difference due to the increased value of the arch perimeters was maintained during the study period.statistically significant difference was obtained using a three-way interaction between week × diameter × application types in terms of the inter-first premolar width change. The transverse interpremolar arch widths increased with both 0.014-in and 0.016-in sequentially applied CuNiTi round archwires than with similarly applied NiTi round archwires.

TITLE: The efficacy and safety of bevacizumab beyond first progression in patients treated with first-line mFOLFOX6 followed by second-line FOLFIRI in advanced colorectal cancer: a multicenter, single-arm, phase II trial (CCOG-0801) ABSTRACT.PURPOSE: The aim of this study was to evaluate the efficacy and safety of the planned continuation of bevacizumab beyond first progression (BBP) in Japanese patients with metastatic colorectal cancer (mCRC). ABSTRACT.METHODS: Previously untreated patients with assessable disease were treated with mFOLFOX6 plus bevacizumab until tumor progression, followed by FOLFIRI plus bevacizumab. The primary endpoint of the study was the second progression-free survival (2nd PFS), defined as duration from enrollment until progression after the second-line therapy. Secondary endpoints of the study were overall survival (OS), survival beyond first progression (SBP), progression-free survival (PFS), response rate (RR), disease control rate (DCR), and safety. ABSTRACT.RESULTS: In the first-line setting, 47 patients treated with mFOLFOX6 plus bevacizumab achieved RR of 61.7 %, DCR of 89.4 %, and median PFS of 13.1 months (95 % CI, 8.7–17.5 months). Thirty-one patients went on to receive a second-line therapy with FOLFIRI plus bevacizumab and achieved RR of 27.6 %, DCR of 62.1 %, and median PFS of 7.3 months (95 % CI, 5.0–9.6 months). Median 2nd PFS was 18.0 months (95 % CI, 13.7–22.3 months). The median OS and SBP were 30.8 months (95 % CI, 27.6–34.0 months) and 19.6 months (95 % CI, 13.5–25.7 months), respectively. No critical events associated with bevacizumab were observed during the second-line therapy. ABSTRACT.CONCLUSION: The planned continuation of bevacizumab during a second-line treatment, BBP strategy, is feasible for the Japanese mCRC patients. BODY.INTRODUCTION: Colorectal cancer is one of the most common cancers worldwide and remains the third leading cause of cancer-related mortality in Japan [1, 2]. For several years, first- and second-line chemotherapy with 5-fluorouracil (5-FU) and folinic acid (FA) in combination with either irinotecan (FOLFIRI) or oxaliplatin (FOLFOX) had been the standard therapy for metastatic colorectal cancer (mCRC) [3, 4]. More recently, these combinations are used together with bevacizumab, a humanized monoclonal antibody that binds to and neutralizes vascular endothelial growth factor (VEGF). Benefits of adding bevacizumab to either the established first-line or second-line chemotherapeutic regimens have been robustly documented in previous clinical trials. Regarding the first-line treatment, Hurwitz et al. reported that addition of bevacizumab to fluorouracil-based combination chemotherapy showed significantly better clinical outcomes as compared with chemotherapy alone (overall survival [OS]: 20.3 vs. 15.6 months [hazard ratio [HR]: 0.66; P < 0.001], progression-free survival [PFS]: 10.6 vs. 6.2 months [HR: 0.54; P < 0.001], and response rate [RR]: 44.8 vs. 34.8 % [P = 0.004]) [5]. Kabbinavar et al. reported that addition of bevacizumab to fluorouracil/leucovorin (FU/LV) improved survival as compared with FU/LV alone (OS: 17.9 vs. 14.6 months [HR: 0.74; P = 0.008], PFS: 8.8 vs. 5.6 months [HR: 0.63; P < or = 0.0001], RR: 34.1 vs. 24.5 % [P = 0.019]) [6]. Furthermore, Saltz et al. reported that addition of bevacizumab to oxaliplatin-based chemotherapy significantly improved PFS, although OS did not reach statistical significance, and the RR was not improved (PFS: 9.4 vs. 8.0 months [HR: 0.83; P = 0.0023], OS: 21.3 vs. 19.9 months [HR: 0.89; P = 0.077]) [7]. In the second-line setting, the RR rate of various chemotherapeutic regimens has not been satisfactory, ranging from 4 % for FOLFIRI after the first-line FOLFOX6 to 15 % for FOLFOX6 after the first-line FOLFIRI and 20 % for XELOX after irinotecan-based therapies [8, 9]. Again, benefit of adding bevacizumab was demonstrated in several clinical trials in this setting. Giantonio et al. reported that bevacizumab plus FOLFOX4 showed significantly better survival data compared with FOLFOX4 alone after the first-line irinotecan-based treatment (OS 12.9 vs. 10.8 months [HR: 0.75; P = 0.011], PFS 7.3 vs. 4.7 months [HR: 0.61; P < 0.001], RR: 22.7 vs. 8.6 % [P < 0.001]) [10]. Bennouna et al. showed that bevacizumab plus irinotecan–based regimens showed efficacy with acceptable safety profile after the first-line oxaliplatin-based treatments (PFS: 7.8, OS: 22.4, and RR: 33 %) [11]. More recently, a survival benefit associated with the continuous use of bevacizumab beyond progression (BBP) was generated by two large studies. A large observational cohort study that evaluated the efficacy and safety of bevacizumab in combination with chemotherapy (BRiTE study) indicated that the BBP could contribute to prolong the OS [12]. The Avastin registry: investigation of effectiveness and safety (ARIES) also looked at the role of BBP and indicated trend toward longer OS among patients who received bevacizumab beyond first progression compared with patients who received bevacizumab only after progression (27.5 vs. 18.7 months) [13]. However, these are observational studies, and true benefits and risks of BBP are yet to be shown in a prospective clinical trial, particularly in Japan. This prompted us to conduct a multicenter phase II study of mFOLFOX6 plus bevacizumab followed by FOLFIRI plus bevacizumab in mCRC to explore the BBP strategy for the first time in the Japanese population. BODY.PATIENTS AND METHODS.PATIENTS: The study inclusion criteria were histologically confirmed colorectal adenocarcinoma; unrespectable metastatic disease; age 20 years or older; Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1; no previous chemotherapy for mCRC; bidimensionally measurable disease; a life expectancy of at least 3 months; adequate organ function (white blood cell count 3,000–12,000 cells per μL, neutrophilic cell count ≥1,500 cells per μL, platelet count ≥100,000 per μL, aspartate aminotransferase [AST] ≤100 IU/L, alanine aminotransferase [ALT] ≤100 IU/L, total bilirubin ≤25.7 μmol/L [≤15 mg/L], and creatinine ≤106.1 μmol/L [≤12 mg/L]). Exclusion criteria were pregnancy or lactation; second non-colorectal cancer; complications such as ileus, uncontrolled diabetes mellitus, or hypertension; severe diarrhea; clinically evident gastrointestinal hemorrhage; and ascites or pleural effusion needing treatment. The protocol of this study was approved by the institutional review board or ethics committee of each institution. The study was conducted in compliance with the Declaration of Helsinki. Written informed consent was obtained from all patients participating in the study. BODY.PATIENTS AND METHODS.TREATMENT PLAN: As the first-line setting for mCRC, the patients received bevacizumab plus mFOLFOX6 therapy (consisting of bevacizumab [5 mg/kg], oxaliplatin [85 mg/m2], and folinic acid [200 mg/m2] followed by bolus infusion of fluorouracil [400 mg/m2] and subsequent continuous infusion of fluorouracil [2,400 mg/m2], repeated every 2 weeks) until disease progression, unacceptable toxicity, or patient's wish to terminate the treatment. In the subsequent second-line setting, the patients received bevacizumab plus FOLFIRI therapy (consisting of bevacizumab [5 mg/kg], irinotecan [150 mg/m2], and folinic acid [200 mg/m2] followed by bolus infusion of fluorouracil [400 mg/m2] and subsequent continuous infusion of fluorouracil [2,400 mg/m2], repeated every 2 weeks) until disease progression, unacceptable toxicity, or patient's wish to terminate the treatment. Surgical treatment of the metastatic lesions was allowed in patients with sufficient objective response that rendered the lesions resectable. BODY.PATIENTS AND METHODS.ASSESSMENTS: The primary objective of this study was the second progression-free survival (2nd PFS), defined as the time duration from the date of initiation of the first-line therapy until investigator-assessed disease progression or patient death due to any cause after starting the second-line treatment. If the patient could not receive second-line treatment for medical reasons or refusal, progression-free survival (PFS) on first-line therapy was used. Secondary objectives were OS (the time duration from the date of initiation of each therapy to death due to any cause), survival beyond first progression (SBP) (the time duration from the date of first disease progression to death due to any cause), PFS (the time duration from the date of initiation of each therapy to disease progression or death due to any cause), RR (the proportion of patients who achieved a best response of either a complete response [CR] or partial response [PR] during each therapy), disease control rate (DCR) (the proportion of patients with CR, PR, or stable disease [SD] during each therapy), and safety. Schematic of patients observation periods is presented in Fig. 1b. Adverse events were assessed using National Cancer Institute Common Toxicity Criteria (NCI-CTC), version 3.0. In addition, the frequency of bevacizumab-related adverse events (gastrointestinal perforation, wound healing complications, bleeding, hypertension, proteinuria, and thromboembolic events) was assessed.Fig. 1Schematic of patient observation periods (a) and consort chart of the patients (b). a The second progression-free survival (2nd PFS) is measured from the start of first-line treatment to disease progression after second-line treatment. Progression-free survival (PFS) of each therapy is measured from the start of each therapy to disease progression. Survival beyond first progression (SBP) is measured from the first progression to death. Overall survival (OS) is measured from the start of first-line treatment to death. b Fifty patients were enrolled in this study. Three patients were excluded from the study. Forty-seven patients who received the protocol treatment were included in the safety evaluation BODY.PATIENTS AND METHODS.STATISTICAL ANALYSIS: Assuming a threshold for 2nd PFS of 10.5 months and an expected 2nd PFS of 15.8 months, referring to data from the previous clinical trials, and a 2-year enrollment period and a 2-year follow-up period, 44 patients in total were required to ensure an alpha error of 0.05 (one-sided) and detection power (1-β) of 80 %. Taking possible dropouts into consideration, the sample size of this study was determined as 50. The 2nd PFS, the primary objective of this study, was estimated using the Kaplan–Meier method, and the median 2nd PFS and its 95 % confidence interval were estimated. Other time-to-endpoint data, PFS and OS, were also estimated in the same manner. RR, DCR, and the toxicities were calculated as proportions with exact confidence intervals. BODY.RESULTS.PATIENT CHARACTERISTICS: Fifty patients from 12 institutions in Japan were enrolled in this study from August 2008 to May 2010. Three patients were excluded from the study: one due to the patient's refusal, one due to the investigator's decision, and one due to no measurable lesions as per the inclusion criteria. Forty-seven patients who received the protocol treatment were included in the evaluation of efficacy and safety. Baseline characteristics and consort chart of the patients are presented in Table 1 and Fig. 1b.Table 1Patient characteristicsParameterNo. of patients (N = 47)%Age, years Median6340–74 RangeSex Male3166.0 Female1634.0Performance status WHO 02961.7 11838.3Primary site Colon3166.0 Rectum1634.0Metastases Synchronous714.9 Metachronous4085.1Metastatic sites Liver2144.6 Lung2144.6 Peritoneum12.1 Lymph nodes1021.3Adjuvant chemotherapy No2757.4 Yes2042.65FU-based2042.6Oxaliplatin-based00No/N number, WHO World Health Organization, 5FU 5-fluorouracil BODY.RESULTS.TREATMENT STATUS: As the first-line treatment, 47 patients received a median of 12 cycles (range 2–39) of bevacizumab plus mFOLFOX6 therapy. Median time-to-treatment failure (TTF) was 6.5 months (95 % CI, 4.0–9.0 months). The median relative dose intensity (RDI) for bevacizumab and oxaliplatin was 88 and 76 %. Treatment was discontinued because of disease progression in 21 patients (44.7 %), adverse events in 14 patients (29.8 %), and patient's refusal in two patients (4.3 %). Secondary surgery to remove metastases was performed in six patients (12.8 %). As for the second-line treatment, 31 patients received a median of eight cycles (range, 2–28) of bevacizumab plus FOLFIRI therapy. Median TTF was 4.4 months (95 % CI, 2.4–6.4 months). The median RDI for bevacizumab and irinotecan was 80 and 76 %. Treatment was discontinued because of disease progression in 20 patients (64.5 %) and adverse events in two patients (6.5 %). Secondary surgery to remove metastases was performed in one patient (3.2 %). After undergoing the second-line protocol treatment, 20 patients (64.5 %) received a third-line chemotherapy, of which the regimen delivered to six patients (19.4 %) was cetuximab. There was no therapy-related death in this study. Treatment status is summarized in Table 2.Table 2Treatment statusFirst-line therapy(mFOLFOX6 + BV)(N = 47)Second-line therapy(FOLFIRI + BV)(N = 31)Treatment cycle (times) Median127 Range2–392–26Time-to-treatment failure (month) Median6.53.8 95 % CI4.0–9.02.7–4.5Median relative dose intensity (%) Bevacizumab8880 Oxaliplatin76– Irinotecan–76Reasons for discontinuation (%) Progression of disease44.764.5 Toxicity29.86.5Secondary surgery for metastasis (%)12.83.2BV bevacizumab, N number, CI confidence interval BODY.RESULTS.CLINICAL OUTCOMES: After a median follow-up period of 35.9 months (range, 24.2–44.8 months), 39 disease progressions (83.0 %) and 26 deaths (55.3 %) occurred in the 47 patients enrolled. Median 2nd PFS, the primary endpoint, was 18.0 months (95 % CI, 13.7–22.3 months) (Fig. 2a).Fig. 2Survival outcomes. a Median second progression-free survival, the primary endpoint, was 17.7 months (95 % CI, 13.4–22.0 months). b Median progression-free survivals were 13.1 months (95 % CI, 8.7–17.5 months) in the first-line setting and 7.5 months (95 % CI, 4.9–10.2 months) in the second-line setting. c Median overall survival was 30.6 months (95 % CI, 13.4–22.0 months). d Median survival beyond the first progression was 17.7 months (95 % CI, 13.4–22.0 months). Survival curves were estimated using Kaplan–Meier methods Median OS was 30.8 months (95 % CI, 27.7–34.0 months) (Fig. 2c), and median SBP was 19.6 months (95 % CI, 13.5–25.7 months) (Fig. 2d). In the first-line bevacizumab plus mFOLFOX6 therapy, RR and DCR of the 47 patients were 61.7 and 89.4 %, respectively (five patients had CR, 24 patients had PR and 13 patients had SD) (Table 3). The median PFS from the initiation of the first-line therapy was 13.1 months (95 % CI, 8.7–17.5 months) (Fig. 2b).Table 3Objective tumor responseResponseFirst-line therapy(N = 47)Second-line therapy(N = 31)No. of patients%No. of patients%CR510.626.5PR2451.1722.6SD1327.71135.5PD510.61135.5RR (%)61.789.429.064.5DCR (%)No/N number, CR complete response, PR partial response, SD stable disease, PD progressive disease, RR response rate (CR + PR), DCR disease control rate (CR + PR + SD) In the second-line bevacizumab plus FOLFIRI therapy, RR and DCR of the 31 patients who went on to the second-line therapy were 29.0 and 64.5 %, respectively (two patients had CR, seven patients had PR, and 11 patients had SD) (Table 3). The median PFS from the initiation of the second-line therapy was 7.3 months (95 % CI, 5.0–9.6 months) (Fig. 2b). BODY.RESULTS.ADVERSE EVENTS: Frequency of common toxicities is presented in Table 4. The incidences of hematologic and non-hematologic > grade 3 toxic events were 44.4 and 16.7 %. The hematologic toxic events (>grade 3) occurred in 10 patients (50 %) in the first-line therapy and six patients (37.5 %) in the second-line therapy. The non-hematologic toxic events (>grade 3) occurred in five patients (25 %) in the first-line therapy and no patient (0 %) in the second-line therapy.Table 4Frequency of common toxicitiesToxicityFirst-line therapy(N = 47)Second-line therapy(N = 31)All grades (%)>Grade 3 (%)All grades (%)>Grade 3 (%)Hematologic toxicity72.327.751.632.3 Neutropenia57.423.441.922.6 Thrombocytopenia12.809.70 Anemia23.409.70 Febrile neutropenia–4.3–3.2Non-hematologic toxicity85.125.551.612.9 Diarrhea0012.93.2 Nausea/vomiting27.74.319.40 Mucositis10.62.112.93.2 Hand-foot syndrome2.1000 Alopecia2.103.20 Fatigue6.403.23.2 Neuropathy72.317.019.43.2 Allergy12.82.13.20Bevacizumab-associated toxicity51.12.145.23.2 Hypertension25.5045.23.2 Proteinuria21.3016.10 Bleeding2.103.20 Infection2.1000 Thrombosis2.1000 GI perforation2.12.100N number, GI gastrointestinal Severe adverse events associated with bevacizumab during the first-line therapy were grade 3 GI perforation in one case (2 %), grade 2 venous thromboembolic event in one case (2 %), and grade 2 bleeding event in one case (2 %). However, no critical events associated with bevacizumab were observed during the second-line therapy. There was a higher incidence of new or worsening hypertension in the second-line therapy as compared with the first-line therapy (26 vs. 45 %). BODY.DISCUSSION: This is the first prospective study to examine the continuous use of bevacizumab in combination with FOLFIRI after failing the first-line treatment with mFOLFOX/bevacizumab combination in the Japanese patients with mCRC. There are several issues regarding the use of BBP that needs to be clarified; the response and survival benefit obtained through adding bevacizumab to each line of chemotherapy, the survival benefit of the BBP strategy per se, and the adverse effect of long-term exposure to bevacizumab among patients who received BBP. Of these, benefits in terms of response rate and survival by adding bevacizumab to either the first-line oxaliplatin-based chemotherapy or second-line irinotecan-based chemotherapy have been well documented in previous clinical trials [7, 11]. In the current study, the response and survival data observed both in the first-line and second-line settings seem to compare favorably with these studies, with a RR of 61.7 % and a PFS of 13.1 months in the first-line setting, and a RR of 29.0 % and a PFS of 7.5 months in the second-line setting. In general, failure to respond to chemotherapy with cytotoxic agents implies inherent or acquired resistance to the therapy and leads to a change in the therapeutic regimen. The mechanisms of the resistance to cytotoxic agents are typically consequences of genetic instability inherent in cancer that renders mutant cells insensitive to chemotherapeutic agents. In contrast, the mechanisms of resistance to biologic targeted agents, including bevacizumab, are not well understood. One hypothesis that forms the basis of BBP is that persistent VEGF suppression continues to have clinical benefit when given in combination with the secondary and tertiary cytotoxic regimens. This hypothesis was supported by the results of several clinical trials exploring benefit of BBP. The first evidence of a survival benefit associated with BBP was generated by a large, observational study, BRiTE study. In this study, the patients who had been treated with BBP had a superior median SBP and OS (19.2 and 31.8 months, respectively) as compared with those who were treated without BBP (9.5 and 19.9 months, respectively) [12]. The ARIES study examined the role of bevacizumab after disease progression in patients who had received first-line bevacizumab and in those who were bevacizumab-naive at the time of second-line treatment. The authors observed a trend toward longer SBP and OS in patients who had received first-line and second-line bevacizumab (median SBP: 14.1 and OS: 27.5 months) when compared with patients who received bevacizumab only after the disease progression (median SBP: 7.5 and OS: 18.7 months), while PFS of the second-line treatment was similar in both groups [13]. The primary objective of the current study was to assess the efficacy of BBP determined in terms of the 2nd PFS, defined as the time duration from the initiation of the first-line therapy until disease progression during the second-line of chemotherapy. Tournigand et al. reported that the median 2nd PFS was 10.9 months when the first-line FOLFOX and second-line FOLFIRI were administered, both without bevacizumab, and this was a historical benchmark to design our study. The median 2nd PFS of 17.7 months as shown in this study met our expectations and clearly pointed to an improvement in the outcome compared with the historical precedent setting without bevacizumab. There could be an argument that the endpoint of a chemotherapeutic strategy such as BBP that constitutes from several lines of treatment should be OS. In this aspect, the median OS and SBP in this study were 30.8 and 19.6 months, respectively. These survival data are potentially comparable with the results observed in the BBP population from the previous studies. Safety of a long-term exposure to bevacizumab among patients who received BBP is another issue explored in this study. The safety outcomes in the BRiTE study showed no apparent increase in serious adverse events reported in the BBP group compared with the no-BBP group [12, 14], with the exception of thromboembolic event in the elderly population [15]. Such thromboembolic event was rare at 2 % in the current Japanese population. Other severe adverse events associated with bevacizumab were grade 2 bleeding event (2 %) and grade 3 GI perforation (2 %), all of which occurred during the first-line chemotherapy. Thus, no critical events associated with bevacizumab were observed during the second-line therapy. It is of note that a higher incidence of new or worsening hypertension was observed during the second-line therapy compared with the first-line therapy. The higher cumulative incidence of hypertension in the BBP group was not unexpected, given that the risk of developing bevacizumab-associated hypertension appears to accumulate over time and that the BBP results in substantially longer bevacizumab exposure. The type and frequency of other grade 3/4 events (including neutropenia, diarrhea, vomiting, and asthenia) were consistent with the known safety profile of the chemotherapy regimens. Our study is merely hypothesis-generating regarding the efficacy of BBP because of the one-arm design and relatively small sample size. However, it does imply that the BBP strategy is beneficial to the Japanese population with the 2nd PFS nearly 10 months longer than that observed in the Tournigand study and SBP and OS that is similar to the survival data observed in the BRiTE study and the ARIES study. Data regarding safety of the BBP strategy was more robust, in which only hypertension was to be carefully taken care of. From these encouraging data, it can now be recommended that a randomized study involving a larger numbers of patients be performed in Japan to obtain hard evidence regarding the efficacy of BBP. In summary, the planned continuation of bevacizumab during the second-line treatment is feasible for the Japanese mCRC patients. A prospective randomized control study to confirm the efficacy is warranted.

TITLE: The comparison of Proseal laryngeal mask airway and endotracheal tube in patients undergoing laparoscopic surgeries under general anaesthesia ABSTRACT: Aims to compare the efficacy of Proseal laryngeal mask airway(PLMA) and endotracheal tube (ETT) in patients undergoing laparoscopic surgeries under general anaesthesia. This prospective randomised study was conducted on 60 adult patients, 30 each in two groups, of ASA I-II who were posted for laparoscopic procedures under general anaesthesia. After preoxygenation, anaesthesia was induced with propofol, fentanyl and vecuronium. PLMA or ETT was inserted and cuff inflated. Nasogastric tube (NGT) was passed in all patients. Anaesthesia was maintained with N2 O, O2, halothane and vecuronium. Ventilation was set at 8 ml/kg and respiratory rate of 12/min. The attempts and time taken for insertion of devices, haemodynamic changes, oxygenation, ventilation and intraoperative and postoperative laryngopharyngeal morbidity (LPM) were noted. There was no failed insertion of devices. Time taken for successful passage of NGT was 9.77 s (6-16 s) and 11.5 s (8-17 s) for groups P and E, respectively. There were no statistically significant differences in oxygen saturation (SpO2) or end-tidal carbon dioxide (EtCO2) between the two groups before or during peritoneal insufflation. Median (range) airway pressure at which oropharyngeal leak occurred during the leak test with PLMA was 35 (24-40) cm of H2O. There was no case of inadequate ventilation, regurgitation, or aspiration recorded. No significant difference in laryngopharyngeal morbidity was noted. A properly positionedPLMA proved to be a suitable and safe alternative to ETT for airway management in elective fasted, adult patients undergoing laparoscopic surgeries. It provided equally effective pulmonary ventilation despite high airway pressures without gastric distention, regurgitation, and aspiration. BODY.INTRODUCTION: In spite of tremendous advances in contemporary anaesthetic practice, advances, airway management continues to be of paramount importance to anaesthesiologists. Till date, the cuffed tracheal tube was considered as the gold standard for providing a safe glottic seal, especially for laparoscopic procedures under general anaesthesia.[1] The disadvantages of tracheal intubation, which involves rigid laryngoscopy, are in terms of concomitant haemodynamic responses and damage to the oropharyngeal structures at insertion. Postoperative sore throat is also a serious concern. This precludes the global utility of the tracheal tube and requires a better alternative.[2] Over a period of time, new airway devices have been added to the anaesthesiologist's armamentarium. Proseal laryngeal mask airway (PLMA) has a dorsal cuff, in addition to the peripheral cuff of LMA, which pushes the mask anterior to provide a better seal around the glottic aperture and permits high airway pressures without leak. The drain tube parallel to the ventilation tube permits drainage of passively regurgitated gastric fluid away from the airway and serves as a passage for gastric tube.[2] The PLMA is a relatively new airway device in developing nations. This study is therefore undertaken to compare PLMA with standard tracheal tube for the number of attempts and time taken for insertion, haemodynamic changes, oxygenation, ventilation and intraoperative and postoperative laryngopharyngeal morbidity (LPM) occurring during general anaesthesia in young healthy adult patients undergoing laparoscopic surgeries. BODY.METHODS: After obtaining the Ethics committee approval and written informed consent, this prospective randomised study was conducted on 60 healthy patients. The patients were of either sex belonging to ASA physical status grade I and II, aged 20-65 years and body weight 40-76 kg, who underwent laparoscopic procedures under general anaesthesia. Patients with anticipated difficult airway, obesity (body mass index > 35 kg/m2), oropharyngeal pathology, cardiopulmonary disease, cervical spine fracture or instability, or at increased risk of aspiration (gastro-esophageal reflux disease, hiatus hernia, and pregnant patients) were excluded from the study. Patients were randomised for airway management with the PLMA or endotracheal tube (ETT) by opening an opaque envelope inside the operation theatre containing the computer-generated random assignment into two groups of 30 each. Patients in group P were to receive a PLMA and patients in group E were to undergo endotracheal intubation. Patients were premedicated with oral alprazolam 0.5 mg the night before surgery and on the day of surgery. After intravenous (IV) access was obtained, ranitidine 50 mg and metoclopramide 10 mg were administered 30 minutes before surgery. In the operation theatre, standard monitors were attachedand baseline parameters were recorded. Injections of midazolam 0.02 mg/kg, glycopyrrolate 0.005 mg/kg, and fentanyl 1-2 μg/kg were administered 1-2 min before induction. After preoxygenation with 100% O2 for 3-5 minutes, anaesthesia was induced with injection of propofol 2-2.5 mg/kg till the loss of verbal commands. Neuromuscular blockade to facilitate placement of device was achieved by vecuronium 0.08-0.1 mg/kg. Following induction and adequate paralysis, the corresponding airway was inserted in each group. The airway devices were inserted by anaesthesiologists with at least 1 year experience with PLMA and ETT. In group P, size 3 or 4 PLMA (according to weight) was used. For the purpose of standardisation, we used the introducer for inserting the PLMA for all cases as recommended by the manufacturer. In group E, endotracheal intubation (7.5 in females and 8 in males) was performed in standard manner. The time interval between holding the airway device to confirmation of correct placement by bilateral air entry on chest auscultation was noted. Correct placement of the devices was confirmed by: Adequate chest movement on manual ventilationSquare wave capnographyExpired tidal volume of more than 8 ml/kgNo audible leak from the drain tube with peak airway pressure (PAP) less than 20 cm H 2O. A leak below 20 cm H 2O was taken as significant and suggested a malpositionThe gel displacement test, done by placing a blob of gel at the tip of the drain tube (DT) and noting the airway pressure at which it was ejectedThe last two tests were specific for group P. Anaesthesia was maintained with oxygen, nitrous oxide, halothane, and vecuronium. The outcomes measured were as follows: Insertion characteristics of the PLMA or ETT and the nasogastric tube (NGT) via the PLMA and the ETT (NGT was introduced in all cases).Easy insertion – insertion at first attempt with no resistance; difficult insertion -insertion with resistance or at second attempt; and failed insertion – insertion not possible.Haemodynamic responses(heart rate and mean arterial blood pressure)were recorded before induction; at the time of insertion; 1 and 3, 5 min after insertion of device; after achieving carboperitoneum, and during removal of devices.Oxygen saturation (SpO2) and end-tidal carbon dioxide (EtCO2); at a tidal volume of 8 ml/kg, fraction of inspired oxygen (FiO2) 0.33, respiratory rate of 12/min and I/E of 1:2 were recorded.The aim was to maintain target SpO2(>95%) and EtCO2 (<45 mm Hg) by adjusting the FiO2, respiratory rate and tidal volume. When SpO2 was 94-90% the oxygenation was graded as suboptimal and failed if it was <90%.Oropharyngeal seal pressure was determined by closing the expiratory valve at a fixed gas flow of 5 l/min and recording the airway pressure at which equilibrium was reached. The airway pressure was not allowed to exceed 40 cm H2O.The PAP was recorded when intra-abdominal pressure (IAP) reached 16 mm Hg. For standardisation, IAP was maintained at 12-16 mm Hg.Incidences of gastric distension (by surgeon), regurgitation, aspiration, intraoperative and postoperative laryngopharyngeal morbidity were noted. BODY.METHODS.STATISTICAL ANALYSIS: Data were analysed using INSTAT 3 (GraphPad Software, California, USA). The primary variables studied were oxygenation and adequacy of ventilation. Secondary variables were time to achieve an effective airway, airway interventions required, haemodynamic parameters, cuff leak pressure, and PAP. Sample size of 60, with 30 patients in each group was determined for primary variables (O2 saturation and EtCO2), using the following information from various previous studies: standard deviations of 5% and 5 mm Hg for the two variables, respectively, were considered statistically significant. If the statistically significant difference in a decrease in oxygen saturation was less than 95% for one of the devices, it was considered to be clinically significant. Sample size was calculated assuming a two-sided test with α = 0.05 and the power of 0.9. Two-sided independent Student's t tests to analyse continuous data, and Fisher's exact test for categorical data. P<0.05 was considered as significant. BODY.RESULTS: The surgical procedures, patient characteristics and details of anaesthesia and airway management are shown in Figure 1. Demographic data were comparable in both groups. Figure 1Demographic data and type of procedures done Size 3 PLMA placement was attempted in 19 patients, size 4 in 11 patients [Table 1]. Insertion success rate was 86.67% for the first attempt, and two attempts were made in 13.33% patients. Insertion was easy in 23 and difficult in 7 patients. In Group E, the insertion success rate was 83.37% for the first attempt; two attempts were made in 13.33% of patients and third attempt was required in 3.33% patients. There was no failed insertion reported in either group. Mean time (range) taken for successful placement was 15.77 s (12-21 s) and 16.93 s (11-28 s) for PLMA and ETT, respectively. Table 1 Details of airway management Airway device details PLMA ETT P value Size of device (3/4,7.5/8) 19/11 23/7 Attempt of insertion (1/2/3/ failed) 26/4/0/0 25/4/1/0 Time taken for insertion of device, Mean (SD) 15.77 (2.97) 16.93 (4.07) 0.209 Attempts at gastric tube insertion (1/2/3/failed) 27/3/0/0 20/7/3/0 Time taken for insertion of gastric tube, Mean (SD) 9.77 (2.44) 11.5 (2.28) 0.006 Oropharyngeal seal pressure, Median 35 cm of H 2 O Time taken for successful passage of NGT was 9.77 s (6-16 s) and 11.5 s (8-17 s) for P and E groups, respectively. On comparing the trends within groups statistically significant (P<0.05) increase in heart rate and the mean blood pressure was observed 10 seconds after intubation and persisted till 3 minutes after intubation and during the time of extubation in the ETT group. However, statistically significant (P<0.05) increase in the heart rate and mean blood pressure in PLMA group was seen only 10 seconds after insertion [Figure 2]. Figure 2Haemodynamic parameters Statistically significant increase in heart rate and mean blood pressure was observed 10 seconds after intubation and persisted till 3 minutes after intubation and also during extubation in the ETT group. However, statistically significant increase in Proseal-LMA group was seen only 10 seconds after insertion. The EtCO2 was comparable in both groups throughout the surgery (P>0.05) and did not increase beyond 45 mm Hg. The PAP in group P showed a statistically significant (P<0.05) increase in value after insertion till 6 minutes after pneumoperitoneum was attained, and thereafter it was insignificant. Both groups maintained oxygen saturation perioperatively except in one patient in the PLMA group where oxygen saturation dropped to 94% (suboptimal oxygenation) after placing patients in the reverse Trendelenburg position. The oxygen saturation returned to normal after the PLMA was repositioned. Oropharyngeal seal pressure for PLMA group observed was 35 mm Hg (median), with no clinically audible leak throughout the surgery. The PAP, however, did not increase beyond the oropharyngeal seal pressure in the PLMA group [Table 1]. In the present study, coughing after removal of PLMA was seen in 6.67% patients, while it was seen in 3.33% patients in the ETT group. Blood staining of device on removal was seen in 10% patients in group P and in 16.67% patients in group E. Minor trauma to the lip and gums was seen in 1 patient (3.33%) in group E. There was no incidence of intraoperative or postoperative laryngospasm, bronchospasm, in either group. There was no incidence of regurgitation or clinically detectable pulmonary aspiration in either group [Table 2]. In 3 patients, gastric distention was successfully decompressed via NGT suction. Sore throat postoperatively was seen in 10% patients in group P and in 20% patients in group E. After 24 hours, no patient in group P but 2 patients (6.67%) in E group complained of sore throat. Table 2 Laryngopharyngeal morbidity PLMA ETT P Intraoperative  1. Leak 1 -  2. Gastric insufflation 3 -  3. Regurgitation, aspiration - - At removal  1. Coughing 2 1 0.556  2. Blood staining of device 3 5 0.45  3. Trauma to lip, teeth, tongue 4 1 0.17 Postoperative  1. Vomiting - -  2. Sore throat 3 7 0.171  3. Dysphagia, dysphonia, dysarthia - - BODY.DISCUSSION: The PLMA is a new entrant to the family of LMA with some added features over the classic LMA.[3] This study was conducted with the aim of comparing PLMA and ETT as a ventilatory device in 60 patients undergoing laparoscopic surgeries. We chose this study because increased intra-abdominal pressure from pneumoperitoneum requires higher airway pressures for adequate pulmonary ventilation, for which the PLMA has proved to be adequate in previous[124] studies. Although PLMA was easier to insert with higher success rate (86.67%) in the first attempt than the ETT (83.33%), this was not statistically significant. Mean time taken for successful placement was 15.77 s and 16.93 s for groups P and E, respectively. Studies by Cook, Shroff and coworkers (median effective time 15 s) corroborated with our study findings.[45] Sharma and coworkers, in their study of 100 and 1,000 PLMA insertions, reported a mean insertion time of 13.51 s and 12 s, respectively.[16] This lesser time could be attributed to the fact that their study was conducted by anaesthesiologists who had more experience in working with PLMA. A NGT was inserted in all patients. The mean insertion time taken to insert NGT through PLMA was significantly less (9.77 s) than via nose (11.5 s) in intubated patients. Similarly, the success rate of NGT in the first attempt was higher (90%) via Proseal than via nasal route in intubated patients (66.67%). These factors may be of clinical relevance in patients with hypertension, head injury, and ischaemic heart disease. There was minimum haemodynamic stress response with PLMA when compared with endotracheal intubation. These findings are similar to those of previous studies.[127] The increase in heart rate during intubation is attributed to sympathetic stimulation during laryngoscopy and the passage of the ETT through the vocal cords.[189] The PLMA being a supraglottic device does not require laryngoscopy and probably does not evoke a significant sympathetic response. Attenuation of this response may be due to diminished catecholamine release.[10] This could be due to the fact that the PLMA is relatively simple and atraumatic to insert and does not require laryngoscopy.[9] Following peritoneal insufflation, CO2 is absorbed transperitoneally, and the rate at which this occurs depends on gas solubility, perfusion of the peritoneal cavity, and duration of the pneumoperitoneum.[11] Both groups maintained adequate oxygenation and ventilation perioperatively, except in one patient in the PLMA group, where oxygen saturation dropped to 94% (suboptimal oxygenation) after placing the patient in reverse Trendelenburg position. We repositioned the PLMA and oxygen saturation returned to normal thereafter. Maltby et al. and Sharma et al. found no statistically significant differences in SpO2 or EtCO2 between the two groups before or during peritoneal insufflations.[711] However, Sharma and colleagues in a later study noted that although all patients had optimal oxygenation, three patients had EtCO2 in excess of 55 mm Hg after CO2 insufflation.[6] This was explained by the fact that the airway tube was narrow and the epiglottis downfolded in some patients. The incidence of epiglottic downfolding has been reported to be as high as 31-66%.[12] The observed oropharyngeal seal pressure for PLMA group was 35 mm Hg (median), with no clinically audible leak throughout the surgery. The PAP did not increase beyond the oropharyngeal seal pressure throughout surgery. This is in accordance with the findings of previous studies.[1367] In three patients, gastric distention was successfully decompressed by suctioning the NGT. There was no incidence of regurgitation or aspiration in either group. Similar results have been reported by others.[11314] The incidence of sore throat was comparatively more in the intubation group E (20%) than in group P (10%). All patients were administered gargles and steam inhalation. After 24 hours, none of the patients in the Proseal group had sore throat; however, two patients in group E had persistent sore throat till 48 hours. Higgins et al. and Shroff et al. also found the greatest incidence of sore throat in patients undergoing intubation than in those in whom a PLMA was used.[414] The virtual absence of sore throat in PLMA group could be explained by the fact that it is a supraglottic device and mucosal pressures achieved are usually below pharyngeal perfusion pressures.[15] Although endotracheal intubation is the gold standard in laparoscopic surgeries done under general anaesthesia, the PLMA proved to be an equally effective airway tool in laparoscopic surgeries in terms of adequate oxygenation and ventilation with minimal intraoperative and postoperative complications. The haemodynamic stress response was also minimal with PLMA when compared to endotracheal intubation. It provided equally effective pulmonary ventilation despite high airway pressures without significant gastric distention, aspiration, and regurgitation. BODY.CONCLUSION: Hence, we conclude that the PLMA proved to be a suitable and safe alternative to ETT for airway management in elective fasted, adult patients undergoing laparoscopic surgeries.

TITLE: Elevated D-dimers in attacks of hereditary angioedema are not associated with increased thrombotic risk ABSTRACT.BACKGROUND: Recommended management of attacks of hereditary angioedema (HAE) due to C1 esterase inhibitor (C1-INH) deficiency (C1-INH-HAE) includes therapy with exogenous C1INH. Thrombotic/thromboembolic events (TEE) have been reported with plasma-derived C1INH, but so far none with recombinant human C1INH (rhC1INH). This phase III, randomized, placebo (saline)-controlled study evaluated the safety of rhC1INH 50 IU/kg for the treatment of acute attacks in 74 patients with C1-INH-HAE. ABSTRACT.METHODS: Monitoring for TEE and assessment of risk of deep vein thrombosis (DVT) by the Wells prediction rule were performed, and levels of fibrin degradation products (plasma D-dimers) were assessed before study drug administration (baseline), 2 h, and 7 days posttreatment. ABSTRACT.RESULTS: Plasma D-dimer levels were elevated in 80% of the patients (median [25th–75th percentiles]: 2149 [480–5105] μg/l; normal ≤250 μg/l) and were higher in patients with submucosal (abdominal, oropharyngeal–laryngeal) attacks (3095 [890–10000] μg/l; n = 29) compared with subcutaneous (peripheral, facial) attacks (960 [450–4060] μg/l; n = 35). Median plasma D-dimer levels were comparable across treatment groups at baseline (1874 [475–4568] μg/l rhC1INH; 2259 [586–7533] μg/l saline) and 2 h postinfusion (2389 [760–4974] μg/l rhC1INH; 2550 [310–8410] μg/l saline); median plasma D-dimer levels were decreased by Day 7 in both groups (425 [232–3240] μg/l rhC1INH; 418 [246–2318] μg/l saline). No increased risk of DVT was identified, nor any TEE reported in rhC1INH treated or controls. ABSTRACT.CONCLUSION: Elevated plasma D-dimer levels were associated with acute C1-INH-HAE attacks, particularly with submucosal involvement. However, rhC1INH therapy was not associated with thrombotic events. BODY: Activation of the contact–kinin pathway in patients with hereditary angioedema (HAE) due to C1 esterase inhibitor (C1INH) deficiency (C1-INH-HAE) results in bradykinin generation, vascular leakage, accumulation of watery fluid in the tissues, and edema formation (1). Additionally, when Factor XII (FXII) becomes activated (FXIIa) in vitro, particularly in the absence of its main serine-protease inhibitor- C1INH, FXIIa, and kallikrein may activate plasminogen, resulting in plasmin formation. Plasmin is the principal enzyme that degrades stable fibrin lattices (2). Moreover, plasmin facilitates the cleavage of high molecular weight kininogen by kallikrein, thus increasing bradykinin generation and edema formation (3). Plasminogen activation by FXIIa may be due to the high homology of FXII and tissue plasminogen activator (tPA), a major fibrinolysis initiator (4), and to similarity in their catalytic sites (both are inhibited by corn trypsin inhibitor—CTI) (5). In addition, endothelial cell activation has been shown to be associated with elevated FXII-dependent fibrinolytic activity (6) and plasminogen activation is triggered by bradykinin-stimulated release of tPA from the surface of endothelial cells (7). Indeed, high levels of procoagulant and fibrinolytic activity have been demonstrated in patients with C1-INH-HAE during acute attacks and remissions (8–11). In clinical practice, elevated plasma D-dimers are considered biomarkers of extensive thrombosis but are also elevated in certain nonpathologic conditions (12). Despite evidence of extensive activation of both coagulation–contact and fibrinolytic systems, relatively low rates of spontaneous thromboembolic events (TEEs) have been reported in patients with C1-INH-HAE. However, recent reports of TEE during treatment with commercially available human plasma-derived C1 inhibitor (pdC1INH) products have raised concerns. Up to ten confirmed and seven possible cases of TEE have been associated with the use of pdC1INH (Cinryze®) in both controlled clinical trials and postmarketing studies; of which, five were considered serious (13). Similarly, TEEs have been associated with recommended and off-label high doses of another pdC1INH (Berinert®) (14). Animal studies have also supported a potential risk of thrombosis from intravenous administration of pdC1INH products (15). Recombinant C1INH (rhC1INH, conestat alfa, Ruconest®) is a novel product homologous to human C1INH and produced in transgenic rabbits (16). To date, no TEE has been observed following repeated treatments with rhC1INH in 250 patients with over 1000 administrations (17–21). Furthermore, animal studies have corroborated lack of thrombotic risk, including a no-observed-adverse-effect level of 2000 IU/kg/day, in a 14-day repeated dose toxicity study in cynomolgus monkeys (22). Here, we present results of a clinical study where plasma D-dimers were measured in patients with C1-INH-HAE during acute attacks and after treatment with rhC1INH, and evaluate their utility as a biomarker of fibrinolyticactivity and potential probe of disease activity. BODY.MATERIALS AND METHODS.PATIENTS AND STUDY DESIGN: Seventy-five patients participated in a randomized, double-blind, placebo-controlled study to evaluate the efficacy and safety of rhC1INH compared with saline, for the treatment of acute angioedema attacks in patients with C1-INH-HAE (https://www.clinicaltrials.gov/ct2/results?term=NCT01188564&Search=Search; identifier NCT01188564). Patients (age ≥13 years), with a laboratory-confirmed diagnosis of C1-INH-HAE, were randomized (3 : 2) to receive an intravenous injection of rhC1INH (50 IU/kg for patients <84 kg, or 4200 IU for patients ≥84 kg) (17) or saline. One patient randomized to the rhC1INH group was withdrawn at the discretion of the investigator, did not receive study medication, and has not been included in the current analysis (safety population, N = 74). Thirteen patients in the saline treatment group received rhC1INH as rescue medication for acute attacks. For all analyses, these patients are included in the saline summaries up until the time they received rescue medication, and are included in the rhC1INH summaries afterward. C1-INH-HAE patients with peripheral (extremities), abdominal, facial, or oropharyngeal–laryngeal attacks were eligible for rhC1INH treatment if the onset of attack was <5 h prior to presentation to the clinic. Overall severity of the attack was rated by the patient to be ≥50 mm on a Visual Analog Scale (VAS, markings made on a 0- to 100-mm horizontal line represent the severity/intensity of each item) (17). For patients with multiple eligible attack locations, the primary attack location was defined as the location with the highest VAS score at baseline. All patients provided written informed consent. The study was approved by the local institutional review board at each site. BODY.MATERIALS AND METHODS.THROMBOTIC RISK ASSESSMENTS: All randomized patients were clinically monitored for TEE including deep vein thrombosis (DVT) and pulmonary embolism (PE). The risk of DVT was also assessed using the Wells prediction rule (23). Patients with an increase in D-dimer levels were to be clinically evaluated for the possible development of TEE, including ultrasound examination as indicated. BODY.MATERIALS AND METHODS.PLASMA SAMPLE COLLECTION: For the determination of D-dimer levels in the plasma, citrated blood samples were collected at baseline (i.e., prior to intravenous injection of study medication or placebo), at 2 h, and at Day 7 (after the attack resolved) following intravenous injection of study medication or placebo. BODY.MATERIALS AND METHODS.PLASMA D-DIMER MEASUREMENT: This was a multicenter study where separated plasma samples were sent immediately to local laboratories for measurement of plasma D-dimer levels, according to standard protocols. D-dimer levels were measured by two latex-based turbidimetric immunoassays: HemosIL D-Dimer HS (Instrumentation Labs, Bedford MA, USA) and Innovance D-Dimer (Siemens AG, Erlangen, Germany). Results in FEU (fibrinogen equivalent units) were converted to DDU (D-Dimer units). Values of ≤250 μg/l were considered normal in the final analysis. BODY.MATERIALS AND METHODS.STATISTICS: Data were analyzed using SAS software version 9.3 (SAS Institute Inc. Cary, North Carolina, USA). All data were summarized by descriptive statistics using the safety population. Descriptive statistics for continuous variables include the mean, standard deviation, median, interquartile range (25th and 75th percentiles), and range (minimum and maximum values); categorical variables were presented as counts (n) and percentages (%). Plasma D-dimer levels were summarized and presented by time point (baseline, 2 h, and Day 7 posttreatment) and treatment group, based on anatomical location (submucosal vs subcutaneous) and baseline severity (moderate: VAS between 50 and 75 mm; severe ≥75 mm) at the primary attack location. The Wilcoxon rank sum test was used to compare medians for plasma D-dimer levels in patients presenting with submucosal vs subcutaneous attacks. BODY.RESULTS.PATIENT DEMOGRAPHICS: Seventy-four patients presenting with eligible acute attacks were randomized and received either 50 IU/kg rhC1INH (N = 43) or saline (N = 31). Patient disposition, key demographics, and HAE attack frequency and severity of the eligible attack were similar between groups (Table1). Attack severity at baseline, as rated by the patients using a 100-mm VAS scale, was similar in both groups (group means: 73.5 mm [rhC1INH] vs 77.3 mm [saline]). The most common primary attack locations were peripheral and abdominal and were similar in the rhC1INH and the saline groups (peripheral: 44% rhC1INH and 45% saline; abdominal: 37% rhC1INH and 39% saline). Table 1 Patient demographics and baseline characteristics for safety population rhC1INH ( N = 43) Saline ( N = 31) Female (%) 65 61 Caucasian (%) 95 97 Age at screening (years)  Mean (SD) 39.1 (12.63) 41.4 (15.38)  Range 17–67 18–69 HAE attacks/year  Mean (SD) 25 (23.9) 31 (27.2)  Range 0–143 3–111 Use of prophylactic maintenance therapy ( n [%]) 21 [49] 15 [48] Primary attack location ( n [%]) *  Peripheral 19 [44] 14 [45]  Abdominal 16 [37] 12 [39]  Facial 6 [14] 2 [6]  Oropharyngeal–laryngeal 2 [5] 3 [10] Overall severity VAS score at baseline for primary attack location (mm) *  Mean (SD) 73.5 (14.13) 77.3 (12.61)  Range 50–100 49–100 HAE, hereditary angioedema; rhC1INH, recombinant human C1 esterase inhibitor; VAS, Visual Analog Scale. * For patients with >1 eligible attack location, the primary attack location was defined as the eligible location with the highest overall severity VAS score at baseline. BODY.RESULTS.THROMBOTIC OR THROMBOEMBOLIC ADVERSE EVENTS: There were no reports of thrombotic or thromboembolic adverse events in patients treated with rhC1INH or placebo. BODY.RESULTS.RISK OF DEEP VEIN THROMBOSIS: Wells scores were available for 39 of 43 rhC1INH-treated patients and 30 of 31 saline-treated patients. None of the patients were identified as having an increased risk of DVT based on these scores (23). Ultrasounds performed on two patients (one rhC1INH and one saline) were normal in both abdomen and lower extremities with no evidence of DVT. BODY.RESULTS.PLASMA D-DIMER LEVELS: Median plasma D-dimer levels were elevated in the patients at baseline (2149 [IQR: 480–5105] μg/l, normal range ≤250 μg/l) (Table2), with 51 of 64 patients (79.7%) having levels above normal. D-dimer levels continued to increase in all patients 2 h after treatment with either rhC1INH or saline, to a median level of 2469 (643–5827) μg/l. By Day 7 posttreatment, D-dimer levels in both treatment groups regressed toward near-normal levels. Median plasma D-dimer levels were not statistically different between the groups at 2 h (P = 0.8706) and Day 7 (P = 0.9753) after treatment with either rhC1INH or saline (Fig.1), suggesting that treatment with rhC1INH did not influence plasma D-dimer production in patients with C1-INH-HAE. Table 2 D-dimer levels over time in safety population Time point D-dimer levels (μg/l) Percentage of patients with D-dimer levels >250 μg/l * Baseline  Mean (SD) 4211 (5622) 79.7  Median (25th–75th percentile) 2149 (480–5105)  Range 6–24634   N 64 2 h after treatment †  Mean (SD) 4421 (5740) 83.8  Median (25th–75th percentile) 2469 (643–5827)  Range 9–5827   N 68 Day 7 after treatment †  Mean (SD) 1842 (2867) 73.4  Median (25th–75th percentile) 425 (241–3240)  Range 1–14250   N 64 * Normal D-dimer levels are ≤250 μg/l. † Thirteen patients who originally received saline solution subsequently received rhC1INH as rescue medication. These patients are included in the saline solution summaries up until the time they received rescue medication, and included in the rhC1INH summaries afterward. Figure 1D-dimer (median, 25th–75th percentile) levels over time in HAE patients treated with rhC1INH or saline. rhC1INH, recombinant human C1 esterase inhibitor. There were 15 submucosal (abdominal, oropharyngeal–laryngeal) attacks and 21 subcutaneous (facial, peripheral) attacks treated with rhC1INH; 14 submucosal and 14 subcutaneous attacks were treated with saline (Table3 and Fig.2). Median plasma D-dimer levels were at least threefold higher at baseline (P = 0.0274) and 2 h posttreatment (P = 0.0126) in patients with submucosal attacks compared to patients with subcutaneous attacks. Table 3 D-dimer levels in HAE patients with submucosal vs subcutaneous locations of the eligible attack Time point/anatomical location * rhC1INH ( N = 43) Saline ( N = 31) Total ( N = 74) Baseline, μg/l  Submucosal † 3095 (250–8676) 3055 (1700–11350) 3095 (890–10000)  Subcutaneous ‡ 1000 (500–4060) 899 (260–3800) 960 (450–4060)   P -value 0.2050 0.1029 0.0274 2 h, μg/l  Submucosal 4100 (1030–7731) 5470 (2550–12500) 4100 (1030–12140)  Subcutaneous 1080 (730–4260) 835 (310–2200) 1070 (600–4100)   P -value 0.1771 0.0308 0.0126 Day 7, μg/l  Submucosal 768 (266–4250) 418 (245–2614) 454 (266–4250)  Subcutaneous 376 (150–1400) 453 (246–2318) 376 (168–1400)   P -value 0.1958 0.9581 0.2699 HAE, hereditary angioedema; rhC1INH, recombinant human C1 esterase inhibitor. Values are presented as median (25th–75th percentiles). * Anatomical location represents the primary attack location (see Materials and methods for description). † Submucosal = oropharyngeal–laryngeal, abdominal. No urogenital attacks were reported. ‡ Subcutaneous = peripheral, facial. Figure 2D-dimer levels over time in HAE patients with submucosal vs subcutaneous locations of the eligible attack. rhC1INH, recombinant human C1 esterase inhibitor; SC, subcutaneous; SM, submucosal. Overall, median baseline plasma D-dimer levels were similar in patients with moderate (1674 [593–5241] μg/l) and severe (2320 [260–5550] μg/l) attacks (Table4). Of note, severe attacks treated with rhC1INH did tend to have lower plasma D-dimer values (280 [109–925] μg/l) by Day 7 than those treated with saline (560 [273–4056] μg/l; P = 0.1323, not significant). Table 4 D-dimer levels by severity at the primary attack location n Moderate (≥50 mm, <75 mm) * n Severe (≥75 mm) * Baseline, μg/l 24 1674 (593–5241) 39 2320 (260–5550) 2 h, μg/l 27 2000 (656–5884) 40 2678 (615–5840) Day 7, μg/l 26 1025 (382–3770) 37 300 (150–1250) VAS, Visual Analog Scale. Values are presented as number of patients who had D-dimer levels at each time point and as median (25th–75th percentiles). * Severity is based on the overall VAS score at each visit at the primary attack location. Sixty-four patients reported single-site attacks and ten reported multiple-affected-site attacks. At baseline and at 2 h, median plasma D-dimer levels were higher in patients with multiple affected locations than those in patients with single locations. By Day 7, D-dimer levels had returned to near-normal for both groups (Fig.3). Figure 3D-dimer levels over time in patients with multiple vs single affected locations. BODY.DISCUSSION: In the absence of normal inhibition by C1INH, kinin–contact pathway activation is accelerated, resulting in bradykinin generation and tissue edema (1). Additionally, increased fibrinolytic activity has been demonstrated in patients during HAE attacks and even in remissions (8–11,24,25). Moreover, activation of coagulation may give rise to prothrombin fragments F1 + 2, Factor VIIa, and thrombin, with similar activation observed during HAE attacks (10,11). In a recent study by van Geffen et al. (25), activated plasma and 'nonactivated' plasma (taken during attacks and remissions) were studied in patients with C1-INH-HAE. Interestingly, F1 + 2, thrombin, and fibrinolysis markers (PAP, D-dimers) were significantly elevated during attacks as compared to remissions and healthy controls. Despite this, spontaneous TEE has been rarely reported in patients with HAE, implying that fibrin degradation occurs in the absence of increased thrombotic risk (11,24). The therapeutic use of pdC1INH is generally considered safe, although concerns have been recently raised regarding the risk of TEE for other C1INHs (13,14). Some events were possibly related to very high off-label doses of pdC1INH (i.e., in capillary leak syndrome), but others occurred during therapy for HAE with recommended doses (13). Therefore, thrombogenicity might have been related to the C1INH protein itself, any impurities in the preparation, its production process, batch-to-batch heterogeneity, delivery systems, or patient factors. For example, human plasma products such as therapeutic immunoglobulins may carry prothrombotic risk, as increased amidolytic activity of kallikrein and FXIa contaminants has been recently demonstrated by Etscheid et al. (26). In view of these reports and the lifelong need for large amounts of pdC1INH for both on-demand and prophylactic purposes, potential risks should be reassessed. As recombinant C1INH (Ruconest®, rhC1INH) is the newest on the market, its potential thrombogenic risk was recently investigated by Relan et al. (27). This study demonstrated that although coagulation and fibrinolysis pathways were indeed activated during acute attacks, treatment with rhC1INH had little or no effect on other coagulation markers, suggesting that it does not exert prothrombotic activity in vivo (16,22). The present study focused on plasma D-dimers because these are considered a reliable and sensitive index of thrombosis in vivo and indicative of a dynamic process of thrombus formation and lysis (12). In clinical practice, D-dimers are elevated in various thrombotic conditions, including venous thromboembolism, disseminated intravascular coagulation, and cerebrovascular accidents (28). However, D-dimers have also been shown to be elevated in conditions without clinical evidence for thrombosis (29–31). Despite such limitations, plasma D-dimers are regarded as useful laboratory markers for the diagnosis of thrombotic conditions (12). Our study corroborates other studies, which have suggested that, despite elevated plasma D-dimers at baseline and during attacks, C1-INH-HAE patients are not at increased thrombotic risk (24,25,27). This seemingly paradoxical situation, where activation of the contact coagulation (i.e., via Factor XI), kinin pathway, and fibrinolytic systems does not lead to increased thrombosis, is reconciled by the observation that while FXII-driven fibrin formation is important for pathologic thrombus formation, it has no important function in fibrin formation during normal hemostasis (32). In this context, it is of interest that bradykinin B2 receptor-deficient mice (a rodent model of HAE) are protected from thrombosis (33). Furthermore, there is recent evidence for a dichotomy between bradykinin formation by activated contact system and lack of procoagulant activity in both human and rodent systems (34). Maas et al. have shown that the activation of FXII by misfolded proteins or heparin initiates contact system activation without triggering the intrinsic (FXIIa/FXIa dependent) coagulation pathway, while activation by naturally occurring polyanions (i.e., polyphosphates released by microorganisms or platelets) triggers fibrin formation in a FXII-dependent manner (32,34). We propose that in patients with C1-INH-HAE, subclinical formation of fibrin degradation products (i.e., D-dimers) may reflect a disturbed balance between coagulation and fibrinolysis. Recently, Koning et al. (11) have shown that there is no preferential activation of the contact over the intrinsic coagulation system in C1-INH-HAE and proposed that the absence of thrombotic complications is due to other regulatory mechanisms controlling the coagulation and fibrinolysis cascades (11). Our results do show higher plasma D-dimer levels in patients with C1-INH-HAE during attacks, as compared to remission, that is, at 7 days after the attack, which may imply that this parameter might be suitable as a biomarker of disease activity, so much desired in this intermittent disease. Such changes were especially prominent in submucosal-type attacks (abdominal, oropharyngeal–laryngeal) as compared with skin involvement, which may indicate an organ-specific association. The contact system can assemble on endothelial surfaces, where C1INH is less capable of inactivating FXII and kallikrein, which may enhance local contact system activity (34). It seems plausible that the extent of endothelial involvement or vascular permeability changes (35) may have contributed to the differences observed in this study, as more extensive attacks at multiple locations were also associated with higher D-dimer levels. Evidently, the administration of rhC1INH does not reduce elevated D-dimer levels (which remained high in the saline-treated cohort as well), suggesting that the major source of ongoing coagulation in C1-INH-HAE is unrelated to activation of the intrinsic pathway, and thrombus formation does not necessarily follow after Factor XII activation (32,34). This indicates that the major source of plasmin activity responsible for D-dimer formation in C1-INH-HAE is unrelated to the Factor XII-dependent intrinsic pathway, which is in line with in vivo studies demonstrating that the large majority of plasminogen activation is attributable to tPA and uPA (6). The source of increased plasma D-dimers in patients with C1-INH-HAE is presently unknown. We can only speculate that local fibrin lattice formation and stabilization may be related to several factors, such as extensive endothelial hyperpermeability, vascular leakage, extravascular tissue factor, or a systemic inflammatory process. We recognize that the main limitation of this study is that it represents the results of a single treatment, whereas in real-life situation, patients with C1-INH-HAE undergo repeated treatments with multiple doses of C1INH. In conclusion, based on the analyses of this cohort, as well as other RCTs and clinical experience (17–21), we suggest that rhC1INH does not carry an increased prothrombotic risk. Moreover, this study indicates that increased plasma D-dimer levels are strongly associated with acute C1-INH-HAE attacks. The value of measuring plasma D-dimers in the decision-making process of C1-INH-HAE treatment needs to be evaluated by RCTs specifically designed for this purpose.

TITLE: Tranexamic acid reduces perioperative blood loss of posterior lumbar surgery for stenosis or spondylolisthesis ABSTRACT.ABSTRACT.BACKGROUND:: A prospective, randomized, double-blind, placebo-controlled study was performed. The routine usage of TA in spinal surgery is controversial. Only a few studies have focused on patients undergoing posterior lumbar surgery for stenosis or spondylolisthesis, although a large clinical cohort exists in the population. This study aimed to evaluate the effect and safety of TA in reducing perioperative blood loss in posterior lumbar surgery for stenosis or spondylolisthesis. ABSTRACT.ABSTRACT.METHODS:: 100 eligible patients out of 126 were randomized to receive either a bolus dose of 30 mg/kg TA i.v, a maintenance dosage of 2 mg/kg/h TA, or an equivalent volume of normal saline. The pedicle screw system was used for fixing in all the patients, followed by decompression and posterior lumbar interbody fusion. The primary outcomes were intraoperative estimated blood loss and total blood loss. The secondary outcomes were receiving packed red blood cells and postoperative hemoglobin and hematocrit levels. ABSTRACT.ABSTRACT.RESULTS:: In total, 4 patients were excluded from the analyses, 50 patients were in the TA group, and 46 in the placebo group. The demographic and baseline data between the groups were not statistically different. The intraoperative estimated blood loss and the total blood loss were 33% and 41% lower in the TA group than the placebo group, respectively. The blood transfusion rate did not vary significantly (P = 0.191). Except a patient with a dural tear in the placebo group, no other complications were observed. ABSTRACT.ABSTRACT.CONCLUSION:: TA significantly reduced the perioperative blood loss in patients undergoing posterior lumbar surgery for stenosis or spondylolisthesis. BODY.INTRODUCTION: 1 Lumbar stenosis and spondylolisthesis are the most common diseases[1,2] in clinical practice. Many patients with this disease are required to undergo posterior decompression interbody fusion surgery,[3,4] and massive blood loss is inevitable in the perioperative.[5–7] Although several methods have been introduced to reduce the perioperative blood loss and transfusion requirements, including preoperative autologous blood donation, hypotensive anesthesia, normovolemic hemodilution, perioperative blood salvage, much of the blood loss was yet inevitable.[4,7,8] Such excessive blood loss may cause severe hypotension, metabolic acidosis, infections, acute lung injury, and cardiac arrest.[6,8,9] Some patients might also have to receive an allogeneic blood transfusion, which increases their risk of transfusion-acquired infectious diseases and immune suppression.[8–10] Recently, a number of studies demonstrated that TA can efficiently decrease the perioperative blood loss and transfusion.[9,11–17] Most of the previous studies focused on patients undergoing spinal correction surgery,[8,9,11,12,16,17] and only a few studies were involved in the patients undergoing posterior lumbar surgery for stenosis or spondylolisthesis.[15,18] Since the surgical procedure for stenosis or spondylolisthesis is not identical to spinal correction surgery, it can be correlated with extensive exposure, long-segment fixation, spinal canal decompression, interbody fusion,[3–5] and massive blood loss.[5,7] Whether TA efficiently reduces the blood loss in these surgical procedures is yet controversial. A large cohort of patients undergoes this surgery, and hence, the surgeon is required to pay more attention to the perioperative blood loss in the patients. TA is a synthetic antifibrinolytic drug and can reversibly bind at lysine sites of plasmin and plasminogen. The proteolytic action of plasmin and plasminogen on fibrin is inhibited by this interaction, which further prevents fibrinolysis and protects the clots, thereby decreasing the blood loss from the surgical wound.[19] The present study evaluated the effect and safety of TA on reducing perioperative blood loss in posterior lumbar surgery for stenosis or spondylolisthesis. BODY.MATERIALS AND METHODS: 2 The study was approved by the First Affiliated Hospital of Chongqing Medical University Review Board (20150401) and registered at WHO International Clinical Trials Registry Platform (ChiCTR-IPR-15006088). All the patients were enrolled between January 2015 and March 2016 at the First Affiliated Hospital of Chongqing Medical University. BODY.MATERIALS AND METHODS.INCLUSION CRITERIA: 2.1 The basic inclusion criteria for recruitment were: (1) patients with lumbar spinal stenosis or lumbar spondylolisthesis who were scheduled to undergo posterior lumbar decompression interbody fusion; the conservative therapy had failed. (2) Patients aged 18 to 80 years. (3) Patients who provided written informed consent. BODY.MATERIALS AND METHODS.EXCLUSION CRITERIA: 2.2 The exclusion criteria were: (1) allergy to TA. (2) History of bleeding disorders or thromboembolic events. (3) Severe cardiac or respiratory disease and renal or hepatic dysfunction. (4) Platelet count <150,000/mm3. (5) Preoperative Hb <10 g/dL. (6) Uncontrolled hypertension; high blood pressure (BP >160/90 mm Hg). (7) ASA physical status >III. (8) Intake of nonsteroidal anti-inflammatory drugs within 7 days before surgery. (9) Pregnancy. BODY.MATERIALS AND METHODS.RANDOMIZATION AND MASKING: 2.3 Each patient eligible for the study was designated a unique sequence number. All the patients were randomly stratified into 3 levels, based on the difficulty of the surgical procedure. Subsequently, the sequence numbers were allocated to either the TA group or the placebo group by the computer-generated random assignment. The protocol assigned for the enrolled patients was also coded. On the day of surgery, a nurse trained in performing the protocol before the study, who was not involved with the trial or was not a personnel caring for the patients, opened the sealed envelope in an isolated room away from the operating room, and prepared the treatment. The bottle with the bolus dose and the syringe with the maintenance intravenous infusion were marked only with the sequence number and the acronym of the patient's name. All the apparatus was indistinguishable between the TA and placebo groups. After the treatment preparation, the envelopes were resealed until disclosed by the statistician. The anesthesiologist, surgeons, researchers, and patients were blinded to the procedure. BODY.MATERIALS AND METHODS.UNBLINDING FOR MEDICAL EMERGENCIES: 2.4 In case, the anesthesiologist or surgeon suspected medical emergencies associated with TA, such as allergic shock, pulmonary embolism, deep vein thrombosis, stroke, or ischemic heart disease, the nature of the treatment was disclosed. BODY.MATERIALS AND METHODS.ADMINISTRATION PROGRAM: 2.5 Patients receiving a loading dose of 30 mg/kg TA over 15 minutes before skin incision followed by a maintenance infusion of TA (2 mg/kg/h) were continued until skin closure in the TA group. Equivalent normal saline was administered in the placebo group. BODY.MATERIALS AND METHODS.ANESTHESIA AND MONITORING: 2.6 The standard general anesthesia applied to all patients was induced with intravenous drugs, including midazolam, sufentanil, propofol, and atracurium. The anesthesia was maintained using sevoflurane and atracurium. Consecutive to standard monitoring, the arterial blood pressure was also continuously monitored by a radial arterial line during operations. The mean arterial blood pressure (MAP) was controlled at about 20% below the preoperative baseline pressure in both the groups, until the procedures of decompression, fusion, and instrumentation were completed following which it was returned to the baseline. The maintenance fluid requirements and third-space losses were regulated by colloid and crystal solution. The transfusion threshold for the intraoperative Hb level was <7.0 g/dL, which was guided by arterial blood gas analysis, arterial blood pressure, central venous catheter, and urinary output. BODY.MATERIALS AND METHODS.SURGICAL PROCEDURE: 2.7 All the surgeries in both the TA group and placebo group patients were performed by 2 surgeons: one with 12 years' experience in spinal surgery and the other with 17 years. The attending surgeons completed a short questionnaire about the difficulty of surgery, a day before the operation. The operations were divided into 3 levels based on surgical difficulty (level I: low difficulty; level II: medium difficulty; level III: high difficulty). Conventionally, the decompression fusion and fixation of 1–2 segments were defined as level I, 3–4 segments as level II, and more than 4 segments as level III.[14,20,21] Surgeries involved revision, extensive decompression, severe ASA physical status, and corpulent patients, which would increase the surgical difficulty,[7,14,22] requiring the surgeons to adjust the assessment of surgical difficulty. All patients were placed in a prone position on the Hall–Relton frame or the Jackson table with abdomen free and underwent posterior decompression interbody fusion and fixation with the pedicle screw. Using the posterior midline approach, the lamina, facet joints, and spinous processes of fixation segment were exposed. After the pedicle screws system had been implanted, and the appropriate location confirmed by radiography, the 2 connecting rods were installed. Next, semilaminectomy and removal of the disc in the decompression segment and a CAGE with autogenous bone granules tamponade was placed into the intervertebral space. Then, radiography was used to confirm the position of CAGE at the appropriate region. The iliac crest bone was not used for grafting. During the procedure of decompression and fusion, it is essential that surgeon carefully protects the nerve root and dura mater spinalis, and avert any injury to venous plexus. Before closing the wound, the bleeding is cautiously stanched, and drainage placed routinely. BODY.MATERIALS AND METHODS.DATA COLLECTION: 2.8 The primary outcomes were intraoperative estimated blood loss and total blood loss. The intraoperative estimated blood loss was estimated by a trained nurse before starting the trial and was blinded to the treatment and patient groups. The intraoperative estimated blood loss was evaluated by adding the weight of gauzes and sponges to the volume of blood in the negative pressure suction bottles, and subtracting the volume of irrigation fluids. The postoperative drainage was recorded daily from the volume of blood accumulated in the negative pressure drainage bottles until the drainage was removed (when the volume of drainage was less than 30 mL/d). The total amount of bleeding was equal to the intraoperative estimated blood loss plus postoperative drainage. The second outcome was receiving packed red blood cells, as well as postoperative hemoglobin, hematocrit levels. Postoperatively, the laboratory parameters such as Hb, HCT, PLT, PT, APTT, as well as, fibrinogen and D-dimer were estimated on postoperative days 1 and 3. When the postoperative Hb level was <70 g/L, 7.0 to10.0 g/dL and patients exhibited symptomatic anemia, such as tachycardia, fatigue, lethargy, poor appetite, and pallor, packed red blood cells were administered. Data were collected on patients' demographics, laboratory values, intra- and postoperative blood loss, the surgical procedure to be performed, and blood products transfused perioperatively. The inpatients were daily observed for putative complications and unexpected events associated with treatment, such as the clinical symptoms of ischemic heart disease, stroke, deep vein thrombosis, and pulmonary embolism. All discharged patients were followed-up each week until the day 35 after the operation. BODY.MATERIALS AND METHODS.STATISTICAL METHODS: 2.9 The data were analyzed by SPSS software version 17 (SPSS Inc., Chicago, IL). A previous study showed that the mean perioperative blood loss for lumbar posterior interbody fusion was 500 ± 350 mL when treated at our institution. Presuming a 30% reduction in the perioperative blood loss, 2-tailed test with 0.05 alpha error, and a statistical power of 0.8, 46 patients were needed for each group. Therefore, we strategized to enroll 50 patients in each group. The qualitative data were expressed numerically and also as a percentage. The parametric quantitative data were expressed as mean and standard deviation, whereas the nonparametric quantitative data were expressed as median (range). The qualitative data were compared with the χ2 test or Fisher's exact test. The parametric quantitative data were analyzed using an unpaired t-test, whereas the nonparametric quantitative data were compared with the Mann–Whitney U test. Correlation analysis was used to explore the potential impact factors for total blood loss. Then, in order to find the weight of potential risk factors for significant bleeding, we used multiple logistic regression analysis to construct the formula model. P < 0.05 was considered statistically significant. BODY.RESULTS: 3 A cohort of 126 consecutive patients was screened, and 100 patients aged 22 to 78 years were enrolled. These patients presented lumbar spinal stenosis or lumbar spondylolisthesis and were undergoing posterior decompression interbody fusion fixation with the pedicle screw. In total, 51 patients were randomly assigned in the TA group and 49 in the placebo group (mean ages, 56 and 54 years, respectively). During the operation, 3 patients changed to the simple procedure (1 in the TA group and 2 in the placebo group), and dural tear occurred in 1 patient in the placebo group. Thus, 4 patients were excluded from all the analyses (Fig. 1). Moreover, any statistical differences were not observed between the 2 groups with respect to age, sex, MAP, weight, ASA physical status, and diagnosis. Additionally, the preoperative parameters of Hb, HCT, PLT, PT, and APTT were also not remarkably different between the 2 groups (Table 1). Figure 1Schematic of the study design. TA indicates tranexamic acid. TA = tranexamic acid. Table 1 Baseline profile of the 2 study groups. However, intraoperative estimated blood loss (170 ± 153 mL vs 255 ± 188 mL, P = 0.016), postoperative drainage (145 ± 84 mL vs 281 ± 165 mL, P ≤ 0.001), and total blood loss (315 ± 205 mL vs 536 ± 261 mL, P < 0.001) were significantly different between the TA and placebo groups (Fig. 2). On the other hand, the operative time, surgeons, transfusion, and total liquid volume of fluid and crystalloid did not exhibit distinct differences between the TA and placebo groups (Table 2). Figure 2The differences between the 2 groups on IEBL, PD, TBL. Data were analyzed by 1-way ANOVA. #P < 0.05; ##P < 0.001, IEBL = intraoperative estimated blood loss, PD = postoperative drainage, TBL = total blood loss. Table 2 Operative data. Similar to the preoperative parameters, the postoperative Hb, HCT, PLT, PT, and APTT were also not statistically different between the groups (Table 3). Except a patient with dural tear in the placebo group, which was excluded from the statistical analysis, no other major complications, such as deep vein thrombosis, pulmonary embolism, stroke, ischemic heart disease, epidural hematoma formation, allergic reaction, liver and kidney dysfunction were observed. Table 3 Postoperative hematocrit and coagulation test. Correlation analysis showed that TA, operative time, difficult level of surgery and prothrombin time were of potential impact on total blood loss (Table 4). Furthermore, the formula model conducted with multiple logistic regression analysis showed that only the operation time and TA were the important factors for significant bleeding (total blood loss >500 mL). TA showed a distinctive protective effect for blood loss (OR 0.002, 95% CI 2.483–25.371, P < 0.001) (Table 5). Table 4 Correlation analysis for potential impact factors to total blood loss. Table 5 Risk factors for significant bleeding (>500 mL) from multiple logistic regression analysis. BODY.DISCUSSION: 4 The present study aimed to evaluate the efficacy and safety of TA in reducing perioperative blood loss in posterior lumbar surgery for stenosis or spondylolisthesis. Our results showed 33% reduced intraoperative estimated blood loss in the TA group and 41% total blood loss as compared to the placebo group. Only 1 patient showed the occurrence of a dural tear in the placebo group; no other adverse events with the use of TA were seen in our study. Previous studies showed that ASA physical status, body weight, different surgical techniques, the number of fusion segment, attending surgeons, duration of operation, and heterogeneous patient populations for various diagnosis may impact the perioperative blood loss.[9,13,20,22,23] In our study, the patient profile, diagnosis, ASA physical status, and level of difficulty did not differ between the 2 groups. Moreover, the operation time and the patients treated by the 2 attending surgeons between the 2 groups did not exhibit any statistical difference. All the surgical procedures were similar among the patients undergoing posterior decompression lumbar interbody fusion fixation with the pedicle screw. Hence, the baseline was identical between the TA and placebo groups. Although correlation analysis showed that tranexamica acid, operative time, surgical difficulty, PT were potential factors for blood loss; the logistic regression model predicted that only TA and operative time were the important factors for significant bleeding. TA was found to exert an impact on decreasing the perioperative blood loss (OR 0.002, 95% CI 2.483–25.371, P < 0.001), which was in agreement with the previous studies.[9,13,20] In order to eliminate the interference of operative time, we had divided patients into subgroups depending on whether their operative time was more than 180 minutes or not. There were 80 patients' operative times less than 180 minutes, and the intraoperative estimated blood loss, total blood loss were significantly different between the TA group and the PL group (P = 0.013, P < 0.001). This meant TA still significantly reduced the blood loss in this subgroup. Nevertheless, the intraoperative time and total blood loss were not significantly different between the 2 groups when the operative time was more than 180 minutes (Table 6). Although TA did not lead to significant reduction in the perioperative blood loss, there were only 16 patients in this subgroup, which may be inadequate to detect a difference in the variables. Table 6 Subgroup analysis of perioperative blood loss in different operative time. The etiology of the perioperative blood loss for spinal surgery is multifactorial.[3,5,8,19] The increased fibrinolytic activity and spinal venous plexus injury are critical factors contributing toward the perioperative blood loss.[8,19,24] TA can competitively inhibit plasmin and plasminogen, thereby impeding the fibrinolysis and stabilizing the blood clot.[19] Previous studies demonstrated that TA could inhibit the fibrinolytic activity and decrease blood loss in spinal deformity surgery or long segment fixation and fusion surgery.[9,11–13,25] Elwatidy et al[25] showed that TA reduced 49% blood loss in the spinal surgery. Berney et al[11] reported that TA decreased nearly 50% intraoperative estimated blood loss in adolescent idiopathic scoliosis surgery. The surgery for lumbar stenosis and spondylolisthesis is not identical to the deformity correction surgery or simple long segment fixation and fusion surgery. The vertebral venous plexus is common in patients with lumbar stenosis or spondylolisthesis,[24,26] when injured during the operation, renders hemostasis difficult.[4,14,27] TA can supposedly stabilize the blood clot to block the epidural venous plexus vascular rupture, thus, reducing the bleeding.[14,19,28] Nevertheless, the hemostatic efficacy of TA is yet unconfirmed in the patients undergoing posterior lumbar surgery for stenosis or spondylolisthesis. Wang et al[15] administered 15 mg/kg TA, which reduced 13% total blood loss in patients undergoing posterior lumbar interbody fusion for stenosis in a randomized controlled trial, and the total blood loss was statistically different between the TA and placebo groups. This study subtly assists the clinicians to make operative decisions. Although the study designed by Wang et al was rudimentary, it was composed on a well-balanced baseline index, and the blood loss was observed to be remarkably different between the treatment and control groups. Moreover, the study did not describe any sample size or test power analysis. Typically, any intervention evaluated in a randomized controlled trial only reduces more than 25%, which calls for attention.[11,13,14] The 13% reduction of the total blood loss could not provide reliable evidence for the usage of TA. The present study manifested administering 30 mg/kg initial dose and 2 mg/kg/h maintenance dosage, which lessened 41% total blood loss. This prospective, randomized, double-blind, placebo-controlled study differed from that of Wang et al as we assumed 30% reduction in blood loss, which was clinically significant. The sample size could be recalculated based on the study. The 41% reduction of total blood loss in the present study could provide reliable evidence for the standard use of TA in the surgery for lumbar stenosis or spondylolisthesis. Another difference between Wang et al and our study was the patient population. Only patients with surgical difficulty levels II and III were enrolled in their study, whereas our study also included the difficulty level I patients. The level I is a huge cohort in clinical practice, and any measures to reduce the bleeding should not be ignored in this population. Therefore, we used stratified randomization to prevent interference by the difficulty levels. The composition of the population between the groups was not statistically different. The subgroup analysis exhibited that TA not only lessened the perioperative blood loss in medium and high-level difficulty surgeries for stenosis or spondylolisthesis but also decreased the blood loss in the low difficulty surgeries for these diseases (Table 7). These results showed the similar effect to those in previous studies.[9,11–14,16] The third difference with Wang et al was the administered dose regimen. More than 15 mg/kg is considered as a high dose regimen.[19] Karski[29] conducted a comparison of different dose regimens of TA in cardiac surgery. They found that 100 mg/kg TA has better hemostasis effect than that of 50 mg/kg TA. Using a initial dose of 30 mg/kg TA i.v. and a maintenance dosage of 2 mg/kg/h TA, Elwatidy et al[25] had a similar result to our study. So our study maybe partly support that the high dose of TA have better effect than the low dose of TA in spinal surgeries. Table 7 Subgroup analysis of perioperative blood loss in different difficulty level surgeries. There was an interesting phenomenon that the perioperative blood loss was significantly different between TA and PL groups, whereas postoperative Hb and HCT were not significantly different. We aboratively analyzed the data, and then found that although there was no statistically significant difference in the blood transfusion requirement between the 2 groups, the patients number received packed red blood cells in the placebo group was obviously larger than that of the TA group (4 patients in PL group vs 1 patient in TA group). This may lessen the difference of Hb/HCT between the 2 groups. This is in line with previous studies.[9,14] The postoperative epidural hematoma is a severe complication of after lumbar decompression surgery.[14,30] TA inhibits fibrinolysis and stabilizes blood clot, thereby decreasing the epidural and subcutaneous hematoma formation.[14,19] All patients were followed-up for more than 35 days after the operation. Any hematoma formation in either of the groups, due to the low incidence of hematoma, was not seen[30]; since the sample size was small, the difference may be not discovered. A common concern related to the use of TA is its potential for inducing thromboembolic events. Several studies have confirmed that TA does not increase the risk of deep vein thrombosis and pulmonary embolism.[8,9,11–16,19] In our study, no clinical symptoms of deep vein thrombosis or signs of pulmonary embolism were revealed. Any complications associated with TA, such as allergic reaction, stroke, ischemic heart disease, and dysfunction of liver and kidney, also did not occur. Thus, TA can be safely administered in patients undergoing posterior interbody fusion surgery for lumbar stenosis or spondylolisthesis. However, the present study conferred some limitations. Due to more than 50% patients belonging to the population with low difficulty surgeries, the effect of the test for patients with the difficulty of level II and III would be lessened. We carried out a subgroup analysis based on the level of difficulty. Patients with only the difficulty levels II and III did not show any statistical difference between the 2 groups in intraoperative estimated blood loss, the reduction trend was visible (less 26%); the total blood loss in the TA group was significantly less than the placebo group (Table 7), which is in agreement with the previous studies.[15,18] Nevertheless, future studies would be designed to adequately decrease the number of patients with difficulty level I, and increase the population of level II and III. The second limitation was single dose treatment regimen, as the optimal dose regimen in this kind of spinal surgery is not known. A simultaneous efficacy comparison of different dose regimens of TA will also be investigated. The third limitation was that there were 2 surgeons included in our study. Some documents reported that surgeons may be a potential factor affecting the amount of bleeding during the perioperative period. However, the operative time, intraoperative estimated blood loss, and total blood loss were not statistically different and the correlation analysis showed the surgeons were not significant factor for total blood loss. The significance of surgeons' effects may be minimal, given the fact the patients were equally distributed in both groups, and the surgeons equally taken part in surgeries between the groups, as well as all procedures were similarly performed in the same institution. The present study agrees with the previous documents.[13,14,25] BODY.CONCLUSION: 5 TA efficacy can safely reduce the perioperative blood loss in patients undergoing posterior decompression interbody instrumentation for stenosis or spondylolisthesis. A simultaneous comparison of different dose regimens of TA should be conducted in order to describe the efficiency of the drug for a wide application in the future. BODY.ACKNOWLEDGMENTS: The authors thank Zhang Junhui (Department of Statistics, Southwest Medical University) who provided statistical support.

TITLE: Auricular Acupressure for Managing Postoperative Pain and Knee Motion in Patients with Total Knee Replacement: A Randomized Sham Control Study ABSTRACT: Background. Postoperative pain management remains a significant challenge for all healthcare providers. A randomized controlled trial was conducted to examine the adjuvant effects of auricular acupressure on relieving postoperative pain and improving the passive range of motion in patients with total knee replacement (TKR). Method. Sixty-two patients who had undergone a TKR were randomly assigned to the acupressure group and the sham control group. The intervention was delivered three times a day for 3 days. A visual analog scale (VAS) and the Short-Form McGill Pain Questionnaire were used to assess pain intensity. Pain medication consumption was recorded, and the knee motion was measured using a goniometer. Results. The patients experienced a moderately severe level of pain postoperatively (VAS 58.66 ± 20.35) while being on the routine PCA. No differences were found in pain scores between the groups at all points. However, analgesic drug usage in the acupressure group patients was significantly lower than in the sham control group (P < 0.05), controlling for BMI, age, and pain score. On the 3rd day after surgery, the passive knee motion in the acupressure group patients was significantly better than in the sham control group patients (P < 0.05), controlling for BMI. Conclusion. The application of auricular acupressure at specific therapeutic points significantly reduces the opioid analgesia requirement and improves the knee motion in patients with TKR. BODY.1. INTRODUCTION: Knee osteoarthritis frequently occurs in elderly people. The overall prevalence of knee osteoarthritis is 23.9%, occurring in 21.0% of men and 27.3% of women [1]. Knee-replacement surgery is frequently performed and is highly successful with respect to pain relief and the improvement in knee function in people with advanced knee osteoarthritis [2]. Postoperatively, patients undergoing total knee replacement would experience significant pain, which can limit their progression in the range of knee motion and ambulatory status. Despite the development of new pain control methods and medication and the adoption of guidelines for pain care by many hospitals, postoperative pain is still a problem for many patients. A substantial proportion of total knee replacement (TKR) patients experience severe postoperative pain during the early recovery period [3]. Improper pain control not only increases the burden on many organs but also limits the patient's activity, increases the postoperative morbidity, affects the physical recovery and the emotional state of the patient after surgery, and is more likely to extend the length of the stay and increase the medical costs [4–8]. Generally, the clinical management of postoperative pain following TKR mainly involves the use of opioids through patient-controlled analgesia (PCA) to provide the patient with satisfactory pain relief [9]. Given the inherent side effects and limitations of conventional opioids, studies have been conducted to investigate the effective use of non-opioids or even nonpharmacological treatment modalities. Acupuncture is a nonpharmacological treatment modality for the management of postoperative pain. Recent studies have found that the application of acupuncture to auricular acupoints is effective in producing analgesia [10–13]. Using auricular acupuncture to reduce postoperative pain is promising but not compelling based on a systematic review [14]. Auricular acupuncture using a combination of auricular Shenmen and other relevant points can relieve postoperative pain after orthopedic surgery [15–18]. Shenmen (TF4) is at the upper part of the posterior 1/3 of the fossa and has analgesic, sedative, and anti-inflammatory effects [19]. In clinical setting, Subcortex (AT4) is frequently applied for managing pain and pain-related anxiety [20]. AT4 is at the medial side of antitragus and serves to regulate the cerebral cortex function. Auricular acupressure is similar to acupuncture, but to our knowledge, there had been no randomized controlled trial (RCT) assessing the effectiveness of auricular acupressure for pain management after TKR surgery. Moreover, routine acupuncture in a clinical setting has its limitations. First, many hospitals are not equipped with an acupuncturist or related resources. Second, the cost for acupuncture is expensive for many patients. Auricular acupressure is a noninvasive intervention and requires less technical expertise. In addition, it may be more acceptable to patients compared with acupuncture. Hence, this study aimed to examine the adjuvant effects of auricular acupressure on reducing analgesic drug requests and improving the range of motion in the knee in postoperative patients after total knee replacement. BODY.2. METHODS: Ethical approval was obtained from the Hospital Authority of Taiwan and the National University of Nursing and Health Sciences. Sixty-two patients scheduled to undergo an elective total knee replacement were recruited from August 1st, 2010 to March 31, 2011. The inclusion criteria were the following: (i) degenerative arthritis and having total knee replacement surgery, (ii) use of patient-controlled analgesia (PCA) after surgery and an American Society of Anesthesiologist physical status of Classes I–III, (iii) ability to communicate in Taiwanese or Mandarin, and (iv) agreement to participate in this study. Patients were excluded if they suffered from pain or injuries in the ears, had a history of neurological disorders that could alter their perception of pain, a prior experience with auriculotherapy, malignant tumors, a serious chronic illness, mental illness, or drug/alcohol addiction, or were unable to give consent or follow instructions during the data collection process. All of the patients received an operation under standardized general anesthesia protocols. The patients received routine pain management at the end of the operation: the intravenous PCA was connected to an IV line and set to deliver a bolus of 1 mg morphine with a lockout interval of 5 minutes and a 4-hour maximum morphine dose of 10 mg. BODY.2. METHODS.2.1. EXPERIMENTAL PROCEDURES: Patients who met the study criteria were interviewed by the investigator before the operation; the purposes of the study were explained, and written consent to participate in the study was then obtained. Throughout the study period, one acupressure therapist performed the auricular acupressure, and the outcome data were collected by a research assistant who had been trained and was blinded to the patient's group. The physicians, anesthesiologists, and nurses had no previous knowledge of acupressure and were blinded to the patients' group allocation. The data collection processes are shown in Figure 1. The study, which was conducted at a regional teaching hospital in central Taiwan, used a sham control group and double-blinded experimental design. Sixty-two patients were recruited in our study during an eight-month period. Based on computer-generated codes, block randomization was used with a block size of 4. The patients were randomly assigned into the auricular acupressure or sham control groups. The auricular acupressure therapy was delivered by the therapist, who was an advanced practice nurse and had been accredited for practicing acupressure. The auricular acupressure involved embedding the magnetic beads within skin-colored adhesive tape that was placed on the auricular acupoints and retained in situ for 3 days (Figure 2). The choice of Shenmen (TF4) and subcortex (AT4) acupoints was based on clinical reports [15–18, 21] and the TCM physician's recommendation. Acupressure then was applied by repeatedly pressing the acupoints with the fingertips for 3 minutes per point, 3 times per day (9 AM, 1 PM, 5 PM). The last treatment was given on the third day after surgery at 5 PM. To validate the auricular acupressure, two Chinese medicine practitioners confirmed the acupoints and the acupressure protocol. The patients in the sham control group received regular care and also received the same skin-colored adhesive tape placed on the acupoints but did not receive any massage or acupressure. The sham control patients were given auricular acupressure after the data collection was completed. The sample size was estimated based on the data of the pilot study. With an effect size f of 0.84, an α error probability of 0.05 and data collection performed in triplicate, a sample size of 62 would achieve statistical power up to 0.90. The pain assessment was conducted using the visual analog scale (VAS) and the Short-Form McGill Pain Questionnaire (SF-MPQ), both of which have good reliability [22]. The VAS was used to measure the pain perceived by the patients through a vertical line scale ranging from 0 to 100 mm. A score of 0 indicates "not painful at all", and a score of 100 indicates "the most severe pain ever experienced". Higher scores indicate more severe degrees of perceived pain [23]. The Cronbach's α was 0.83 in this study. The SF-MPQ was modified from the MPQ by Melzack in 1987 [24]. The scale covers the nature of pain and the quantity of pain. The sensory dimension comprises 11 questions, the affective dimension comprises 4 questions, and the present pain intensity (PPIS) dimension comprises one question. The SF-MPQ has been used to assess postoperative pain, labor pain, bone pain, and muscle pain. Similar to the McGill Pain Questionnaire (MPQ), the short form of the questionnaire has good validity, and the correlation between the two reaches 0.65~0.94 [25]. This study adopted the Chinese version of the SF-MPQ, which has been shown to have good internal reliability (Cronbach's α range, 0.78 to 0.83) [26]. Cronbach's α was 0.76 in this study. The range of motion is measured with the flexion mobility of the knee at day 3 after the surgery using the goniometer integrated in the dynamometer [27]. Non-weight-bearing passive ROM values were obtained with the patient in the supine position to allow free hip flexion. BODY.2. METHODS.2.2. STATISTICAL ANALYSIS: Data management and analyses were conducted using the SPSS for Windows statistical package, version 17.0. The patients' demographic profiles and baseline data were compared using the t-test, χ 2, or one-way analysis of variance (ANOVA). A generalized estimation equation (GEE) modeling was applied to investigate the effect of auricular acupressure for the outcomes of pain and opioid consumption. A t-test was used to compare differences in knee motion between the acupressure and sham control groups. A P value of less than 0.05 was considered to indicate statistical significance. BODY.3. RESULTS: A total of 62 patients completed the study, 53 women and 9 men, with a mean age of 70.98 (range 46–88) years. The operations included 31 left knees and 31 right knees and were performed by 2 surgeons. There were no differences between the acupressure and sham control groups in terms of the demographic and clinical characteristic data obtained at the baseline (all P > .05) (Table 1). None of the patients who received auricular acupressure showed any complications or adverse reactions after the therapy. BODY.3. RESULTS.3.1. THE CHANGES IN THE PAIN SCORES: The descriptive data of the postoperative pain scores (VAS-pain and Short-form McGill Pain Questionnaire) reported by the patients are shown in Table 2. Overall, the patients experienced moderately severe pain on the 1st day after surgery (VAS 58.66 ± 20.35) while being on the routine patient-controlled analgesia. However, the pain scores decreased gradually over time (Figure 3) but did not reach significant between-group differences in either of the pain scores (P > .05). BODY.3. RESULTS.3.2. THE CHANGES IN PAIN MEDICATION USE: The results of the analgesic (morphine) usage of the patients are summarized in Figure 4. The analgesic drug usage (38.49 ± 15.53 mg of morphine) in the acupressure group patients was significantly lower than in the sham control group patients (53.07 ± 19.90 mg) (P < 0.05). Additionally, after controlling for BMI, age, and pain scores (VAS) in the generalized estimated equation model analysis, analgesic drug consumption in the acupressure group patients remains significantly lower than in the sham control group (P = .002) patients over time (Table 3). The dose of morphine in the sham control group was 8.38 times higher than in the acupressure group. BODY.3. RESULTS.3.3. THE CHANGES IN THE PASSIVE FLEXION MOTION OF THE KNEE: On the 3rd day after surgery, the passive knee flexion motion in the acupressure group patients (71.68 ± 6.90°) was significantly better than in the sham control group patients (66.94 ± 7.15°) (P = .01). The results of Pearson's correlation analysis found a negative correlation between the knee flexion motion and BMI (P < 0.05). Therefore, further regression analysis was conducted, and after controlling for BMI, the results remained significant between the groups (P = .02). BODY.4. DISCUSSION: Our patients reported postoperative pain as moderately severe during the 1st day (baseline) following TKR while the patients were on PCA for pain management. This result corresponded with previously reported results [1] and those of Norkin et al. [27], who reported that patients are still in pain while being treated with analgesic drugs and techniques, indicating that the management of postoperative pain remains a challenge for medical practitioners. Subsequently, alongside the conventional opioid analgesics, an adjuvant treatment involving a nonpharmacological modality may offer some benefits in the management of postoperative pain in TKR patients. Similar to acupuncture, we found that 3 days of auricular acupressure can significantly lower the postoperative opioid consumption after a total knee replacement surgery. Our results showed that the morphine consumption in the sham control group patients was 8.38 times higher than that in the acupressure group. This finding is consistent with that of previous studies, which showed that auricular acupuncture is effective in managing postoperative pain [14, 15]. Usichenko et al. [15–17] demonstrated that auricular acupuncture significantly reduced the consumption of analgesics by patients after they had undergone a total hip arthroplasty and an ambulatory knee arthroscopy. The analgesic effect of acupressure in the reduction of required postoperative morphine can most likely be explained through the stimulation of acupoints, which can adjust organs, correct imbalances of Qi, stabilize the body, strengthen the functions, and cure diseases [28]. Acupoints stimulation also increases endorphin secretion and serotonin production, thereby suppressing the transmission of pain messages and its perception [28]. Our present findings illustrate that auricular acupressure applied to the Shenmen (TF4) and Subcortex (AT4) acupoints can also improve the range of motion of the knee after the operation. This effect can be explained primarily because auricular acupressure produced an analgesic effect, allowing patients to have a better compliance with the treatments designed to improve the range motion in the knee. A literature search revealed no acupressure studies and only one acupuncture study in a TKR population. Tsang et al. concluded that acupuncture was no better than sham acupuncture in pain relief and in improving the range of knee motion in patients who had undergone bilateral total knee arthroplasty [29]. Nevertheless, that study suffered from small sample size. Thus, more studies need to be conducted and documented to support the effectiveness of acupuncture or acupressure in total knee replacement patients. The acupressure procedure was safe and easy to perform under the perioperative clinical conditions using finger press and massage on the auricular acupoints after the operation. The study protocol was based on the experts' recommendations for the practice of acupuncture. Although the randomization in the groups was successful as indicated by the balance regarding age, gender, and BMI, which can confound the postoperative analgesic requirement, we improved the control over BMI in the statistical analysis to increase the validity of our results. Our double-blinded study design minimized the potential biases. The blinding of patients and data collectors was adequate, which strengthened the credibility of the main result. We selected a randomized sham control design and used adhesive tape on the acupoints as the sham control. We also provided acupressure to the sham control group patients at the end of this study. With this design, all participants eventually received the therapeutic auricular acupressure. Our design may avoid the ethical dilemma of withholding a potentially helpful treatment for half of the participants. Noticeably, our study had no actual placebo group for comparing the study effects. According to TCM theory, the body contains 14 meridians (''Jing", like boulevards) that bear countless branches ("Luo", like byways). As the theory notes, finding a body site without the influence of any Jing and Luo would be difficult. Considering that most of the meridians are densely distributed, especially in the ear, massage anywhere on the body can produce the same therapeutic effect [28]. Consequently, using a nonacupoints placebo group in this study may be inappropriate. Thus, our design has no true placebo group, and the sham control group was used to try to distinguish the auricular acupressure effects from the psychological effects. A potential limitation of our research is that those who chose to be involved in the trial were limited to one hospital, and the findings may therefore not be indicative of the changes likely to occur with the general TKR population. Furthermore, the followup period of 3 days after the TKR surgery was short, and we have no way to determine the long-term effects of auricular acupressure. Furthermore, the dose of acupressure may have been inadequate; for example, more frequent treatments or a different treatment protocol may have generated different results. Given that reasonable evidence on the course effectiveness has been established in the current study, further research should focus on longer-term followup and collecting data from multiple centers to determine whether the changes and positive effects of acupressure are maintained over time. In conclusion, a program of auricular acupressure applied to therapeutic auricular acupoints significantly reduces the opioid analgesic consumption and improves the passive range motion of the knee postoperatively after total knee replacement. However, both groups of patients experienced a similar degree of pain, based on their reported scores, while on the routine PCA for pain management.

TITLE: Cost-Effectiveness Analysis of a Transparent Antimicrobial Dressing for Managing Central Venous and Arterial Catheters in Intensive Care Units ABSTRACT.OBJECTIVE: To model the cost-effectiveness impact of routine use of an antimicrobial chlorhexidine gluconate-containing securement dressing compared to non-antimicrobial transparent dressings for the protection of central vascular lines in intensive care unit patients. ABSTRACT.DESIGN: This study uses a novel health economic model to estimate the cost-effectiveness of using the chlorhexidine gluconate dressing versus transparent dressings in a French intensive care unit scenario. The 30-day time non-homogeneous markovian model comprises eight health states. The probabilities of events derive from a multicentre (12 French intensive care units) randomized controlled trial. 1,000 Monte Carlo simulations of 1,000 patients per dressing strategy are used for probabilistic sensitivity analysis and 95% confidence intervals calculations. The outcome is the number of catheter-related bloodstream infections avoided. Costs of intensive care unit stay are based on a recent French multicentre study and the cost-effectiveness criterion is the cost per catheter-related bloodstream infections avoided. The incremental net monetary benefit per patient is also estimated. ABSTRACT.PATIENTS: 1000 patients per group simulated based on the source randomized controlled trial involving 1,879 adults expected to require intravascular catheterization for 48 hours. ABSTRACT.INTERVENTION: Chlorhexidine Gluconate-containing securement dressing compared to non-antimicrobial transparent dressings. ABSTRACT.RESULTS: The chlorhexidine gluconate dressing prevents 11.8 infections /1,000 patients (95% confidence interval: [3.85; 19.64]) with a number needed to treat of 85 patients. The mean cost difference per patient of €141 is not statistically significant (95% confidence interval: [€-975; €1,258]). The incremental cost-effectiveness ratio is of €12,046 per catheter-related bloodstream infection prevented, and the incremental net monetary benefit per patient is of €344.88. ABSTRACT.CONCLUSIONS: According to the base case scenario, the chlorhexidine gluconate dressing is more cost-effective than the reference dressing. ABSTRACT.TRIAL REGISTRATION: This model is based on the data from the RCT registered with www.clinicaltrials.gov (NCT01189682). BODY.INTRODUCTION: Catheter-related bloodstream infections (CRBSIs) are associated with attributable mortality rates of up to 11.5% and additional intensive care unit (ICU) length of stay of up to 12 days [1,2]. The broadly accepted method for minimizing CRBSIs is a bundle of care combining maximal sterile barrier precautions for insertion, an appropriate antiseptic solution for skin antisepsis and line access, preferential subclavian catheterization, and immediate removal of unnecessary catheters [3,4]. Combining this catheter-care bundle with continuous quality improvement programs can decrease the CRBSI rate below 2 per 1,000 central venous catheter (CVC)-days [5,6]. In Europe, the incidence of CRBSIs ranges from 1 to 3.1 per 1,000 patient-days [7] and according to the French surveillance network, less than one CRBSI occurred per 1,000 CVC-days in 2010 [8]. However, rates below 2 per 1,000 CVC-days are difficult to achieve in all ICUs [9,10] and in the long term [11]. Most organisms responsible for short-term CRBSIs originate from the insertion site [12]. It has been previously demonstrated that the risk of developing CRBSIs can be dramatically reduced (60% decrease) by the systematic use of a new antimicrobial transparent dressing [13] containing a Chlorhexidine Gluconate (CHG) gel even though bundles of care are appropriately followed and CRBSI level is lower than 1.5 per 1,000 catheter-days in the control group. The purpose of this work is to evaluate the advantages of the routine use of the new CHG dressing to secure central lines of patients in ICU from a medico-economic viewpoint compared to non-antimicrobial transparent dressings, in settings where bundles of care practices are appropriately followed and where incidence of infection is already low (below 2 per 1,000 catheter-days [13,14]). Both medical and economic criteria are embedded into a decision-analytic model to support the choice of the best dressing strategy from an ICU perspective. BODY.METHODS.STUDY DESIGN: The adopted modeling approach complies with the guidelines of French National Authority for Health (Haute Autorité de Santé—HAS) [15]. The 30-day ICU-time non-homogeneous Markov model [16–18] structure was based on observed data of a multicentre randomized controlled trial (RCT) [13], conducted by the Grenoble University Hospital—CHU Grenoble (ClinicalTrials.gov Identifier: NCT01189682). The model has been programmed using Visual Basic Application with the Excel 2007 software. BODY.METHODS.DATA COLLECTION: The main data source was the database assembling all patient data collected during the RCT [13]. This multicentre randomized-controlled study compared the impact of the antimicrobial 3M Tegaderm CHG (referred in the current study as CHG dressings) and of non-antimicrobial transparent dressings (referred as non-CHG dressings) on the rate of catheter related infections. The main objective of the RCT transposed in this cost-effectiveness analysis was to determine if the use of the new transparent CHG dressing decreased CRBSI rates. The RCT was not blinded to the investigators or ICU staff due to the obvious visual differences between the dressings, but was blinded to the microbiologists processing the skin and catheter cultures and to the committee adjudicating on the CRBSI cases. The two groups receiving different types of non-antimicrobial transparent dressings in the RCT were pooled together as "non-antimicrobial transparent dressings" for the purpose of the modeling presented in this paper. BODY.METHODS.STUDY POPULATION: The multicentre RCT [13] enrolled adult patients (>18 years) admitted to 12 French ICUs in seven universities and four general hospitals, from 31 May 2010 to 29 July 2011, and expected to require intravascular catheterization for 48 hours. Patients with known allergies to chlorhexidine or transparent dressings were excluded. Of 2,054 screened patients with at least one catheter, 1,898 could be enrolled in the study and 1,879 were assessable for the intention-to-treat analysis, for a total of 4,163 catheters and 34,339 catheter-days. Patients and catheters characteristics are reported in the Results section. BODY.METHODS.STUDY CATHETERS: In the RCT [13], all central venous catheters inserted at subclavian, jugular and femoral veins, as well as arterial catheters inserted at radial and femoral arteries for a given patient, were managed according to the randomized dressing assignment. Pulmonary arterial, hemodialysis, and peripherally-inserted venous catheters and catheters inserted before ICU admission were excluded from the study. All study centers followed French recommendations for catheter insertion and care, which are similar to Center for Disease Control (CDC) recommendations [19]. BODY.METHODS.ENDPOINTS: The final health outcome of the cost-effectiveness analysis is the number of CRBSIs avoided and the cost-effectiveness criterion is the cost per patient with CRBSI avoided resulting from chlorhexidine dressing use. BODY.METHODS.MODELING AND STATISTICAL ANALYSIS: Markov models simulate the trajectory of patients among distinct states of health over time [20–23]. The main assumption of state-transition Markov models is that the next health state depends only on the present state and not on the sequence of events that preceded it. Eight health states were considered (Table 1), four combining either occurrence, or no occurrence, of CRBSI, and the need, or no need, of a new central line (CT); one for contact dermatitis; one for changing to an alternative dressing (gauze and tape) in case of dermatitis, and two absorbing states (death and discharge from the ICU). 10.1371/journal.pone.0130439.t001 Table 1 Health states defined from a multicentre randomized controlled trial [ 13 ]. Health States Definition 1. No CRBSI / No new CT needed Insertion of a first catheter, no diagnosed CRBSI and no contact dermatitis 2. No CRBSI / new CT needed * No diagnosed CRBSI, no contact dermatitis and a new catheter inserted (not as a replacement) 3. CRBSI / No new CT needed CRBSI diagnosed without neither contact dermatitis nor the need for inserting a new catheter 4. CRBSIs / new CT needed * CRBSI diagnosed without contact dermatitis but the need for inserting a new catheter 5. Contact dermatitis No diagnosed CRBSI, and no need for new catheter inserted but occurrence of contact dermatitis 6. Dressing Gauze and Tape Change to an alternative dressing strategy (gauze and tape) 7. Discharge Patient leaves the ICU alive 8. Death Patient dies during the ICU stay * New CT needed can mean either the replacement of the existing catheter, or the need for an additional catheter at a new site. CRBSI, Catheter-related Bloodstream Infections; CT, Catheter (Central venous or radial / femoral arterial). The statistical unit of the study is the ICU patient within a time horizon of 30 days (including patients discharged alive from the ICU, alive but still in the ICU, or deceased during the ICU stay). Patient data from the multicentre RCT (source study) [13], comparing the 3M Tegaderm Chlorhexidine Gluconate (CHG) Securement dressing to non-antimicrobial transparent dressings, were translated into a daily patient transition matrix among the different possible health states, for both the antimicrobial and non-antimicrobial dressing groups (see transition matrices in S1 and S2 Tables in Supporting Information). Data was censored beyond 30 days. The transition matrixes were used to perform non-homogeneous Markov-Chain Monte Carlo (NH-MCMC) simulations [24] representing the observed daily evolution of patients in ICU. The possible transitions among health states from one day to the next are represented in the Markov diagram (Fig 1). The Markov cycle duration corresponds to one day. One thousand Monte Carlo simulations of 1,000 patients were used for probabilistic sensitivity analysis and 95% confidence intervals (CI) calculations. 10.1371/journal.pone.0130439.g001Fig 1Structure of the Markov Model showing the possible transition between health states from one Markov cycle to the next cycle.The costs per patient for each health state were calculated in both CHG and No-CHG dressing as respectively: State 1: €1,270 and €1,266; State 2: €1,364 and €1,361; State 3: €13,661 and €13,658; State 4: €13,756 and €13,752; State 5: €1,388 and €1,385; State 6: €1,266 and €1,266; State 7: €0 for both groups; State 8: €0 for both groups; CHG: chlorhexidine gluconate; CRBSI: catheter-related bloodstream infection; CT: catheter. BODY.METHODS.MAIN ASSUMPTIONS: In the cases where the "Discharge" state was reported, and a CRBSI was observed for this patient up to two days after the event, the infection was considered to occur the day of discharge from the ICU.The transitional probability from the health state "Contact Dermatitis" to "Dressing Gauze and Tape" state was considered the same for both groups. By entering to the "Dressing Gauze and Tape" the patient followed probabilities of transition corresponding to the non-CHG dressings arm.The cost of CRBSI is independent from the outcome (survival or death or discharge).Catheter colonization with or without CRBSIs was considered as having negligible diagnosis costs and was excluded from the model for not being considered as a "health state" per se.The costs related to replacement of a catheter suspected to be colonized (and causing CRBSI) were comprised in one of the health states including the need for a new central line. The cost per ICU day was considered as identical for each dressing group. The cost of a gauze and tape dressing is identical in both groups.Health states including CRBSIs were assumed to last a single day because it was not technically possible to identify the termination of a CRBSI in the patient database. However, the costs of treating the complete episode, as well as the total costs associated with the extra length of stay due to the CRBSI were accounted on the day when the CRBSI was diagnosed. BODY.METHODS.MAIN ASSUMPTIONS.BASE CASE INPUT PARAMETERS CONSIDERED IN THE COST ANALYSIS: The base case analysis is the most representative case of the real life, considering French ICU settings, and depending on expert opinions, literature, and RCTs. The main input parameters considered in the cost analysis are the following, in €2013: Dressing costs per day: CHG dressing, €3.59 [13]; non-antimicrobial transparent film, €0.18; gauze and tape, €0.06.Cost of treating contact dermatitis (mean/episode): catheter removal, €23.62 [25]; four gauze and tape dressings, €0.24; catheter insertion, €94.87. Note that the skin lesions themselves healed spontaneously upon removal of the transparent dressings, without further negative health impact or treatment costs.Direct cost of treating CRBSI (mean/episode) [25]: €580.26.Cost per ICU [26]: €1,265.93 per day.Additional ICU Length of stay (LOS) due to CRBSI: 9.33 days (NH-MCMC calculation).Cost of added ICU LOS due to CRBSI: €11,811.13 (NH-MCMC calculation).Cost per catheter change (venous + arterial: 50%-50%) [25]: €94.97.Overall cost of one CRBSI (direct cost of treating one CRBSI plus cost of additional ICU LOS due to CRBSI): €12,391.40 (calculation). Direct costs for the treatment of CRBSIs were obtained from a micro-costing study [25]. ICU costs were based on an observational (real life) study [26] that assessed all resources consumed during a patient day in the ICU. This twenty-four hours multicentre prospective medico-economic study provides a complete overview and estimation of the actual average cost for medical and surgical ICUs in different hospital types in France: Hospitals (CH), University Hospitals (CHU) and Regional Hospitals (CHR). Twenty-two ICUs were selected randomly and all costs for 109 patients were estimated. For patients with CRBSI, an additional cost [27] due to an extra ICU length of stay (LOS) was calculated (see next section). BODY.METHODS.MAIN ASSUMPTIONS.ADDITIONAL ICU LOS DUE TO CRBSIS AND COMPARABILITY OF PATIENTS’ SUBGROUPS WITH OR WITHOUT CRBSIS: In order to assess the impact of CRBSIs on extending ICU LOS, a subgroup analysis was performed comparing patients having developed a CRBSI during the ICU stay with those who did not have a CRBSI. The comparison was made through independent non-homogeneous MCMC simulations for each dressing group (CHG and Non-CHG). The non-homogeneous Markov Chain Monte Carlo simulation in each group estimated additional ICU LOS due to CRBSIs as of 8.55 days and 10.1 days for the CHG and Non-CHG strategies, respectively. For the base case scenario, we set an extra ICU LOS of 9.33 days, which was an average between the two strategies. BODY.METHODS.MAIN ASSUMPTIONS.COSTS PER MARKOV STATE PER PATIENT: The calculation of the cost for each Markov state per patient was done as follows (using the base case input parameters listed above): Dressing costs (including time needed per dressing, number of nurses involved, and materials used [25]) and cost per ICU day [26] were taken into account for health states 1–6;Cost of treating contact dermatitis [25]–(including catheter removal, four alternative dressings, and insertion of a new catheter) was taken into account only for health state 5;Cost of treatment of CRBSI [25] and additional ICU-LOS due to CRBSI [13,25] were taken into account for health states 3 and 4;Cost per catheter change (venous, arterial) [25] was taken into account for health states 2 and 4. BODY.METHODS.MAIN ASSUMPTIONS.ADJUSTMENTS ON COVARIATES BETWEEN THE SUBGROUPS: A statistical analysis for all confounding covariates, such as age, sex, Sequential Organ Failure Assessment severity score (SOFA, a score predicting ICU mortality based on lab results and clinical data [28]), duration of catheterization, number of dressing change per day, was performed in order to demonstrate the comparability between the subgroups. Four subgroups of patients (CHG/CRBSI, CHG/No-CRBSI, Non-CHG/CRBSI, Non-CHG/No-CRBSI) were compared with these covariates. Mann-Whitney tests between subgroups were performed. BODY.METHODS.MAIN ASSUMPTIONS.SENSITIVITY ANALYSES: Sensitivity analyses are performed to vary each parameter of the model in order to determine what levels will result in a change of preference for the therapeutic strategy. This is a way to test the boundaries of the model and identify the main parameters driving cost differences. One-way sensitivity analyses were performed varying the main input parameters (additional ICU LOS due to CRBSI (days), 3M Tegaderm CHG Dressing cost (€2013), number of CHG dressing per day, number of Non-CHG dressing per day, and cost per ICU day) of the model around the base case assumptions. A probabilistic sensitivity analysis [29] was performed with 1,000 non-homogeneous MCMC simulations of 1,000 patients for both CHG dressing and non-CHG dressing groups. Each group of 1,000 patients depicts an average patient representing all patients for each dressing group studied in the RCT [13] The method used was the Gibbs sampling [30], a commonly used Markov Chain Monte Carlo algorithm. It allowed to retrace 106 health trajectories (1,000 x 1,000 patients for each dressing strategy), based on the probabilities observed in the RCT [13] day-after-day (during 30 days) for each patient to change from one health-state to another. Repeating the algorithm 1,000 times allows the calculation of 95% confidence intervals for the cost-effectiveness criterion (here, number of CRBSI avoided and cost per patient). The health states including CRBSI (CRBSI/No new catheter and CRBSI/new catheter) as rare events for both strategies are in the area of low probabilities. On the other hand, the "discharge" and "death" states as frequent events for both strategies are in the area of high probabilities. This corresponds to the reality observed in the RCT (higher frequency of discharge and death than CRBSI). BODY.RESULTS.IMPACT OF THE COVARIATES: PATIENTS AND CATHETERS CHARACTERISTICS: The 4 subgroups of patients (CHG/CRBSI, CHG/No-CRBSI, Non-CHG/CRBSI, Non-CHG/No-CRBSI) were similar in regards to the SOFA score, age, sex, exposure to the risk factor "duration of catheterization" and daily number of dressing(s) needed (Table 2). The results of Mann-Whitney tests on these covariates between subgroups (CRBSI/No CRBSI) show no statistically significant difference at the 0.05 level. 10.1371/journal.pone.0130439.t002 Table 2 Comparability of subgroups on covariates. Dressing group CHG * Mean (std) Non-CHG ** Mean (std) Comparison p-value ⱡ SOFA score (severity) CRBSI 7.89 (4.08) 10.29 (3.39) 0.1459 No CRBSI 8.17 (3.76) 8.17 (3.83) 0.8737 Age (years) CRBSI 58.78 (13.73) 62.57 (19.08) 0.5262 No CRBSI 61.97 (15.71) 62.17 (16.42) 0.6043 Number of males CRBSI 5 (55.56%) 12 (57.14%) 1.0000 No CRBSI 630 (68.11%) 603 (65.97%) 0.3460 Catheterization time (days) CRBSI 39.67 (22.58) 28.43 (31.56) 0.0984 No CRBSI 11.01 (11.52) 10.92 (11.01) 0.9934 Number of dressings per day CRBSI 0.59 (0.29) 0.73 (0.37) 0.2675 No CRBSI 0.67 (0.52) 0.65 (0.58) 0.2653 * CHG group frequencies: 9 patients with CRBSI, 925 patients without CRBSI. ** Non-CHG group frequencies: 21 patients with CRBSI, 914 patients without CRBSI. ⱡ The results (p value) of Mann-Whitney tests on these covariates between subgroups show no statistically significant difference if p>0.05 (at a 0.05 level). CHG, chlorhexidine gluconate; SOFA, Sequential Organ Failure Assessment; CRBSI, catheter-related bloodstream infection. BODY.RESULTS.COST-EFFECTIVENESS ANALYSIS: A ratio of 1 to 5 was observed for the average number of CRBSIs between CHG and non-CHG dressing groups. CRBSI occurred for 3 and 14 patients in each CHG and non-CHG groups respectively (1,000 patients in each group; Table 3). This difference was highly statistically significant as indicated by the non-overlapping 95% confidence intervals (CI). The number of ICU-days as well as the number of days before discharge and death occurred was comparable in the two groups. Moreover, the number of patients entering both absorbing states, coded as 7 (discharge from ICU) and 8 (death), was comparable in both groups of dressings. 10.1371/journal.pone.0130439.t003 Table 3 Occurrences per 1,000 patients as generated through 1,000 NH-MCMC of 1,000 patients in each dressing group, according to the base case scenario. Study arm CHG dressing Non-CHG dressing Statistics Mean (%ₒ) Lower 95%CI Upper 95%CI Mean (%ₒ) Lower 95%CI Upper 95%CI State 1 no CRBSI / no new CT (at the beginning of the simulation) 1,000 1,000 1,000 1,000 1,000 1,000 State 2 no CRBSI / new CT 278.2 241.8 314.5 251.6 218.8 284.4 State 3 CRBSI / no new CT 0.00 0.00 0.00 5.3 0.7 9.8 State 4 CRBSI / new CT 3.1 0.00 64.8 9.5 3.3 15.7 State 5 Contact Dermatitis 28.8 14.6 43.0 12.7 4.4 20.9 State 6 G+T dressing 28.8 14.6 43.0 12.7 4.4 20.9 State 7 (ICU Discharge) 604.1 574.1 633.8 613.4 582.8 644.1 State 8 (Death) 263.7 234.7 292.7 270.7 242.4 299.0 Number of ICU-days 12.91 12.30 13.52 12.72 12.12 13.32 Number of days before State 7 Discharge 18.74 18.05 19.43 18.43 17.72 19.16 Number of days before State 8 Death 25.17 24.49 25.85 25.28 24.64 25.92 CHG, Chlorhexidine Gluconate; CI, Confidence Interval; CRBSI, Catheter-related bloodstream infection; CT, Catheter; ICU, Intensive Care Unit; NH-MCMC, Non-Homogeneous Markov Chain Monte Carlo. For a 30-day time horizon in ICU, the mean cost per patient for CHG group was of €16,461, versus €16,320 for the non-CHG strategy. The mean cost per patient with CRBSI was of €39,071 and €41,424 in CHG and non-CHG dressing groups while the mean cost per patient without CRBSI was of €16,385 and €15,946 in CHG and non-CHG dressing groups, respectively (Table 4). Subgroup analyses supported by the comparability test (Table 2) compared the average total costs for patients with CRBSI versus patients without CRBSI for each study group (CHG and Non-CHG dressings). This comparison revealed no significant differences in costs among the subgroups (Table 4). 10.1371/journal.pone.0130439.t004 Table 4 Mean Cost for one patient in each dressing group. Groups /Statistics Mean Lower 95%CI Upper 95%CI ALL PATIENTS CHG (1) €16,461 €15,659 €17,265 Non-CHG (2) €16, €15,538 €17,103 Diff. Cost (1–2) €141 €-975 €1,258 PATIENTS with CRBSI in ICU CHG (1) €39,071 €17,384 €60,758 Non-CHG (2) €41,424 €36,213 €46,635 Diff. Cost (1–2) €-2,353 €-24,984 €20,277 PATIENTS without CRBSI CHG (1) €16,385 €15,584 €17,186 Non-CHG (2) €15,946 €15,177 €16,715 Diff. Cost (1–2) €439 €-664 €1,542 Time Horizon: 30-days ICU—1,000 NH-MCMC simulations of 1,000 patients (€2013). CHG: Chlorhexidine Gluconate; CI: Confidence Interval; ICU: Intensive Care Unit; NH-MCMC: Non-Homogeneous Markov-Chain Monte Carlo simulation BODY.RESULTS.ONE-WAY SENSITIVITY ANALYSIS: A tornado diagram (Fig 2) shows the variation in the mean cost difference between the CHG and non-CHG strategies around the one calculated for the base-case (€141). The model was most sensitive to the variation of the number of extra ICU LOS due to CRBSIs. The cost difference varied of approximately €370, when accounting from a single extra ICU day (cost difference of €251) to 26 extra ICU days (cost difference of €-115). The next three influential parameters were the CHG-dressing cost, the interval for dressing change, and the cost per ICU-day. However, the variation in the cost differences obtained by changing these parameters was less pronounced (differences between upper and lower limits of €88, €85 and €83, respectively). 10.1371/journal.pone.0130439.g002Fig 2Tornado diagram for the One-way Sensitivity Analysis.This diagram illustrates the impact of the variation in some parameters of the model on the cost difference between the strategies. The base case is average cost difference (€+141) between the two dressing strategies for the parameter's values indicated on the "y" axis. The tested range for each parameter is indicated by the arrows. The main driver parameter for cost difference is the Extra LOS associated to CRBSI. ICU: Intensive Care Unit; CRBSI: Catheter-related bloodstream infection; CHG: Chlorhexidine Gluconate; LOS: Length of Stay. BODY.RESULTS.PROBABILISTIC SENSITIVITY ANALYSIS (PSA): The PSA cost-effectiveness plan (Fig 3) describes the difference in the effectiveness on the x-axis and the difference in cost on the y-axis between the two groups of dressings, for 1,000 NH-MCMC simulations of 1,000 patients in each group. The (0,0)-point indicates the reference dressing strategy (Non-CHG group). All other points observed on the graph represent the incremental cost-effectiveness ratio (ICER: Difference in costs / Difference in effectiveness [15]) of CHG-dressing strategy versus reference dressing. This PSA supports the decision to adopt the CHG dressing for critically ill patients since the strategy is 99.7% more effective than the comparator at the same cost per patient in the intensive care unit (only 3 points over 1,000 were observed in the cost-effectiveness plan where the assessed product was less effective than the reference dressing). The incremental cost-effectiveness ratio (ICER) is of €12,046 per CRBSI prevented, which is far less than the cost of caring for an infected patient in the ICU setting, estimated here to be around €40,000 (Table 4). 10.1371/journal.pone.0130439.g003Fig 3Cost-effectiveness results for the probabilistic sensitivity analysis.The analysis uses 1,000 non-homogeneous Markov-Chain Monte Carlo simulations of 1,000 patients for each dressing strategy. The x axis represents the difference in effectiveness (number of CRBSI events in CHG versus non CHG dressing) and the y axis represents the difference in cost (mean cost per patient with CHG versus non CHG dressing) in €2013. The (0,0)-point indicates the reference dressing strategy (Non-CHG group). Each point in the graph represents the Incremental Cost-Effectiveness Ratio (ICER) of CHG-dressing strategy versus reference dressing. All but three points are at the left side of the graph, showing that CHG dressing strategy was 99.7% more effective than the comparator at the same costs per patient. The squared point in the center of the cloud represents the average CE ratio of all 1,000 simulations. CHG: chlorhexidine gluconate; CRBSI: catheter-related bloodstream infection. The average incremental net monetary benefit (iNMB) per patient induced by using CHG- instead of non-CHG dressings in ICU can be calculated by valuing the net health gain in monetary terms, based on the current willingness to pay (WTP) minus the difference in cost per patient between each compared strategy, as follows: iNMB = difference in effectiveness per patient x WTP – difference in cost per patient. In the current context, the willingness to pay (WTP) was considered as the mean cost for treating one patient with CRBSI included in the reference dressing arm. The mean "incremental net monetary benefit" obtained was positive (€344 (95%CI: [€-883; €1,573])), indicating that the assessed technology is cost-effective. BODY.DISCUSSION: Several studies highlighted the clinical and economic impact of catheter-related infections [31, 32]. Others pointed out the medical and economic benefits of using CHG dressings [33, 34] or antiseptic impregnated central venous catheters for preventing these infections [35, 36]. Crawford et al. [33] estimated that the potential annual U.S. net benefits from using chlorhexidine sponge dressing ranged from $275 million to approximately $1.97 billion. According to Ye et al. [34] the systematic use of chlorhexidine gluconate (CHG)-impregnated sponge dressing could avoid 35 CRBSIs, 145 local infections, and 281 intensive care unit days and save about $895,000 annually in a hypothetical 400-bed hospital inserting 3,078 central venous catheters (CVCs) per year. Schwebel et al. [25] showed that the expected savings per catheter when using CHG dressings were of US $117 with a 3-day dressing change schedule and US $98 with a 7-day dressing change schedule. In the current study, we estimated that CHG dressing prevents 11.74 infections per 1,000 patients via probabilistic cost-effectiveness sensitivity analysis. Through the systematic use of the CHG dressing, the adjusted cost per patient was on average €16,461, the cost difference per patient was of €141, and incremental net monetary benefit per patient of €344. Beyond the "cost saving" aspects pointed out by authors cited above, our analysis demonstrates that the use of CHG dressings in ICUs is cost-effective as indicated by a positive iNMB [37,38]. The one-way sensitivity analysis identified the co-variables impacting most the cost-effectiveness calculations. Additional ICU LOS due to CRBSIs appears as the main driver of the model. The next more important variables are the CHG dressing unit price, the number of dressings per day and the cost per ICU day. These results are consistent with the health-economics analysis for a CHG sponge dressing published by Schwebel et al. [25]. The non-homogeneous Markov Chain Monte Carlo (NH-MCMC) simulation represents an innovative analytical approach for modeling healthcare-acquired infections. The literature in this field offers only examples based on static decision tree models, used for both cost-effectiveness or cost-benefit studies [25,33,34]. The more remarkable feature of the current NH-MCMC simulation relates to the fact that it is based on daily real-life raw data, and not on published mean values found in the literature. The time-dependence addressed here (i.e. evolution of the risk of developing a CRBSI with increased catheterization time) corroborates that the "micro" simulation approach chosen is suitable considering the nature of the available data (daily observations). This model has some limitations. First, it was built on a single clinical study because it was the only RCT available with this particular product. Second, the cost-effectiveness analysis was based on a scenario specific to French ICUs, where the CRBSI rates are rather low (below 2 per 1,000 catheter-days [13,14]). As a consequence, the NH-MCMC model cannot be directly transposed to other settings or other countries with different CRBSI baseline rates. This transposition would require local individual data on time-dependent probabilities of transition among health states at the daily level, which are not available in general. Further studies involving other countries are needed to generalize our results and therefore our findings do not necessarily predict similar cost effectiveness of CHG dressings in other countries or in specific patients' subgroups. Third, the NH-MCMC model very likely underestimated the costs for the non-antimicrobial dressing group, where the number of occurrences of discharge and death (absorbing states with associated null cost) was higher, with 9 discharged patients and 7 deceased patients. As a consequence, the calculated average cost per patient with CHG dressings increased and the corresponding effectiveness decreased. This third limitation is on the positive side since it ensures a conservative cost-effectiveness approach, as recommended by the HAS [15] for the base case analysis. A subgroup analysis based on living patients only, not discharged from the ICU within the specified time horizon, was not possible since there were no CRBSI events in this sub-population. International guidelines for prevention of catheter-related infections were followed in all study centers participating in the source RCT and the rate of infection was low also in the control group. Furthermore, there was no difference between treatment groups in the covariates (see Table 2). Some studies have shown an increase in infection rate for the femoral insertion site, but this was not observed in our source study (see electronic supplement in [13]). According to the probabilistic sensitivity analysis, which addresses the level of uncertainty of the results, the CHG-dressing strategy passed the test for cost-effectiveness even in the conservative scenario of very low CRBSI incidence and frequent dressing changes. The transparent antimicrobial dressing is significantly more efficacious to prevent CRBSIs when compared to the reference dressing without any additional cost for the ICU. This study also has the non-technical limitation of being sponsored by industry (the 3M Company). However, an external research organization (Statesia) was hired to handle independently the development of the simulation model and the data analysis to remove any possible bias. Two employees of the 3M Company worked alongside with non-3M authors for the preparation of the manuscript, with the final version being approved by all non-3M authors prior to submission. BODY.SUPPORTING INFORMATION: S1 TableTransition Matrix for CHG dressings.AE, Adverse Event; CRBSI, Catheter-related bloodstream infections; CT, Catheter; G+T, Gauze and Tape; ICU, Intensive Care Unit.(XLSX)Click here for additional data file. S2 TableTransition Matrix for non-CHG dressings.AE, Adverse Event; CRBSI, Catheter-related bloodstream infections; CT, Catheter; G+T, Gauze and Tape; ICU, Intensive Care Unit.(XLSX)Click here for additional data file.

TITLE: Management of oral submucous fibrosis by two different drug regimens: A comparative study ABSTRACT.BACKGROUND:: Oral Submucous Fibrosis (OSF) is a precancerous condition of the oral mucosa. Existing treatments give only temporary symptomatic relief. Colchicine is an ancient drug with anti-fibrotic and anti-inflammatory properties. We planned to study the effects of colchicine in the management of oral submucous fibrosis. ABSTRACT.MATERIALS AND METHODS:: Fifty OSF patients were divided randomly into two groups and treated for 12 weeks. Group 1-Patients were administered tablet colchicine orally, 0.5 mg twice daily and 0.5 ml intralesional injection Hyaluronidase 1,500 IU into each buccal mucosa once a week. Group 2-Patients were administered 0.5 ml intralesional injection Hyaluronidase 1,500 IU and 0.5 ml intralesional injection Hydrocortisone acetate 25 mg/ml in each buccal mucosa once a week alternatively. Student's t test and analysis of variance (ANOVA) were used to compare pre and post treatment results. P<0.05 was considered as significant. ABSTRACT.RESULTS:: Thirty-three percent in group 1 got relief from burning sensation in the second week. Inter group comparisons of increase in mouth opening and reduction in histological parameters indicated that group 1 patients responded better than group 2. ABSTRACT.CONCLUSION:: These encouraging results should prompt further clinical trials with colchicine alone on a larger sample size to broaden the therapeutic usefulness of the drug in the management of OSF. BODY.INTRODUCTION: Oral Submucous Fibrosis (OSF) is a chronic condition almost exclusively occurring among Indians and to a lesser extent in the other Asiatic people.[1] However, with the increase in immigration of people from the Indian subcontinent, dental professionals in many developed countries will encounter this disease in the near future.[2] A wide range of treatment including drug management, surgical therapy, and physiotherapy have been attempted till date, with varying degrees of benefit, but none have been able to cure this disease.[3] This is mainly due to the fact that the etiology of the disease is not fully understood and the disease is progressive in nature.[4] Instead of continuing the limited available modes of therapy, the idiopathic nature of this condition indicates new avenues for its management.[5] Colchicine is an alkaloid found in the crocus like plant, Colchicum Autumnale, named for the land of Colchis at the eastern tip of Black Sea.[6] Chemically, it is Colchicinum-N-(5, 6, 7, 9-Tetrahydro-1, 2, 3, 10-tetramethoxy-9-oxobenzo [a] heptalen-7-yl) acetamide. Colchicine is an ancient drug-at least 2,000 years old, which is attracting renewed interest because of its actions at a subcellular level. Many studies during the past years have elucidated a variety of previously unsuspected drug actions and have demonstrated the varying effectiveness of colchicine therapy for a surprisingly broad array of diseases including recurrent aphthous stomatitis, Behcet's disease, familial Mediterranean fever, polymyositis, and scleroderma.[7] The pharmacodynamics of colchicine as an anti-fibrotic agent is well-established by various in vitro and in vivo studies [Table 1][8910] warranting its use in the treatment of various diseases associated with fibrosis. The long held view that colchicine's anti-inflammatory actions are specific for gout is no longer tenable. Perhaps the most important anti-inflammatory properties of colchicine are related to the drug's effect on polymorphonuclear leukocytes and monocyte chemotaxis, leukocyte adhesiveness, the drug's action in potentiating factors that increase leukocyte cyclic adenosine monophosphate (cAMP) levels, thereby, inhibiting lysosomal degranulation that accompanies phagocytosis and its effect on the release of prostaglandin E, which suppresses the leukocyte function.[11] Table 1 Effects of colchicine on fibroblasts and collagen fibers[ 8 9 10 ] Hence, the exciting combination of an anti-fibrotic agent along with anti-inflammatory properties of a drug that is surprisingly well tolerated, easily available, and cost effective, prompted us to embark on this study. This study was planned to compare the effectiveness of colchicine and intralesional hyaluronidase with intralesional corticosteroid and hyaluronidase in the management of OSF. BODY.MATERIALS AND METHODS: This study was conducted in Government Dental College, Bangalore, between 2002 and 2004. The patients for the study were selected from those who visited our department. A formal ethical clearance to conduct this study was given by the Ethical Committee of the institute. An informed consent was obtained from the patients before including them in the study. A detailed case history of the patient with emphasis on their habits (chewing betel nut, pan parag, etc.) and a thorough clinical examination was recorded on a standard proforma. A clinical diagnosis of OSF was made based on the World Health Organization (WHO) criteria and the patients were graded clinically according to Gupta Dinesh Chandra S. et al.[12] The diagnosis was confirmed histopathologically by a punch biopsy of the lesion. Hematoxylin and Eosin and van Gieson's stains were used to analyze the staining intensity of the inflammatory infiltrate and the density of collagen fibrils, respectively, in the pre and post treatment biopsy specimens. Fifty patients (41 males and nine females) in the age group of 15 years to 55 years, thus diagnosed as having OSF, were included in the study and were divided randomly into two groups for the purpose of treatment. Group 1-Patients were administered orally tablet colchicine (Tablet Zycolchine, Zydus Cadila Healthcare Ltd., Ahmedabad, Gujrat, India) 0.5 mg twice daily. Hyaluronidase 1,500 IU was mixed in 1 ml of lignocaine. 0.5 ml of this solution was injected intralesionally in each buccal mucosa once a week. Baseline liver function tests, serum urea, and creatinine were done for these patients to rule out any existing hepatic pathology and the tests were repeated once every month during the study period and one month following cessation of treatment. Group 2-Patients were administered intralesional injection of Hyaluronidase 1,500 IU as in group 1 and 0.5 ml of injection Hydrocortisone acetate 25 mg/ml in each buccal mucosa once a week alternatively. Patients in both the groups were asked to discontinue all betel nut chewing habits during the entire study period. Both groups were treated for 12 weeks. Post-treatment punch biopsy was taken one week after the cessation of treatment for histopathological evaluation. Patients were asked to observe for any local allergic symptoms as itching, redness or ulcerations at the site of injection or if any constitutional symptoms developed and to report the same immediately. Treatment was discontinued in such patients. During the subsequent visits the patient's response to the treatment procedures was recorded with emphasis on ascertaining the amelioration of specific symptoms like burning sensation, trismus, and intolerance to hot and spicy foods. Clinical examination of the oral mucosa, site of lesion, margins, extension, color and surface texture and presence of fibrotic bands was recorded. The interincisal opening of the mouth was recorded during each visit [Figures 1 and 2]. Pre and post treatment histopathological specimens were compared for the inflammatory cells and fibrous tissue [Figures 3a, 3b, 4a, and 4b]. Student's t-test and analysis of variance (ANOVA) were used to compare pre and post treatment results. P<0.05 was considered as significant. Figure 1Mouth opening recorded before treatment in a patient belonging to Group I Figure 2Clinical improvement of mouth opening recorded in the same patient after treatment of 12 weeks Figure 3(a) Photomicrograph of tissue section taken before treatment showing atrophic epithelium, relatively avascular hyalinized collagen with abundant inflammatory infiltrate (low-power view: H and E ×40), (b) Photomicrograph of the same tissue specimen stained with van Gieson's showing dense avascular hyalinized collagen (low-power view: ×40) Figure 4(a) Photomicrograph of tissue section taken after treatment showing reduced density of collagen and inflammatory infiltrate (low-power view: H and E ×40), (b) Photomicrograph of the same tissue specimen stained with van Gieson's showing reduced density of collagen (low-power view: ×40) BODY.RESULTS: The mean age of the patients in the study was 28.40 ± 8.6 (Mean ± SD) years, with 68 per cent in the age group of 20-29 years. Sixty-four percent of the patients complained of both trismus and burning sensation. All the patients had an areca nut chewing habit, either in the form of betel nut or other commercially available products like Pan Parag, Pan masala, Gutka etc. Additional habit of smoking was observed in only one male patient. The buccal mucosae were affected in all the patients, however, only five showed tongue involvement. The clinical grading of the patients and the inter incisal distance measured in them are shown in Tables 2 and 3, respectively. Table 2 Distribution of the clinical grading of the patients in both the groups Table 3 Distribution of inter-incisal distance measured in patients in the study groups Almost all the patients in both the groups were relieved from burning sensation after treatment. However, 33% in group 1 got relief in the second week itself as against 21% in group 2 [Table 4]. For inter group comparison of the effectiveness of treatment in increasing the mouth opening parameters, student's t test was done that resulted in a value P < 0.05 which indicated that group 1 patients responded better than group 2. To know the effect of treatment in increasing the mouth opening parameters in the clinical grades, a test of significance, analysis of variance was carried out that resulted in P < 0.05 for grade 3 and P > 0.05 for grade 4 which indicated that clinical grade 3 responded better than grade 4. To compare the effectiveness of treatment in reducing the histological parameters in between the two groups after treatment, Student's 't' test was done which resulted in a value of P < 0.05 which is statistically significant and indicated that group 1 responded better than group 2. To know the effect of treatment in reducing the histological parameters in clinical grades (3 and 4) in between two groups, a test of significance of analysis of variance (ANOVA) was done which resulted in P < 0.05 for grade 3 and P > 0.05 for grade 4 which indicates that clinical grade 3 responded better than grade 4. Table 4 Week-wise reduction in burning sensation in patients of both groups after treatment BODY.DISCUSSION: OSF is a morbid, crippling, and a premalignant condition of the oral mucosa associated with the areca nut chewing habit.[1314] It is commonplace in various Indian states to use pan quid with tobacco and lime.[15] Several medical and surgical approaches have been tried for the management of OSF over the decades. The results are not predictable with some therapies and none has been consistently successful.[12] In 2001, Haque et al., successfully treated 29 OSF patients with recombinant interferon gamma (rhIFN-γ) and concluded that IFN-γ may reverse OSF. They tried IFN-γ in OSF based on the studies which showed that systemic recombinant IFN-γ improved the mouth opening, musculoskeletal, and pulmonary efficiency in patients with scleroderma.[16] In view of the female predilection, its presentation in middle life and histological similarities, the analogy that OSF is an "idiopathic scleroderma of mouth" seems reasonable.[17] Collagen fibrils in OSF are more embryonic in nature with a defective maturation similar to scleroderma.[18] Canniff et al., showed that OSF and scleroderma were similar with an increased frequency of HLA — DR3 and haplotypic pairs B 8/DR3 in the latter and HLA — DR3 A10 and B 7 in the former.[17] However, there is difficulty in the availability of IFN-γ and the cost of this agent is high in developing countries. Considering the shortcomings of all previous treatment modalities for OSF and the positive observations of the study by Haque et al., we have studied the effects of colchicine in the management of OSF. The patients in group 1 showed an early reduction in the burning sensation. There was also a significant improvement in the mouth opening and in the movement of the tongue (clinical grade 5). The histopathological findings also showed a marked reduction in the inflammatory cell infiltrate and density of collagen fibrils. The mechanism by which colchicine improved the clinical status of OSF patients in our study was difficult to ascertain since the drug was used in combination with Hyaluronidase. Also since this was the first study where cochicine had been used in the treatment of OSF, we could not compare our results. However, we attributed them to the effect of colchicine which is both an anti-fibrotic and anti-inflammatory agent. Like IFN-γ, colchicine also reduces collagen synthesis, down regulates fibroblast proliferation and upregulates anti-fibrotic cytokine and collagenase synthesis in the basal layer of the epithelium and lamina propria.[16] One of our patients, however, showed relapse of the restricted mouth opening during the follow-up of 6 months after the cessation of treatment. The cause of the relapse could be due to a number of factors like the dosage being less for that particular individual or the non-compliance of treatment by the patient. Colchicine has been reported to be beneficial in the treatment of diseases associated with fibrosis in animals and human beings.[19] The short and long term administration of colchicine therapy in moderate dosages is surprisingly well tolerated. None of our patients reported any local or systemic adverse reactions during treatment and also after the cessation of drug intake. The most common toxic side effect reported reflect the drug's action on rapidly proliferating gastrointestinal tract epithelial cells and include nausea, vomiting, diarrhea, and abdominal pain. These symptoms are especially frequent at dosage levels 2-3 mg/day although they are rapidly and completely reversible.[6] It is possible that the dosage we used in our study, i.e. 0.5 mg colchicine administered orally twice a day, is an acceptable dosage, because at this dosage the patients achieved significant therapeutic results with no adverse reaction.[20] Large amounts of the drug and its metabolites enter the liver and then the bile and, hence, should not be administered to patients with hepatic disease.[21] Follow-up blood cell count should also be performed periodically and medication stopped if the toxic effects develop. Tests to determine baseline serum urea nitrogen, creatinine levels, a complete blood cell count, and liver function test, performed prior to initiating the treatment and during follow-up of our patients also recorded values within the normal range. BODY.CONCLUSION: The encouraging results should prompt a clinical trial on more number of OSF patients to broaden the therapeutic usefulness and applications of one of our most ancient treatment agents. This baseline study gives scope for further studies with the systemic use of colchicine alone in the treatment of OSF, and, also for research in the use of the drug as a formulation that can be administered locally into the fibrous bands to confirm the above results.

TITLE: Effects of Prophylactic and Therapeutic Paracetamol Treatment during Vaccination on Hepatitis B Antibody Levels in Adults: Two Open-Label, Randomized Controlled Trials ABSTRACT: Worldwide, paracetamol is administered as a remedy for complaints that occur after vaccination. Recently published results indicate that paracetamol inhibits the vaccination response in infants when given prior to vaccination. The goal of this study was to establish whether paracetamol exerts similar effects in young adults. In addition, the effect of timing of paracetamol intake was investigated. In two randomized, controlled, open-label studies 496 healthy young adults were randomly assigned to three groups. The study groups received paracetamol for 24 hours starting at the time of (prophylactic use) - or 6 hours after (therapeutic use) the primary (0 month) and first booster (1 month) hepatitis B vaccination. The control group received no paracetamol. None of the participants used paracetamol around the second booster (6 months) vaccination. Anti-HBs levels were measured prior to and one month after the second booster vaccination on ADVIA Centaur XP. One month after the second booster vaccination, the anti-HBs level in the prophylactic paracetamol group was significantly lower (p = 0.048) than the level in the control group (4257 mIU/mL vs. 5768 mIU/mL). The anti-HBs level in the therapeutic paracetamol group (4958 mIU/mL) was not different (p = 0.34) from the level in the control group. Only prophylactic paracetamol treatment, and not therapeutic treatment, during vaccination has a negative influence on the antibody concentration after hepatitis B vaccination in adults. These findings prompt to consider therapeutic instead of prophylactic treatment to ensure maximal vaccination efficacy and retain the possibility to treat pain and fever after vaccination. ABSTRACT.TRIAL REGISTRATION: Controlled-Trials.com ISRCTN03576945 BODY.INTRODUCTION: Paracetamol (acetaminophen) is an analgesic and antipyretic drug widely used in children and adults [1]. In many European countries, paracetamol is used prophylactically (preventative treatment) to reduce pain and fever associated with vaccination [2]. In the Netherlands, the advice is to be cautious with the use of paracetamol during vaccination and only children who experienced fever or persistent screaming after vaccination are advised to use paracetamol prior to further vaccinations [3]. Despite this advice to restrict paracetamol to those children with previous reactions, many parents give paracetamol to their children prior to or just after the vaccine administrations. Estimates of paracetamol use made by the Dutch National Immunization Program (NIP) range from 19–27% prophylactically, and up to 49% prophylactically and therapeutically [4]. Data on use of paracetamol by adults during vaccination are not available. Paracetamol is generally regarded as safe, besides the hepatotoxic effects at higher doses, and is abundantly used as an over the counter drug [5]. Paracetamol was long considered to be a drug without anti-inflammatory effects and the immunomodulatory properties of paracetamol have only recently been described. Paracetamol suppressed several immune parameters in animal studies, such as T-cell dependent antibody response [6], [7]. Toxicogenomic studies revealed an influence on gene expression in lymphocytes consistent with inhibition of cell proliferation of immune cells [8]. This information is in line with older studies that suggested inhibitory effects on clearance of chickenpox in children and rhinovirus [9], [10]. Furthermore, several epidemiologic studies suggested an association between paracetamol use in children and adults and development of asthma. Asthma is a disease characterized by deregulated inflammatory responses and possible interference of paracetamol with these immune processes was proposed to underlie this association.[11]–[14]. Recently published results suggest a negative influence on vaccination response, i.e. a decrease in antibody levels, in infants who received paracetamol prior to vaccination (ten-valent pneumococcal vaccine) in order to prevent fever [15]. The decreased antibody levels were only observed after paracetamol treatment prior to the first vaccination, the priming of the immune response, and not when paracetamol was given prior to booster immunisations. The potential immunosuppressive effects of paracetamol that were recently shown to affect the efficacy of vaccination could result in increased susceptibility to infections, especially when paracetamol is applied shortly before active immune processes are induced. To date, it has not been studied whether use of paracetamol also affects the immune response after a primary vaccination in adults. In addition, it is not known to what extent timing of paracetamol intake, i.e. prophylactic or therapeutic, affects the response. The present study was performed to investigate possible effects of prophylactic and therapeutic paracetamol use in adolescents, on the quantitative antibody response to hepatitis B vaccination. Effects of paracetamol are highly relevant for health authorities who advise on the use of paracetamol as a treatment for vaccination-induced adverse responses. BODY.METHODS.STUDY DESIGN: The present study was composed of two phases. First a pilot study (phase 1) was performed to investigate effects of prophylactic use of paracetamol on the immune response in adults after a hepatitis B vaccination. Thereafter, phase 2 was performed to confirm prophylactic effects of paracetamol found in phase 1, and to evaluate the effect of therapeutic use of paracetamol on the immune response to hepatitis B vaccination. The two (phase 1 and phase 2) randomized, controlled, open-label studies were performed at the Hogeschool of Utrecht from October 3, 2011 to April 20, 2012 (phase 1), and from October 8, 2012 to April 26, 2013 (phase 2). Both studies were undertaken according to Good Clinical Practice and the Declaration of Helsinki (Somerset West, 1996 version). The protocol (NL36577.041.11) was approved by the ethics review committee of UMC Utrecht, and is available as supporting information as well as the CONSORT checklist; see Protocol S1 and Checklist S1. The study was registered at the European Clinical Trials database (EudraCT number: 2011–000923–33) prior to recruitment of participants. Due to a communication error this study was registered at the ISRCTN register (ISRCTN03576945) after start of recruitment. The authors confirm that all ongoing and related trials for this drug/intervention are registered. BODY.METHODS.STUDY POPULATION: Study participants were healthy young health care students of 18 years or older, who are routinely vaccinated against hepatitis B. The students were approached by the Hogeschool Utrecht for a hepatitis B vaccination and at the same time participation in the study was offered, as described in the Consort 2010 Flow Diagram (Fig. 1). Students were enrolled in the study after written informed consent was obtained. Participants were not included in case they used NSAIDs or paracetamol within 48 hours before the first vaccination; or if they had a history of acute or chronic hepatitis B; earlier vaccination against hepatitis B, medical immunosuppressive treatment, primary or secondary immunodeficiency, or allergic reactions to components of the hepatitis B vaccination or paracetamol. In case participants had fever (>38.5°C), vaccination or blood collection were postponed. Participants that did not take paracetamol according to the protocol they were assigned to, were excluded during the study and blood was not collected. Therefore, the analysis was a per-protocol analysis. 10.1371/journal.pone.0098175.g001Figure 1Consort 2010 Flow Diagram.The participation flow during the vaccination, intervention and blood collection. BODY.METHODS.INTERVENTION: Hepatitis B vaccines (Engerix-B) were manufactured by GlaxoSmithKline Biologicals (GSK), Rixensart, Belgium. Hepatitis B vaccines contained 20 μg recombinant hepatitis B surface antigen (HBsAg). Vaccines were administered intramuscularly into the right or left deltoid muscle into a strict schedule at 0 (primary vaccination), 1 month (5 weeks±4 days; first booster vaccination) and 6 months (23 weeks±4 days; second booster vaccination). The timeline of the vaccination, intervention and blood collection in phase 1 and 2 is shown in figure 2. In phase 1 the study participants were randomly assigned (IBM SPSS Statistics version 19) to two groups (1∶1): prophylactic paracetamol group and control group. In phase 2 the study participants were randomly assigned to three groups (1∶2∶1): prophylactic paracetamol, therapeutic paracetamol and control group. The paracetamol treatment consisted of three doses of paracetamol purchased from Omega Pharma (The Netherlands) administrated orally within the first 24 hours directly (prophylactic; t = 0 h, t = 8 h, and t = 16 h) or 6 hours after (therapeutic; t = 6 h, t = 14 h, and t = 22 h) the primary and first booster vaccination (Fig. 3). The first administration of paracetamol in the prophylactic group was performed immediately after vaccination in the vaccination clinic. The second and third administrations were done at home every 8 h. The therapeutic group took all the administrations at home every 8 h. 10.1371/journal.pone.0098175.g002Figure 2Timeline of the vaccination, intervention and blood collection.The study was composed of two phases. In phase 1, one prophylactic and one control group were investigated. In phase 2, one prophylactic paracetamol, one therapeutic paracetamol and one control group were investigated. The paracetamol groups received paracetamol after the primary and first booster hepatitis B vaccine doses, at 0 and 1 month. Participants received no paracetamol after the second booster vaccine dose at 6 months. The control group received no paracetamol or placebo during the hepatitis B vaccination procedure. Blood (14–21 mL) was taken of all participants prior to and one month after the second booster vaccination. 10.1371/journal.pone.0098175.g003Figure 3Timeline of the paracetamol treatment after the primary and first booster vaccination.The prophylactic paracetamol group received three doses of paracetamol (1000 mg/dose) starting immediately after vaccination, 8 hours and 16 hours after the vaccination. The therapeutic paracetamol group received three doses of paracetamol (1000 mg/dose) starting 6 hours after vaccination, 14 hours and 22 hours after vaccination. The control group received no paracetamol or placebo during the hepatitis B vaccination procedure. The dose of paracetamol was the maximal allowed paracetamol dose per administration (1000 mg/8 hours) for adults. The control group received no paracetamol or placebo. None of the participants received paracetamol after the booster vaccine dose at 6 months. Furthermore, participants were asked to restrain from use of more paracetamol or NSAIDs 48 hours after the vaccination in the treated and control groups. Participants that reported paracetamol use other than that prescribed in this study were excluded. Blood samples (14 ml) were collected prior to and one month after the second booster vaccination in blood collection tubes (Ref#367955, Becton Dickinson, US). BODY.METHODS.LABORATORY METHODS: Qualitative and quantitative HBsAg antibody levels in the sera were measured on the ADVIA Centaur XP system (Siemens Healthcare Diagnostics Inc., USA) by using the ADVIA Centaur Anti-HBs assay according to the manufacturer's protocol. In short, 100 μl sample was added to 100 μl of inactivated human HBsAg (subtype ad and ay, about 2 μg/ml) coupled to magnetic latex particles (solid phase) in a cuvette. Then 50 μl inactivated HBsAg labeled with acridinium ester was added. After 7.5 minutes of incubation at 37°C, the cuvette was washed with PBS. Chemiluminescence was initiated by adding 300 μl acid reagents and 300 μl base reagents were added. Antibody levels were measured as International Units per Liter (IU/L). Values above the 10.0 IU/L were considered to be positive and give sufficient protection against hepatitis B, according to the World Health Organization. Measurement of the anti-HBs level prior to the second booster vaccination was used to exclude participants that had an earlier vaccination against hepatitis B. Participants with values above the 10,000 IU/L were asked for all their vaccination certificates to control the hepatitis B vaccination status, and, if necessary, these participants were excluded from further participation in this study. BODY.METHODS.STATISTICAL ANALYSIS: The power calculation is based on the Z-test for the difference between expected values of log10 concentrations in two groups, taking into account the different variance in the two groups. A ratio of 1.65 was used, as the significant effects of the pneumococcal antibody concentrations in the Prymula study gave this average ratio [15]. More details of the power calculation are available as supporting information; see Protocol S1. The effect of paracetamol on the vaccination response was evaluated in two different manners; the number of participants whose antibody levels were above the protected level (10 IU/L), and the geometric mean concentrations (GMCs) of the anti-HBs. GMCs of the anti-HBs levels with 95% confidence interval (CI) were calculated for the control, prophylactic paracetamol and therapeutic paracetamol group. The two phases of the present study were performed according to the same protocol, same inclusion and exclusion criteria and corresponding time between the vaccine administrations and blood collections. Therefore, the data of phase 1 and phase 2 have been combined into one dataset (supporting information; see Table S1) to increase the power of the study. Small year differences between the two phases were adjusted by correcting for the difference between the median anti-HBs levels of the phase 1 and phase 2 control group; the prophylactic and therapeutic groups were adjusted with the same value correction as their corresponding control group. The GMCs of the anti-HBs levels of phase 1 and phase 2 before and after correction are presented in the supporting information; see Table S2. Further analyses were performed on the combined dataset. Statistical differences were calculated using the Mann–Whitney test, as the data were not normally distributed. Based on the study of Prymula et al., 2009 it was expected that the paracetamol groups would have a lower antibody concentration, as significant decreasing effects of paracetamol on the vaccination response on pneumococcal and tetanus antibody levels were found. Although, the effect of paracetamol on hepatitis B was not significantly different, a decrease in antibody levels was still apparent. These effects point in the same direction, lower antibody concentration after paracetamol use during vaccination. Therefore, an unpaired, one-sided test was performed.[15]. Statistical differences in drop-out percentages were calculated using the Chi-Square test. A P-value of <0.05 was considered statistically significant. BODY.RESULTS.STUDY PARTICIPANTS: The numbers of participants and their characteristics in the different groups of phase 1 and phase 2 studies are shown in table 1 and in the Consort Flow Diagram (Fig. 1). 177 participants were enrolled and vaccinated in phase 1 (pilot study) and 319 participants in phase 2. Study participants were randomly divided in a control, prophylactic or therapeutic paracetamol group. 10.1371/journal.pone.0098175.t001 Table 1 Number of participants and characteristics during phase 1 and phase 2. Paracetamol Treatment Primary Vaccination(N = ) First boostervaccination (N = ) Second booster vaccination/Blood collection 1 (N = ) Blood collection 2 (N = ) Drop-out(%) Age range(years) Male (%) Phase 1 Control 85 83 67 65 23.5 18–45 29.2 Prophylactic 92 88 77 71 22.8 18–25 33.8 Total 177 171 144 136 23.2 18–45 31.6 Phase 2 Control 83 78 64 59 28.9 18–48 27.1 Prophylactic 86 70 52 49 43.0 18–32 30.6 Therapeutic 150 136 112 107 28.7 18–47 33.6 Total 319 284 238 215 32.6 18–48 31.2 Phase 1+2 Control 168 161 133 124 26.2 18–48 28.2 Prophylactic 178 158 129 120 32.6 18–32 32.5 Therapeutic 150 136 112 107 28.7 18–47 33.6 Total 496 455 374 351 29.2 18–48 31.3 Overall there was a drop-out percentage of 27.6% (23.2% in phase 1 and 32.0% in phase 2). The drop-out percentage was not significantly different (p = 0.4187) between the control, prophylactic and paracetamol group. Dropping out occurred for several reasons; students that discontinued their education and therefore stopped the vaccination procedure or students that stopped the vaccination procedure without stating reasons (n = 112); students that reported paracetamol use, unrelated to the present study, in the 48 hours prior to or after the booster vaccination (n = 6); students that forget to take a dose of paracetamol at the right time (n = 11); students that no longer wished to participate in the present study (n = 16). The mean age of the vaccinated cohort at the time of the primary vaccination was 21.09 years with a minimal age of 18 years and a maximal age of 48 years. The distribution of the age of the study cohort was divided in typical student ages (18–25 years) and individuals with ages scattered up to 48 years. Therefore an outlier test based on age was performed. The outcome of this test was 25.575 years and this number was round up to 26 years. Only 5.4% of the study participants were 26 years or older (median = 20.05, min-max = 18.03–48.56). No significant differences were found in the anti-HBs levels of the students ≥26 years compared to the anti-HBs levels of the students <26 years. The vaccinated cohort comprised 68.7% female and 31.3% male participants; the gender distribution was similar and not significantly different in the control, prophylactic and therapeutic paracetamol group. There were no significant differences in anti-HBs levels between female and male participants. Anti-HBs levels were not statistically different for female and male participants and between different age groups (<26 years and ≥26 years). Therefore, further data analysis of the anti-HBs levels between study groups was based on data of all participants and performed without age and gender as confounding factors. BODY.RESULTS.ANTI-HBS LEVELS: Anti-HBs levels were measured just before and one month after the second booster vaccination. Before the second booster no differences were observed between the groups (Fig. 4, panel A). After completion of the study. i.e. one month after the second booster hepatitis B vaccination, none of the students displayed a response below the protection level. Subsequently, we focused on comparison of the GMC of the different treatment groups. The anti-HBs level in the prophylactic paracetamol group was significantly lower (p = 0.048) than the level in the control group (Fig. 4, panel B) by using the Mann-Whitney U test. The reduction in anti-HBs level in the prophylactic paracetamol group was 26% as compared to the control group (4257 mIU/mL vs. 5768 mIU/mL) indicating that paracetamol decreases the induction of antibody response in adults. The anti-HBs level in the therapeutic paracetamol group was not different (p = 0.34) from the level in the control group (4958 mIU/mL vs. 5768 mIU/ml) indicating the timing of paracetamol determines its immunomodulatory effect. 10.1371/journal.pone.0098175.g004Figure 4Anti-HBs levels prior (A) to and one month after (B) the second booster vaccination.Line at GMC. A) The anti-HBs levels in the groups were not significantly different. B) The anti-HBs level in the prophylactic paracetamol group (4257 mIU/mL) is significantly lower (p = 0.048) from the anti-HBs level in the control group (5768 mIU/mL). The anti-HBs level in the therapeutic paracetamol group (4958 mIU/mL) was not significantly different from the control group. Significant differences in anti-HBs levels were tested by using the Mann-Whitney U test. * = p<0.05. Furthermore, the increase in individual antibody concentration after the second booster vaccination was calculated and expressed as the ratio of the two (Fig. 5). The ratio in the prophylactic paracetamol group is significantly lower (p = 0.005) from the ratio in the control group. The ratio in the therapeutic paracetamol group was not different from the ratio in the control group (p = 0.17). These results indicate that only prophylactic paracetamol treatment during the primary vaccinations affects the initial immune response resulting in a diminished effect of the second booster vaccination. 10.1371/journal.pone.0098175.g005Figure 5Ratio between the anti-HBs levels prior to and one month after the second booster vaccination.The increase in antibody concentration after the second booster vaccination is expressed in ratio. The ratio in the prophylactic paracetamol group (20.3) is significantly lower from the ratio in the control group (34.9). The ratio in the therapeutic paracetamol group (27.4) was not significantly different from the control group. * = p<0.01. BODY.DISCUSSION: Our study showed, in accord with earlier studies published by Prymula et al. [15], that exposure to paracetamol can suppress immune function to antigens derived from bacterial and viral pathogens, and this might have consequences for resistance to infectious agents. The effects noted were modest, but modest suppression on a population basis may have considerable consequences as has been noted with exposure to environmental pollutants inducing similar levels of immune suppression [16]–[18]. The effects of prophylactic and therapeutic paracetamol treatment were observed in a human model in which specific antibody responses to hepatitis B antigen were assessed. The main finding of this study is that prophylactic use of paracetamol exerts a negative effect on the primary antibody response after hepatitis B vaccination in adults. In addition, we show that such an inhibitory effect is not observed when paracetamol is given therapeutically. Apparently, the timing of paracetamol determines its immunomodulatory effects. Our finding extends the data recently published by Prymula et al. that showed that prophylactic use of paracetamol inhibited the induction of antibodies to a combination of child-hood vaccines in infants [15]. Infants were vaccinated with the hexavalent diphtheria-tetanus-3-component acellular pertussis-hepatitis B-inactivated poliovirus types 1, 2, and 3-H influenza type b and oral human rotavirus vaccines. Prymula et al. aimed to study the effect of paracetamol use on the occurrence of side-effects to vaccination. While pain and fever were reduced, an inhibitory effect of paracetamol on the induction of antibodies to these vaccines was observed. The decrease in GMC that was observed was approximately thirty-five percent. In the present study a decrease of twenty-six percent was measured in the prophylactic paracetamol group indicating that the effects of paracetamol in adults is equivalent to those in infants. In contrast to the inhibitory effects of prophylactic paracetamol treatment on antibody responses, paracetamol did not exert similar significant effects when given therapeutically. Nevertheless, a slightly non-significant decrease in antibody response was visible in the therapeutic paracetamol group compared to the control group. To our knowledge our study is the first in which the effects of prophylactic and therapeutic paracetamol treatment on response to vaccination were compared. The antibody levels of the control and both paracetamol groups display large variation, reflecting the variation in the human vaccine responses and thus the unique immune response of every individual after vaccination and the various physical and genetic factors that influence the response to vaccination [19], [20]. Although the antibody concentrations in this study were significantly lower in the prophylactic group, all study participants were considered to be protected against hepatitis B after the full schedule since all titers were above 10.0 IU/L, the threshold for protection against hepatitis B, according to the World Health Organization [21]. Since anti-HBs levels are measured quantitative and anti-HBs levels are standardized against an international reference and expressed in International Units, hepatitis B vaccination is a good model to investigate the effects of paracetamol on immune responses after vaccination in a safe manner. However, there is clear evidence that paracetamol has an effect on the functionality of the immune system, which could be relevant under conditions when the immune response to either vaccination or pathogen is already suboptimal. However, the suppressive effect of paracetamol was not found in a comparable study with influenza vaccinations in elderly people [22]. Participants in the influenza study were many times exposed during their life to influenza viruses before vaccination, whereas infants had their primary contact with the viruses/bacteria vaccine antigens in the study of Prymula et al. Participants of the present study also encountered hepatitis B antigens for the first time. These results indicate that inhibitory effects of paracetamol are especially evident during the vaccinations that prime the immune response. The development of vaccine antigen-specific memory after vaccination is a slow multistep process taking several days. The effects of paracetamol on the vaccination response in adults found in this study suggest that paracetamol plays an important role in the first six hours after the primary vaccinations. This is exemplified by our observation that the vaccination response was only significantly decreased in the prophylactic paracetamol treatment group and not in the therapeutic treatment group. The finding that paracetamol influences the vaccine response only when given directly at the time of vaccination, point to an effect during the early stages of the immune response, when APC-T cell interaction take place in lymphatic tissues [23]–[25]. Paracetamol may decrease the amount of glutathione thereby impairing respiratory antioxidant defenses [26]. Decreased glutathione levels could influence lymphocyte activation mechanisms [27], [28]. Another potential mechanism is the capacity of paracetamol to suppress fever by influencing the COX-2 activity and the production of prostaglandin E2 that stimulate the accompanying immune cell recruitment [29]. It is tempting to speculate that decreased recruitment of APC to the site of vaccination, and decreased activation of these cells during the early stages of induction of immunity underlie our observation, a mechanism which was already previously proposed by Prymula et al. although their study only included a prophylactic treatment group. In the present study, we showed that prophylactic paracetamol treatment during vaccination has a negative influence on the antibody concentration after hepatitis B vaccination in adults. In the Netherlands, paracetamol is mostly used therapeutically after vaccination when side effects start to occur. Since therapeutic paracetamol treatment starting 6 hours after vaccination did not significantly inhibit the response to vaccination, our data indicate that treatment of fever and pain may be without disadvantages. These results are not only important for hepatitis B vaccination procedures, but also for other primary vaccinations in the national immunization program, although we cannot exclude effects of therapeutic paracetamol treatment on vaccines other than hepatitis B. Most important, these findings prompt to consider therapeutic instead of prophylactic treatment to ensure maximal vaccination efficacy and retain the possibility to treat pain and fever after vaccination. BODY.SUPPORTING INFORMATION: Table S1 Anti-HBs levels of all participants. (DOCX)Click here for additional data file. Table S2 Geometric concentrations (GMC) with 95% conficence intervals of the treatment groups. (DOCX)Click here for additional data file. Checklist S1 CONSORT Checklist. (DOC)Click here for additional data file. Protocol S1 Trial Protocol. (DOC)Click here for additional data file.

TITLE: Acute Peanut Consumption Alters Postprandial Lipids and Vascular Responses in Healthy Overweight or Obese Men ABSTRACT: Background: Postprandial hyperlipidemia is associated with impaired endothelial function. Peanut consumption favorably affects the lipid and lipoprotein profile; however, the effects on endothelial function remain unclear. Objective: The purpose of the study was to evaluate the effects of acute peanut consumption as part of a high-fat meal on postprandial endothelial function. Methods: We conducted a randomized, controlled, crossover postprandial study to evaluate the effect of acute peanut consumption on postprandial lipids and endothelial function as assessed by flow-mediated dilatation (FMD) of the brachial artery in 15 healthy overweight or obese men [mean age: 26.7 y; mean body mass index (in kg/m2): 31.4]. Participants consumed, in a randomized order, a peanut meal containing 3 ounces (85 g) ground peanuts (1198 kcal; 40.0% carbohydrate, 47.7% fat, 19.4% saturated fat, 13.2% protein) and a control meal matched for energy and macronutrient content. Meals were in the form of a shake, scheduled ≥1 wk apart. Lipids, lipoproteins, glucose, and insulin were measured at baseline (0 min) and at 30, 60, 120, and 240 min after shake consumption. FMD was measured at baseline and at 240 min. Results: Acute peanut consumption blunted the serum triglyceride (TG) response 120 and 240 min after consumption compared with the control meal (means ± SEMs—120 min: 188.9 ± 19.4 compared with 197.5 ± 20.7 mg/dL; 240 min: 189.9 ± 24.3 compared with 197.3 ± 18.4 mg/dL; P < 0.05 for both). Total, LDL, and HDL cholesterol and glucose and insulin responses were similar between the test meals. Compared with baseline, only the control meal significantly decreased FMD at 240 min (control: −1.2% ± 0.5%; P = 0.029; peanut: −0.6% ± 0.5%; P = 0.3). Participants with higher baseline total (>150 mg/dL) and LDL (>100 mg/dL)-cholesterol concentrations showed a significant decrease in FMD after the control meal (−1.8%, P = 0.017; −2.0%, P = 0.038), whereas the peanut meal maintained endothelial function in all participants irrespective of total- and LDL-cholesterol concentrations. Conclusion: The inclusion of 85 g peanuts (3 ounces) as part of a high-fat meal improved the postprandial TG response and preserved endothelial function in healthy overweight or obese men. This trial was registered at clinicaltrials.gov as NCT01405300. BODY.INTRODUCTION: Cardiovascular disease (CVD)7 remains the leading cause of morbidity and mortality in the United States and globally. Several prospective studies have shown that the postprandial increase in nonfasting serum TGs increases the risk of atherosclerosis and ischemic stroke and is an independent risk factor for coronary artery disease (1–4). Evidence from the Women's Health Study, a prospective study in 26,509 participants followed for a median of 11.4 y, reported that TGs measured 2–4 h after the last meal was a stronger predictor of events than were fasting TG concentrations (5). In addition, postprandial hypertriglyceridemia is involved in proinflammatory cytokine production and oxidative stress, thereby impairing vascular endothelial function, which also contributes to increased CVD risk (6–8). Nuts contain many bioactive compounds with cardiometabolic benefits. Epidemiologic evidence from 3 large cohort studies (total n = 206,029) showed that nut and peanut consumption was inversely associated with total mortality (9). In the Nurses' Health Study, the consumption of peanuts ≥2 times/wk led to a 34% reduction in relative risk of coronary artery disease (10). Peanuts are a popular snack in the United States and a source of unsaturated FAs, plant protein, and bioactive compounds (resveratrol, arginine, vitamin E, magnesium, fiber, and other antioxidants) (11, 12). Moderate [1–1.5 ounces/d (28–42 g/d)] consumption of peanuts has been shown to improve glucose metabolism (13, 14) and the blood lipid and lipoprotein profile (15), with no increase in body weight (16). A recent clinical study in obese men showed that acute consumption of conventional peanuts and high-oleic peanuts (56 g, providing 25% of total energy) attenuated postprandial inflammatory and insulin responses compared with a control meal with no peanuts (17). The cardioprotective effects of nuts are also thought to be achieved by maintaining endothelial function. Endothelial dysfunction is a precursor for atherosclerosis and an independent predictor of cardiac events (18). Endothelial dysfunction can limit the production and/or availability of NO, leading to impaired vasodilation (18). A previous study reported that the consumption of an ad libitum diet that included 56 g (366 kcal) walnuts/d for 8 wk improved endothelial function measured by flow-mediated dilatation (FMD) compared with a control diet without walnuts in individuals with type 2 diabetes (change in FMD: 2.2% ± 1.7% compared with 1.2% ± 1.6%; P = 0.04) (19). In a randomized crossover trial, FMD improved in hypercholesterolemic subjects after 4 wk of consumption of walnuts (contributing 32% of total energy) compared with a Mediterranean-type control diet without walnuts (change in FMD: 5.9% ± 3.3% compared with 3.6% ± 3.3%; P = 0.04) (20). Postprandial hyperglycemia and hyperlipemia can initiate a cascade of biochemical events that lead to endothelial dysfunction (21). With the evidence we have to date that peanuts improve postprandial glucose (22, 23), lipid (17), and inflammatory (17) responses, it is possible that acute peanut consumption may improve vascular responses. However, there is limited evidence about whether peanut consumption affects postprandial vascular function. Therefore, in the present study we evaluated the effect of acute peanut consumption as part of a high-fat meal on postprandial vascular function, plasma glucose, insulin, and lipid and lipoprotein profiles. We hypothesized that acute peanut consumption would result in an improved lipid and lipoprotein profile and vascular function as assessed by FMD, a commonly used noninvasive assessment of vascular endothelial function. BODY.METHODS.PARTICIPANTS.: We recruited generally healthy, overweight or obese [BMI (in kg/m2) 28–39] men (to avoid hormone cycle effects on vascular function in women) between 20 and 65 y of age, with fasting TGs <350 mg/dL, LDL cholesterol <160 mg/dL, glucose <99 mg/dL, and blood pressure ≤140/90 mm Hg. Participants were free of diabetes, CVD, kidney disease, and other metabolic diseases. The study was approved by the Ethics Committee of The Pennsylvania State University and carried out in accordance with the Helsinki Declaration (clinicaltrials.gov, NCT01405300). BODY.METHODS.STUDY DESIGN.: A randomized crossover trial was conducted. Participants were evaluated on 2 occasions separated by a washout period of a minimum of 7 d between interventions. Participants were randomly assigned to consume the control or peanut shake during visit 1 and the alternate shake was consumed during visit 2. On each occasion, fasting (10–12 h) blood samples were collected at baseline (t = 0 min) and then at 30, 60, 120, and 240 min after the ingestion of either a control shake or a peanut shake (consumed within 15 min). Participants were asked not to consume alcohol 48 h before the blood draw and to avoid vigorous exercise 2 h before the blood draw. FMD and blood pressure were measured at baseline (t = 0 min) and at 240 min. BODY.METHODS.NUTRIENT PROFILE AND COMPOSITION OF THE PEANUT AND CONTROL SHAKES.: The nutrient profile of the test shakes is presented in Table 1. The control and peanut shakes were matched for macronutrient content. The control shake provided 24.8% carbohydrate, 64.7% fat (27.6% SFAs, 26.0% MUFAs, 11.1% PUFAs), 8.4% protein, 8.2 g fiber, and 1229 kcal. The peanut shake provided 25.9% carbohydrate, 66.2% fat (28.2% SFAs, 26.7 MUFAs, 11.3% PUFAs), 8.5% protein, 8.2 g fiber, and 1198 kcal. The ingredient composition of the test shakes is presented in Table 1. Sunflower oil, safflower oil, powdered egg white, and fiber were used in the control shake to match the nutrient profile of 3 ounces (85 g) of peanuts. The amount of whipping cream and glucose in the peanut and control shakes was adjusted to ensure that all shakes were matched for carbohydrate and SFA contents. The control shake provided 34.8 g glucose, 150 g heavy whipping cream, 39 g chocolate syrup, 20 g sunflower oil, 17 g safflower oil, 27 g powered egg white, and 9.6 g fiber supplement (Benefiber; Novartis Consumer Health, Inc.). The peanut shake provided 34.8 g glucose, 137 g heavy whipping cream, 39 g chocolate syrup, and 85 g (3.0 ounces) ground peanuts with skins. Ingredients were blended with 150 g water and 60 g crushed ice in a Vitamix blender (Vita-Mix Corporation). TABLE 1 Nutrient profile and composition of the test meals Control Peanut 1 Macronutrients  Carbohydrate, % 24.8 25.9  Protein, % 8.4 8.5  FAs, % 64.7 66.2   SFAs 27.6 28.2   MUFAs 26.0 26.7   PUFAs 11.1 11.3  Fiber, g 8.2 8.2  Total energy, kcal 1229 1198 Composition, g  Glucose 34.8 34.8  Heavy whipping cream 150 137  Chocolate syrup 39 39  Sunflower oil 20  Safflower oil 17  Powdered egg white 27  Fiber supplement 9.6 1 Provided 3.0 ounces of ground peanuts with skins. BODY.METHODS.BLOOD SAMPLING.: A flexible catheter was inserted in the antecubital vein of the left arm, and blood samples were collected into evacuated tubes. Blood samples were collected in the fasted state (0 min) and at 30, 60,120, and 240 min after consumption of the test meals. Serum samples were sent to Quest Diagnostics (Chem 24 panel; Quest Diagnostics) for analysis. Lipids and lipoproteins, glucose, insulin, and C-reactive protein were assayed at all time points. BODY.METHODS.FMD.: Participants were asked to lie down for 15 min before the FMD assessment. Endothelial function was assessed by FMD with high-frequency ultrasound in a quiet, temperature-controlled room (21–24°C). Longitudinal images of the brachial artery 5–10 cm above the elbow of the right arm were acquired with external B mode ultrasound imaging (Acuson Aspen 128XP equipped with a 10-mHz linear array transducer; Acuson) and recorded during the following procedure: baseline (1 min), arterial occlusion (5 min) via inflation of a cuff on the forearm (distal to the segment being imaged) set at 250 mm Hg (Hokanson), and reactive hyperemia (2 min). Images were graded by using R-wave detection so that scans were assessed at the end of diastole. Two independent, blinded technicians used automated edge detection software (Brachial Analyzer; MIA) to quantify artery diameter continuously throughout the test. Resting diameters were the average of all images collected over the 1-min baseline period. Peak artery diameter was determined as the largest diameter recorded in the 2-min reactive hyperemia segment. The reported FMD score was calculated as the percentage change from resting arterial diameter to peak reactive hyperemia diameter. Scores presented are the average of the 2 technicians' scores. BODY.METHODS.STATISTICAL ANALYSES.: Statistical analysis was performed by using SAS (version 9.2; SAS Institute). The mixed-models procedure (PROC MIXED) was used to test the effects of treatment, time, and treatment by time interactions on each outcome. Subject was included as a random effect, and the remaining factors were fixed effects. Tukey-Kramer–adjusted P values were used for post hoc comparisons. The 4-h incremental AUC (iAUC) of postprandial concentrations of TGs, glucose, and insulin were analyzed by using a mixed model with treatment as a fixed effect. Changes were calculated by using values from the 240-min time point minus the baseline value (0 min). As part of a secondary analysis, we categorized participants into 2 groups on the basis of their baseline total cholesterol (TC) concentration at 150 mg/dL (mean of baseline TC) and baseline LDL-cholesterol concentration at 100 mg/dL (optimal LDL-cholesterol concentration: ≤100 mg/dL). For this stratification, we determined baseline concentrations to be the value taken at 0 min on the first day of testing. Baseline lipid concentrations were added as a categorical variable to the mixed model as a fixed effect to determine whether participants initially presenting with elevated cholesterol responded differently to peanut and control meals than did participants with lower baseline cholesterol concentrations. Means are reported as least-squares means ± SEMs. A histogram and residual plots were used to verify normality of data. The study sample size was estimated with power set to 0.90 and α set to 0.05, which predicted a sample size of 15 participants to detect a 1% change in FMD (24). BODY.RESULTS.PARTICIPANTS AND BASELINE CHARACTERISTICS.: Fifteen nonsmoking male participants met all of the inclusion criteria and completed the study. None of the enrolled participants withdrew from the study. Participant baseline characteristics are presented in Table 2. In the present study, 40% of participants were overweight and 60% were obese. TABLE 2 Anthropometric and metabolic characteristics of the men at screening 1 Clinical measurements Value Age, y 26.7 ± 1.6 Weight, kg 100.0 ± 3.7 BMI, kg/m 2 31.4 ± 0.8 SBP, mm Hg 125 ± 2 DBP, mm Hg 83 ± 1 Serum biochemistry  Glucose, mg/dL 92.5 ± 0.7  Total cholesterol, mg/dL 159.7 ± 7.1  HDL cholesterol, mg/dL 42.4 ± 1.9  LDL cholesterol, mg/dL 99.1 ± 6.4  TGs, mg/dL 90.6 ± 8.9  TG-to-HDL-cholesterol ratio 2.26 ± 1.15 1 Values are means ± SEMs. n = 15. DBP, diastolic blood pressure; SBP, systolic blood pressure. BODY.RESULTS.POSTPRANDIAL INSULIN, GLUCOSE, AND LIPIDS.: Postprandial glucose, insulin, lipid, and lipoprotein concentrations in response to the test meals are presented in Table 3. A mixed linear model showed a treatment by time interaction for TGs (P < 0.0001). Both control and peanut meals increased postprandial TGs after 60 min of ingestion compared with baseline (P < 0.05 for both treatments). Peak TG responses to the 2 treatments were observed at 120 and 240 min (P < 0.05 for both treatments compared with baseline). At these time points, postprandial changes in plasma TG concentrations were greater (P = 0.033) after the control meal than after the peanut meal (Figure 1). The postprandial changes in plasma TG concentrations were also calculated as iAUCs. There was a 31.9% reduction in the TG iAUC after consumption of the peanut meal compared with the control meal (P = 0.047). TABLE 3 Serum glucose, insulin, lipid, and lipoprotein responses to the control and peanut test meals in overweight or obese men 1 Control test meal Peanut test meal 0 min 30 min 60 min 120 min 240 min 0 min 30 min 60 min 120 min 240 min P -meal Glucose, mg/dL 88.7 ± 1.2 a 103.2 ± 2.3 b 99.8 ± 4.1 b 88.2 ± 3.9 93.4 ± 3.14 86.3 ± 1.73 c 103.2 ± 2.3 d 99.8 ± 4.1 d 99.9 ± 3.61 d 88.9 ± 1.9 0.38 Insulin, μU/mL 6.3 ± 0.82 a 30.78 ± 6.24 b 36.7 ± 6.9 b 24.8 ± 4.2 b 13.2 ± 1.1 b 5.2 ± 0.82 c 46.3 ± 8.7 d 35.2 ± 5.9 d 29.4 ± 4.5 d 12.9 ± 1.7 0.11 TGs, mmol/L 82.8 ± 7.4 a 100.5 ± 9.5 124.1 ± 12.1 197.5 ± 20.7 b 197.3 ± 18.4 b 93.0 ± 11.4 c 106.3 ± 10.5 132.1 ± 10.8 188.9 ± 19.4 d * 189.9 ± 24.3 d * 0.04 HDL-C, mmol/L 41.7 ± 2.1 42.9 ± 2.1 42.7 ± 2.1 40.4 ± 2.1 38.9 ± 2.1 41.1 ± 1.8 43.1 ± 2.1 41.7 ± 2.1 40.1 ± 2.1 39.1 ± 2.1 0.32 LDL-C, mmol/L 95.1 ± 6.6 98.2 ± 6.7 92.3 ± 6.7 80.6 ± 6.3 82.8 ± 6.9 95.1 ± 6.8 96.5 ± 7.8 92.7 ± 6.8 80.9 ± 6.6 86.4 ± 8.2 0.36 CRP, mg/dL 1.4 ± 0.4 1.4 ± 0.5 1.4 ± 0.4 1.4 ± 0.4 1.5 ± 0.4 1.7 ± 0.7 1.7 ± 0.7 1.8 ± 0.7 1.7 ± 0.7 1.6 ± 0.6 0.34 Non-HDL-C, mmol/L 111.5 ± 7.1 118.5 ± 7.2 117.1 ± 7.1 120.3 ± 6.9 122.5 ± 7.2 113.8 ± 7.8 117.8 ± 8.5 119.4 ± 7.6 118.9 ± 8.1 124.4 ± 8.2 0.37 Total cholesterol, mmol/L 153.1 ± 7.1 161.2 ± 7.3 159.8 ± 7.1 160.5 ± 6.9 161.2 ± 7.2 154.8 ± 7.7 160.8 ± 8.4 160.9 ± 7.6 158.9 ± 8.1 163.6 ± 8.2 0.28 TG:HDL-C 2.09 ± 0.23 2.48 ± 0.29 3.12 ± 0.42 5.25 ± 0.69 5.37 ± 0.57 2.42 ± 0.35 2.66 ± 0.34 3.37 ± 0.35 5.06 ± 0.58 5.30 ± 0.8 0.37 1 Values are means ± SEMs. n = 15. *Different from the control meal at that time, P < 0.05. Within-meal: means in a row with different superscript letters differ, P < 0.05. Unlabeled means are intermediate and not significantly different. CRP, C-reactive protein; HDL-C, HDL cholesterol; LDL-C, LDL cholesterol. FIGURE 1Changes in serum TGs in overweight or obese men in response to control and peanut test meals. Values are means ± SEMs. n = 15. *Different from the control meal at that time, P < 0.05. Within-meal: means with different letters differ, P < 0.05. Unlabeled means are intermediate and not significantly different. The TG-to-HDL-cholesterol ratio was used as a surrogate marker for insulin resistance (25). At 120 and 240 min, there was a trend for an increased TG-to-HDL-cholesterol ratio in response to the control meal compared with the peanut meal (P = 0.09). There were no treatment, time, or treatment-by-time interactions for TC, LDL cholesterol, or HDL cholesterol. As expected, glucose and insulin increased immediately after shake consumption (effect of time, P < 0.001), but no difference between treatments was observed. BODY.RESULTS.ENDOTHELIAL FUNCTION.: The control shake significantly reduced FMD by 1.2% at 240 min postconsumption compared with baseline (P = 0.029). There were no significant differences in FMD between baseline and 240 min postconsumption of the peanut shake (Figure 2). Baseline (0 min) cholesterol concentration predicted change in FMD. Participants with higher baseline TC concentrations (>150.0 mg/dL; n = 9) showed a significant decrease in FMD (−1.8%; P = 0.017) 240 min after consumption of the control meal compared with baseline (Figure 3A). In contrast, FMD in response to the peanut meal remained unchanged in participants with both lower and higher baseline TC concentrations. Similar results were observed in participants with a higher baseline LDL-cholesterol concentration. Participants (n = 6) with a higher baseline LDL-cholesterol concentration (>100 mg/dL) showed a significant decrease in FMD (−2.0%; P = 0.038) 240 min after the control meal (Figure 3B). Furthermore, the peanut meal did not decrease FMD compared with baseline in participants, irrespective of baseline LDL cholesterol concentration. FIGURE 2FMD responses of overweight or obese men 4 h after the control and peanut test meals. Values are means ± SEMs. n = 15. *Different from 0 h, P < 0.05. FMD, flow-mediated dilatation. FIGURE 3FMD responses of overweight or obese men by baseline TC (A) or LDL cholesterol (B) concentration 4 h after the control and peanut test meals. Values are least-squares means ± SEMs. *Different from 0 min, P < 0.05. FMD, flow-mediated dilatation; LDL-C, LDL cholesterol; TC, total cholesterol. BODY.DISCUSSION: In the present study, we assessed the effects of peanut consumption on metabolic responses and endothelial function in generally healthy overweight or obese men. Acute peanut consumption attenuated postprandial TG responses (31.9% reduction in TG iAUC) compared with a calorie and macronutrient-matched control meal. We showed that acute consumption of 3.0 ounces of peanuts as part of a high-fat meal preserved postprandial endothelial function, particularly in participants with unfavorable baseline lipid profiles. In addition, these findings suggest that the inclusion of peanuts in the context of a high-fat meal may have favorable postprandial cardiometabolic effects. Most individuals spend the majority of their nonsleeping hours in a postprandial state. Postprandial lipemia, a well-recognized risk factor for atherosclerosis, is associated with impaired endothelial function. During the postprandial period, an increase in TG-rich lipoproteins after a high-fat load increases oxidative stress. This resulting reduced NO bioavailability leads to endothelial dysfunction, which is indicated by a suppressed FMD response (26). Previous studies have shown that increases in postprandial TG-rich lipoproteins after a high-fat meal temporarily impair endothelial function in young (23 ± 2 y) participants (27, 28). In the present study, the consumption of a high-fat control meal significantly decreased endothelial function, whereas it was maintained after the consumption of a macronutrient-matched meal with peanuts, and was accompanied by a blunted postprandial TG response. These findings suggest that peanut consumption preserves endothelial function in the presence of postprandial TG-rich lipoprotein increases, which may be related to a reduction in oxidative stress. We hasten to add that other mechanisms may be involved in mediating this effect, and additional research is needed to clarify this. This cardioprotective effect may be due to the bioactive compounds in peanuts and peanut skins. For example, NO is an important vasodilator that is synthesized from l-arginine in endothelial cells (29). Peanuts are a rich source of l-arginine, which reflects their high protein content (25% of total energy) (30). Dietary l-arginine could enhance NO synthesis and protect the endothelium against postprandial oxidative damage. Marchesi et al. (24) reported that 10-d administration of a low-dose of l-arginine (6 g/d) attenuated postprandial endothelial dysfunction in young healthy men. Oral l-arginine supplementation (7 g, 3 times/d for 4 wk) improved endothelial dysfunction in hypercholesterolemic young adults (31). In our study, 85 g peanuts were provided to the participants, which provided 2.74 g l-arginine. In addition to l-arginine, other bioactive compounds in peanuts may offer other cardioprotective effects. Wien et al. (32) reported that the daily consumption of peanuts (46 g/d) over 24 wk improved the nutrient profile of the diet (increased unsaturated fat, α-tocopherol, niacin, and magnesium) and reduced body weight in participants with type 2 diabetes. Each kilogram of weight loss was associated with a 0.11-mmol/L reduction in fasting blood glucose and a 0.07% reduction in glycated hemoglobin (32). Moreover, peanuts (including peanut skins) are a source of phenolic compounds that have high antioxidant capacity, which may prevent endothelial damage by blunting postprandial oxidative reactions associated with a high-fat meal. The phenolic compounds in peanuts and nuts may also contribute to an anti-inflammatory postprandial response (33). There is evidence that postprandial inflammation and oxidative stress can exaggerate postprandial lipemia, which leads to vascular dysfunction and damage (34). Moreira Alves et al. (17) reported that acute consumption of 56 g peanuts (with skins) significantly reduced the postprandial inflammatory biomarkers TNF-α and IL-10 3 h after consumption in overweight men. Thus, the bioactive compounds in peanuts may attenuate inflammation and the resulting oxidative stress (caused by meals high in SFAs), thereby benefiting endothelial health, especially in the postprandial state. The role of antioxidants and l-arginine in improving FMD and the lipid and lipoprotein profile has been investigated in other nuts. Ros et al. (20) reported that the consumption of a walnut diet (18% of total energy) for 4 wk attenuated endothelial dysfunction in individuals with hypercholesterolemia. Acute consumption of a walnut meal (90 g walnuts) increased postprandial γ-tocopherol and resulted in a lower concentration of malondialdehyde postprandially, suggesting less lipid peroxidation (35). Hudthagosol et al. (36) reported that, after consumption of a pecan test meal (90 g pecans), concentrations of γ-tocopherol doubled and oxidized LDL decreased by 26% at 8 h. Collectively, the findings of these studies indicate that bioactive constituents in nuts contribute to postprandial antioxidant defenses. Although walnuts are the most-studied nuts relative to the evaluation of vascular reactivity, other nuts and peanuts would be expected to have similar benefits. In the present study, we performed a stratification analysis on the basis of participants' baseline lipid and lipoprotein profile. Participants with higher baseline cholesterol and LDL-cholesterol concentrations are more prone to endothelial dysfunction after a high-fat meal. We have shown that peanut consumption maintained vascular function in participants who were at greater cardiometabolic risk on the basis of their lipid and lipoprotein profile. This robust study design directly compared peanut consumption with the consumption of a macronutrient-matched control meal without peanuts. Thus, we were able to evaluate the contribution of the bioactive components in peanuts. In addition, the postprandial design allowed us to characterize the metabolic time course of the endpoints we assessed after peanut consumption. A limitation of this study is the small sample size (n = 15). The results from the present study need to be verified in a larger sample that includes both women and men. In addition, the fat content of the test meal may have been greater than the typical fat content of a Western diet, which may limit the generalizability of the study results. However, it is not uncommon that milkshakes that are high in calories are consumed in the context of a high-fat meal. The use of shakes as a delivery vehicle may somewhat represent the unhealthy diet pattern that may contribute to the postprandial hyperlipidemia responses that have deleterious effects on endothelial function. Postprandial effects also may be more apparent in individuals with diabetes who have impaired glycemic control. In conclusion, the acute consumption of 3.0 ounces (85 g) of peanuts as a part of a high-fat meal reduced the postprandial TG response and maintained endothelial function in overweight and obese men, particularly in those who were at greater cardiometabolic risk due to elevated baseline TC and LDL-cholesterol concentrations. Therefore, frequent consumption of peanuts may improve CVD risk via mechanisms that involve postprandial cardiometabolic responses, including those that affect vascular function.

TITLE: A randomized clinical trial investigating the efficacy of targeted nutrition as adjunct to exercise training in COPD ABSTRACT.ABSTRACT.BACKGROUND: Evidence regarding the efficacy of nutritional supplementation to enhance exercise training responses in COPD patients with low muscle mass is limited. The objective was to study if nutritional supplementation targeting muscle derangements enhances outcome of exercise training in COPD patients with low muscle mass. ABSTRACT.ABSTRACT.METHODS: Eighty‐one COPD patients with low muscle mass, admitted to out‐patient pulmonary rehabilitation, randomly received oral nutritional supplementation, enriched with leucine, vitamin D, and omega‐3 fatty acids (NUTRITION) or PLACEBO as adjunct to 4 months supervised high intensity exercise training. ABSTRACT.ABSTRACT.RESULTS: The study population (51% males, aged 43–80) showed moderate airflow limitation, low diffusion capacity, normal protein intake, low plasma vitamin D, and docosahexaenoic acid. Intention‐to‐treat analysis revealed significant differences after 4 months favouring NUTRITION for body mass (mean difference ± SEM) (+1.5 ± 0.6 kg, P = 0.01), plasma vitamin D (+24%, P = 0.004), eicosapentaenoic acid (+91%,P < 0.001), docosahexaenoic acid (+31%, P < 0.001), and steps/day (+24%, P = 0.048). After 4 months, both groups improved skeletal muscle mass (+0.4 ± 0.1 kg, P < 0.001), quadriceps muscle strength (+12.3 ± 2.3 Nm,P < 0.001), and cycle endurance time (+191.4 ± 34.3 s, P < 0.001). Inspiratory muscle strength only improved in NUTRITION (+0.5 ± 0.1 kPa, P = 0.001) and steps/day declined in PLACEBO (−18%,P = 0.005). ABSTRACT.ABSTRACT.CONCLUSIONS: High intensity exercise training is effective in improving lower limb muscle strength and exercise performance in COPD patients with low muscle mass and moderate airflow obstruction. Specific nutritional supplementation had additional effects on nutritional status, inspiratory muscle strength, and physical activity compared with placebo. BODY.INTRODUCTION: It is well established that extra‐pulmonary pathology enhances the disease burden and mortality risk in patients with COPD. Muscle wasting is common, in particular in emphysema, and associated with a high prevalence of osteoporosis, impaired exercise performance, and higher mortality risk.1 Furthermore muscle wasting often coincides with a decreased oxidative metabolism due to a shift in muscle fibre type composition.2 Treatment of muscle dysfunction to alleviate progressive disability has been subject of intensive research in COPD. Exercise training is considered as cornerstone of pulmonary rehabilitation (PR) for improving lower limb muscle function.3 The latest Cochrane meta‐analysis by Ferreira et al. 4 concludes that nutritional supplementation promotes weight gain and fat‐free mass as proxy of muscle mass among patients with COPD, especially if undernourished and when combined with exercise training. However, the potential of nutritional supplementation to enhance efficacy of exercise training is not well established. Limited studies are available that differ in COPD target population and nutrient composition, and most of them are underpowered.5 In this randomized placebo‐controlled multi‐centre NUTRAIN‐trial, we aimed to determine the efficacy of specific nutritional supplementation targeting muscle derangements as adjunct to exercise training in COPD patients with low muscle mass. Based on available evidence regarding the mode of action of specific nutrients on skeletal muscle maintenance combined with reported deficiencies in COPD, we chose a multimodal approach including high quality protein enriched with leucine,6, 7 vitamin D,8 and omega‐3 (n‐3) poly‐unsaturated fatty acids (PUFA).9 We hypothesize that COPD patients with low muscle mass, eligible for out‐patient PR, show more pronounced improvement in muscle strength, endurance, and nutritional status after an exercise training program including targeted nutritional support. BODY.METHODS: A detailed methodology can be found in the Supporting Information. BODY.METHODS.STUDY DESIGN: This double blind placebo controlled multi‐centre NUTRAIN‐trial was integrated in the out‐patient PR program performed in seven hospitals of the Centre of Expertise for Chronic Organ failure (CIRO) network in the South East of the Netherlands. The study was registered at clinicaltrials.gov (NCT01344135) and the Medical Ethics Committee from Maastricht University Medical Centre granted ethical approval (NL34927068.10/MEC 11‐3‐004). The NUTRAIN‐trial also comprised an exploratory maintenance program with a wider scope which will be reported separately. BODY.METHODS.PATIENTS: Patients with COPD (post‐bronchodilator FEV1/FVC <0.7) referred for PR between September 2011–April 2014 were eligible for participation when they had low muscle mass (defined as FFMI under the sex‐ and age‐specific 25th percentile FFMI values10), were admitted to out‐patient PR, did not meet any of the exclusion criteria (see Supporting Information), and gave written informed consent. The target sample size (alpha 0.05, power 80%) was based on data from the INTERCOM‐study11 assuming a 10% difference in peak torque between the groups assuming maintenance of skeletal muscle strength in PLACEBO during follow‐up and a SD of 5 Nm in peak torque, revealing n = 60 in each group including 25% drop‐out. Patient inclusion was prematurely discontinued because the test product could not be produced within the appropriate quality specifications due to discontinuation of the supply of one of the ingredients, but justifiable based on other RCTs published in the meantime as argued in the discussion. To explore if the nutrient supplementation treated a deficient status or not, baseline plasma nutrient status was compared with a healthy control group (see Supporting Information for details). BODY.METHODS.PROCEDURES: The nutritional intervention was integrated in a 4 month standardized outpatient PR program (see Supporting Information for PR components). Patients from both the PLACEBO and NUTRITION group were advised to consume two to three portions of the supplement daily. Randomization and masking procedures are described in the Supporting Information. Per volume serving of 125 mL unit, the NUTRITION product provided 187.5 kCal in a distribution of 20 energy percent (EN%) protein, 60EN% carbohydrate (CHO), and 20EN% fat, and was enriched with leucine, n‐3 PUFA, and vitamin D (Nutricia NV, Zoetermeer, the Netherlands, for details see table E1). The PLACEBO product did not comprise the investigated active components but consisted of a flavoured non‐caloric aqueous solution. BODY.METHODS.OUTCOMES: Measurements were performed at CIRO before entering PR, as part of a 3 day baseline assessment and after completion of the PR during a 2 day outcome assessment3 with quadriceps muscle strength (QMS) as primary outcome. Secondary outcomes included body composition measured by dual energy X‐ray absorptiometry (DEXA), cycle endurance time (CET), inspiratory muscle strength (IMS), and daily steps by a tri‐axial GT3X Actigraph accelerometer (Health One Technology, Fort Walton Beach, FL) worn on an elastic belt firmly attached around the waist for 7 consecutive days before and after the intervention period, habitual dietary intake using a validated cross‐check dietary history method and fasting plasma levels of vitamin D (25‐hydroxycholecalciferol), branched‐chain amino acids (BCAAs), and fatty acid profile in phospholipids. Furthermore, post‐bronchodilator forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), static lung volumes, and diffusing capacity of the lung for carbon monoxide (DLCO) were assessed. Exploratory outcomes included 6 min walk distance (6MWD) and mood assessment (HADS: Hospital Anxiety and Depression scale). BODY.METHODS.STATISTICAL ANALYSES: Intention‐to‐treat (ITT) analysis was performed using the Statistical Package for Social Sciences (SPSS version 20.0 for Windows, Inc.), using all available change‐values (every subject completing the PR and outcome assessment), independently of dropping out of the nutritional intervention. Within group treatment outcomes were compared by paired samples T‐test for continuous normally distributed data or Wilcoxon Signed Rank Test for continuous non‐normally distributed data. Between‐group differences were compared by ANCOVA taking pre‐treatment value as covariate, the 4 month post‐treatment value as response, and considering treatment as a factor.12 Two‐sided P‐values <0.05 were considered statistically significant. Results are presented as mean ± standard error of the mean (SEM). BODY.RESULTS.PATIENTS: Between September 2011 and April 2014, 81 patients were enrolled in the trial and randomized to NUTRITION or PLACEBO. A patient flowchart is shown in (Figure 1). Figure 1NUTRAIN flowchart. A total of 1640 patients referred for pulmonary rehabilitation were assessed for trial eligibility. COPD patients (post‐bronchodilator FEV1/FVC <0.7) were eligible when they had low muscle mass (FFMI < sex‐ and age‐specific 25th percentile FFMI values) and referred for outpatient rehabilitation. A total of 1420 patients were excluded and 139 eligible patients declined to participate; 81 patients were enrolled in the trial and randomized to NUTRITION or PLACEBO. Two of the 81 randomized patients did not start the treatment. During the PR, the drop‐out rate was 9.5% (4 patients) in NUTRITION and 5.4% (2 patients) in PLACEBO. At baseline, the study population (51% male, aged 43–80) were characterized by low diffusion capacity (DLCO: 49.4 ± 1.7%), on average moderate airflow limitation (FEV1: 55.1 ± 2.2%predicted), a BMI of 22.7 ± 0.3 kg/m2, and fat‐free mass index (FFMI) of 15.8 ± 0.2 kg/m2. The majority of the patients (88.8%) had vitamin D insufficiency/deficiency, and average plasma DHA level was decreased compared with controls (−12.4%), but the plasma level of total BCAA was comparable with healthy age matched volunteers (Figure 2a). Mean protein intake was well above recommended daily intake (RDI) (1.4 ± 0.1 g/kg BW), whereas intake of vitamin D and calcium were below RDI in, respectively, 92.4 and 72.2% of the participants. Although baseline CET and 6MWD tended to be higher in NUTRITION, no significant differences in baseline characteristics were found between the NUTRITION and PLACEBO group (Table 1). Figure 2Plasma status of supplemented nutrients. A: Baseline plasma nutrient levels compared with healthy controls. Dark grey bars represent NUTRAIN patients with COPD. White bars represent healthy controls. B: Mean change from baseline in plasma concentrations of supplemented nutrients. Light grey bars represent patients that received PLACEBO. Mid grey bars represent patients that received NUTRITION. * P < 0.05; **P < 0.01; *** P < 0.001. Table 1 Baseline characteristics of the randomized study population PLACEBO ( n  = 39) NUTRITION ( n  = 42) N Mean  ±  SEM N Mean  ±  SEM General Gender, % male 59.0% 42.9% Current smokers, % 31.6% 19.0% Age, years 39 62.2 ± 1.3 42 62.8 ± 1.3 Self‐reported co‐morbidities, n 39 2.5 ± 0.3 42 2.5 ± 0.2 Exacerbations in last 12 months, n 39 1.4 ± 0.3 42 1.1 ± 0.2 Lung function DLCO, %predicted 36 47.1 ± 2.5 42 51.4 ± 2.2 FEV 1 , %predicted 39 53.0 ± 2.8 42 57.0 ± 3.3 FVC, %predicted 39 100.6 ± 2.7 42 102.8 ± 2.9 FEV 1 /FVC, % 39 41.6 ± 1.8 42 44.4 ± 2.0 FRC, %predicted 36 146.7 ± 5.9 42 139.1 ± 4.3 Residual volume, %predicted 36 153.1 ± 8.5 42 143.4 ± 6.3 Plasma nutrient levels BCAA, μmol/L 39 466.6 ± 10.9 41 448.0 ± 10.8 Vitamin D, nmol/L 39 45.3 ± 3.1 41 54.0 ± 4.5 Vitamin D insufficiency, % 23.1% 39.0% Vitamin D deficiency, % 69.2% 46.3% AA, % of total FA 37 11.3 ± 0.4 36 10.4 ± 0.4 EPA, % of total FA 37 1.2 ± 0.1 36 1.1 ± 0.1 DHA, % of total FA 37 3.2 ± 0.2 36 2.9 ± 0.2 N‐3 FA, % of total FA 37 5.5 ± 0.3 36 5.2 ± 0.2 N‐6 FA, % of total FA 37 34.8 ± 0.4 36 34.7 ± 0.4 Body composition Total body mass, kg 39 65.0 ± 1.7 42 64.3 ± 1.6 BMI, kg/m2 39 22.6 ± 0.5 42 22.9 ± 0.4 FM, kg 39 19.0 ± 1.3 42 20.0 ± 1.0 Lean mass, kg 39 43.6 ± 1.2 42 42.0 ± 1.3 SMM, kg 39 18.4 ± 0.6 42 17.4 ± 0.6 BMC, g 39 2414.7 ± 82.3 42 2331.3 ± 73.0 Dietary intake Total energy, kcal/day 39 2361.7 ± 161.3 40 2188.8 ± 111.4 Protein, g/kg BW/day 39 1.4 ± 0.1 40 1.4 ± 0.1 Protein <1.0 g/kg BW/day 25.6% 25.0% Vitamin D, μg/day 39 5.1 ± 0.5 40 5.7 ± 0.5 Vitamin D < RDI, % 89.7% 95.0% Calcium, mg/day 39 947.8 ± 62.4 40 998.1 ± 56.9 Calcium <RDI, % 74.4% 70.0% Lower limb muscle strength QMS, Nm 37 118.0 ± 6.6 39 121.5 ± 6.4 Exercise performance Peak workload, Wmax 39 72.5 ± 3.8 42 84.6 ± 5.2 Peak workload, %predicted 39 57.0 ± 3.4 42 69.5 ± 3.3 ** CET, s 37 231.5 ± 12.0 41 319.0 ± 35.2 6MWD, m 39 484.30 ± 13.0 42 501.4 ± 13.1 6MWD, %predicted 39 72.1 ± 1.8 42 77.2 ± 1.7 * Respiratory muscle strength IMS, kPa 38 7.0 ± 0.3 42 6.8 ± 0.4 IMS, %predicted 38 77.3 ± 2.7 42 79.4 ± 3.8 Physical activity level PAL, steps/day 37 4516.6 ± 379.3 38 4716.7 ± 327.2 Data are mean ± SEM or %. BCAA, branched‐chain amino acids; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; n‐3 FA, omega 3 fatty acids; n‐6 FA, omega 6 fatty acids; BMC, bone mineral content; SMM, skeletal muscle mass; FM, fat mass; QMS, quadriceps muscle strength; CET, cycle endurance time; 6MWD, 6 minwalking distance; IMS, inspiratory muscle strength; PAL, physical activity level. * P  < 0.05. ** P  < 0.01. Two of the 81 randomized patients did not start the treatment. During the PR, the drop‐out rate was 9.5% (four patients) in NUTRITION and 5.4% (two patients) in PLACEBO. Mean supplement intake reached 2.1 ± 0.1 portions/day. Reported side‐effects included stomach‐ache (n = 3 in NUTRITION, n = 1 in PLACEBO), constipation (n = 2 in NUTRITION), and undesirable weight loss (n = 5 in PLACEBO). Nonetheless, neutral or positive product rating was revealed in the majority of patients regarding taste, sweetness, mouthfeel, and thickness of the NUTRITION and PLACEBO supplement. The aftertaste of the NUTRITION supplement was equally rated pleasant or unpleasant. A high satiety after consuming the supplement was reported by ±70% in NUTRITION vs. ±17% in PLACEBO (P < 0.001). Outcomes of the intervention are presented in Table 2. Table 2 Outcomes of the intervention PLACEBO ( n  = 35) NUTRITION ( n  = 38) Between group differences (NUTRITION − PLACEBO) Pre Post Pre Post Mean  ±  SEM Mean  ±  SEM Mean  ±  SEM Mean  ±  SEM Adj. difference  ±  SEM a Plasma nutrient levels BCAA, μmol/L 471.6 ± 11.6 483.9 ± 15.5 445.3 ± 11.6 450.1 ± 10.3 −14.4 ± 14.2 Vitamin D, nmol/L 44.6 ± 3.3 49.9 ± 4.0 54.2 ± 4.9 68.0 ± 3.6 *** 12.8 ± 4.3 ** AA, % of total FA 11.3 ± 0.4 11.0 ± 0.5 10.6 ± 0.5 9.5 ± 0.3 * −1.2 ± 0.5 * EPA, % of total FA 1.2 ± 0.1 1.1 ± 0.1 1.1 ± 0.1 2.1 ± 0.2 *** 1.0 ± 0.2 *** DHA, % of total FA 3.2 ± 0.2 2.9 ± 0.1 * 3.0 ± 0.2 3.8 ± 0.1 *** 0.9 ± 0.2 *** N‐3 FA, % of total FA 5.5 ± 0.3 5.2 ± 0.2 5.3 ± 0.3 7.3 ± 0.3 *** 2.2 ± 0.4 *** N‐6 FA, % of total FA 34.8 ± 0.4 35.5 ± 0.4 35.1 ± 0.3 33.4 ± 0.5 *** −2.2 ± 0.5 *** Body composition Total body mass, kg 65.7 ± 1.7 66.0 ± 1.7 63.8 ± 1.7 65.7 ± 1.7 *** 1.5 ± 0.6 * BMC, g 2427.2 ± 85.9 2428.5 ± 84.1 2326.7 ± 78.7 2339.2 ± 80.3 10.0 ± 17.7 SMM, kg 18.5 ± 0.6 18.8 ± 0.6** 17.2 ± 0.6 17.8 ± 0.7** 0.3 ± 0.2 FM, kg 19.4 ± 1.4 19.2 ± 1.4 19.8 ± 1.1 21.0 ± 1.1 *** 1.6 ± 0.5 ** Lower limb muscle function QMS, Nm 121.2 ± 6.9 132.0 ± 7.2 ** 121.7 ± 6.9 135.3 ± 8.2*** 2.8 ± 4.6 Exercise performance CET, s 237.9 ± 12.3 482.4 ± 62.5 *** 323.2 ± 38.8 467.2 ± 54.7 *** −109.7 ± 70.4 6MWD, m 492.5 ± 14.0 492.0 ± 16.6 504.0 ± 14.5 500.3 17.9 −3.9 ± 12.2 Respiratory muscle function IMS, kPa 7.1 ± 0.3 7.5 ± 0.3 6.7 ± 0.4 7.2 ± 0.4** 0.0 ± 0.3 Physical activity level PAL, steps/day 4664.7 ± 415.9 3841.9 ± 393.4 ** 4790.1 ± 352.2 4866.4 ± 479.0 929.5 ± 459.2* Mood HADS total score 11.1 ± 1.2 8.5 ± 0.9 ** 12.2 ± 1.0 9.2 ± 1.1 *** −0.2 ± 1.0 HADS anxiety score 6.0 ± 0.7 4.1 ± 0.5 ** 6.3 ± 0.7 4.8 ± 0.6 *** 0.4 ± 0.6 HADS depression score 5.1 ± 0.6 4.4 ± 0.5 5.9 ± 0.5 4.4 ± 0.6 ** −0.5 ± 0.6 Data are mean ± SEM or %. BCAA, branched‐chain amino acids; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; n‐3 FA, omega 3 fatty acids; n‐6 FA, omega 6 fatty acids; BMC, bone mineral content; SMM, skeletal muscle mass; FM, fat mass; QMS, quadriceps muscle strength; CET, cycle endurance time; 6MWD, 6 min walking distance; IMS, inspiratory muscle strength; PAL, physical activity level; HADS, Hospital anxiety and depression scale. * P  < 0.05. ** P  < 0.01. *** P  < 0.001. a Between‐group differences were compared by ANCOVA (taking pre‐treatment value as covariate, the 4 month post‐treatment value as response, and considering treatment as a factor in the statistical model). BODY.RESULTS.OUTCOME ASSESSMENT: Change in plasma status of supplemented nutrients is shown in Figure 2B. Compared with baseline, plasma levels of vitamin D, EPA, DHA, and total n‐3 FA significantly increased in NUTRITION (respectively, +26%, P < 0.001; +91%, P < 0.001; +27%, P = 0.001; and +38%, P < 0.001), while AA and total n‐6 FA significantly decreased (−10%, P = 0.012 and −5%, P < 0.001). In PLACEBO, DHA significantly decreased (−9%, P = 0.030), resulting in significant between‐group difference in favour of NUTRITION in plasma vitamin D (+24%, P = 0.004), EPA (+91%, P < 0.001), DHA (+31%, P < 0.001), and n‐3 FA (+42%, P < 0.001). Arachidonic acid and n‐6 FA were significantly lower in NUTRITION than PLACEBO (−1.2 ± 0.5 (−11%), P = 0.019; and −6%, P < 0.001). No differences were found in plasma BCAAs. Body composition results showed significantly increased body mass, skeletal muscle mass (SMM,) and FM in NUTRITION compared with baseline (Table 2). Also in PLACEBO, SMM increased significantly. After PR, NUTRITION showed significantly higher body mass and FM than PLACEBO. No between‐group differences were found in SMM. Assessment of lower limb muscle strength and exercise performance showed comparable improvements in quadriceps muscle strength and CET after PR (Figure 3). No differences were found in 6MWD within or between groups (Table 2). Figure 3Mean pre‐ and post‐ values of A: Lower limb muscle strength; B: Exercise performance. Light grey bars represent patients that received PLACEBO. Mid grey bars represent patients that received NUTRITION. * P < 0.05; **P < 0.01; *** P < 0.001. Inspiratory muscle strength improved significantly only in NUTRITION (Figure 4). After PR, daily steps decreased significantly in PLACEBO by 822.8 ± 283.8 steps/day (−18%; P = 0.005) but remained stable in NUTRITION (76.4 ± 385.1 steps/day, P = 0.767). This resulted in a significant between‐group difference in favour of NUTRITION. Assessment of mood showed significantly improved HADS depression score in NUTRITION but not in PLACEBO (Table 2). Figure 4Mean pre‐ and post‐ values of A: Respiratory muscle strength; B: Physical activity level. Light grey bars represent patients that received PLACEBO. Mid grey bars represent patients that received NUTRITION. * P < 0.05; **P < 0.01. BODY.DISCUSSION: The NUTRAIN‐trial showed that COPD patients with low muscle mass and moderate airflow obstruction respond well to high intensity exercise training. In this population, no additional effect of nutritional intervention was shown on lower limb muscle strength as primary outcome measure, but respiratory muscle strength (which was not a training target) improved in NUTRITION only. Between‐group differences favouring NUTRITION were shown in body weight, plasma nutrient status, and physical activity. Earlier research on nutritional rehabilitation has mainly focused on providing enough energy and protein to improve or maintain body weight and muscle mass. Recent research has shifted towards the quality of dietary protein in terms of protein sources and supplementation of specific anabolic amino acids. Casein and whey, both high‐quality proteins because of their high essential amino acid (EAA) content, are shown to comparably and efficiently stimulate muscle protein synthesis.13 Leucine is one of the BCAAs known to increase insulin secretion and influence molecular regulation of muscle protein synthesis (via the mTOR pathway), thereby stimulating anabolism and muscle protein synthesis, provided sufficient supply of essential amino acids (EAA) to the muscles.6 The current supplement therefore provided equal amounts of casein and whey (4.2 g) for a balanced amino acid profile, enriched with 1.8 g leucine per serving volume of 125 mL. Skeletal muscle mass increased after exercise training, but this response was not augmented by protein supplementation. Possibly, protein supplementation is mainly effective in subjects with a deficient state of protein. Indeed, patients with advanced COPD and muscle wasting are reported to have low plasma levels of BCAAs (especially leucine) compared with age‐matched controls,7, 14 which could reflect a deficient state in muscle,7 but no evidence for a deficient BCAA state was found in our NUTRAIN patients with less advanced COPD. In a sample of 88 patients with severe COPD (GOLD 3–4) and BMI <23 kg/m2 not receiving exercise training, 12 weeks of supplementation with an EAA (4 g/day) mixture high in leucine led to higher FFM and muscle strength compared with an isocaloric placebo.15 Nonetheless, these patients were characterized by lower protein intake at baseline (1.0 ± 0.2 g/kg) than the current study population (1.4 ± 0.1 g/kg BW). Constantin et al. 16 randomly allocated 59 COPD patients (FEV1: 46.9 ± 17.8%predicted) to receive 19 g protein and 49 g carbohydrate or placebo supplements during 8 weeks of resistance training. They found a strong response to the training component (lean mass and strength gain), but nutritional supplementation did not augment functional responses to resistance training. In line, we found a strong response to the exercise training in the present trial, reflected as significantly increased muscle mass, lower limb muscle strength, and CET in both groups. All in all, it might be concluded that the augmenting value of protein fortification on skeletal muscle mass and quadriceps muscle strength is minimal in COPD with less advanced airflow obstruction when habitual protein intake is already adequate. Moreover, previous literature has suggested that further improvements on top of the effects of exercise training via 'add‐on' modalities may be challenging to obtain. Particularly, muscle composition and function may have realistic ceilings in terms of the magnitude of expected improvement within a short time period.17 So‐called pharmaco‐nutrients have recently been proposed to enhance efficacy of nutritional supplementation on physical performance by targeting muscle metabolism.5 Poly‐unsaturated fatty acids are the natural ligands of peroxisome proliferator‐activated receptors (PPARs) and PPAR‐gamma coactivator (PGC)‐1α, which are involved in regulation of skeletal muscle morphology and oxidative metabolism. A decreased expression of these regulators is reported in the skeletal muscle of advanced COPD patients compared with healthy controls.18 Broekhuizen et al. 9 demonstrated that PUFA modulation during an 8 week inpatient rehabilitation program was able to improve exercise capacity (peak workload and CET) to a larger extent than placebo in patients with severe COPD independent of lower limb muscle mass and strength. In the current NUTRAIN‐trial peak workload was not assessed post‐PR, but no group differences were found in CET. However, a comparison between studies is limited because of differences in CET protocol. Furthermore, the population by Broekhuizen et al. consisted of patients with more advanced disease (FEV1 37.3 ± 13.8%predicted) and with severe exercise impairment (41.3 ± 19.3%predicted). The majority of the NUTRAIN patients were vitamin D insufficient/deficient (88.8%) providing a clear rationale for supplementation. In line with our study, a published post‐hoc subgroup analysis of 50 patients with COPD and vitamin D deficiency following a 3 month outpatient PR,19 reported a similar dissociation between improvements in peripheral and respiratory muscle strength after vitamin D supplementation. Patients receiving a monthly dose of 100.000 IU vitamin D had significantly larger improvements in inspiratory muscle strength and peak exercise tolerance, but not in quadriceps strength and 6 min walking distance (6MWD) compared with placebo. Median 25‐hydroxycholecalciferol level increased from 15 ng/mL (37.4 nmol/L) to 51 ng/mL (127.3 nmol/L), which exceeded the increase observed in the NUTRAIN trial (+25%), presumably by the 5–6 times higher administered dose. As respiratory muscle training was not part of the PR program in both trials, this might suggest a differential response of respiratory muscle following vitamin D and/or n‐3 fatty acids supplemented nutrients than peripheral muscle possibly related to maintenance of muscle oxidative phenotype in respiratory muscles.20 Exercise training significantly increased quadriceps muscle strength and CET in both groups. Cycle endurance time tended to increase to a smaller extent after supplementation, presumably because of a trend towards higher baseline value compared with the placebo group. Still, the increase in CET exceeded the proposed minimal clinically important difference of 100–105 s.21 No changes were observed in 6MWD in the current study, but average 6MWD did not seem rigorously impaired at baseline (almost 500 m) and also in the INTERCOM trial, investigating a comparable group of COPD patients with less advanced disease, 6MWD maintained similar in the intervention group but deteriorated in muscle wasted patients receiving usual care.11 That was also the reason why 6MWD was only included as exploratory outcome in this trial. Recently, a more active lifestyle, reflected by objective assessment via accelerometry, has been proposed as novel and clinically important outcome measure of PR. According to the sparse literature available, the effects of current PR programs in terms of physical activity level are heterogeneous, which could be related to differences in studied population, type, and intensity of the exercise training and incorporation of other interventions like nutritional or psychological interventions.22 The exercise training part of the NUTRAIN trial primarily focused on high intensity resistance and endurance exercises, but no standardized behavioural change intervention for physical activity was incorporated. Despite concomitant increase in exercise capacity, the number of steps/day decreased in the placebo group, indicating a compensatory response to recover from the intensive exercise training sessions in daily physical activity. The observed dissociation between physical performance and daily physical activity has previously been reported in studies evaluating the effect of PR on daily physical activity22 and suggests that changing physical activity behaviour requires a more comprehensive approach than only targeting exercise capacity.23 In contrast to the placebo group, no adaptive decrease in daily steps was shown by patients receiving nutritional supplementation. This could be related to specific nutrient effects on lower limb muscle metabolism resulting in decreased muscle fatigue or via the observed improvement in respiratory muscle function. Dal Negro et al. 15 reported increased number of steps after EAA supplementation in domiciliary severe COPD patients. Alternatively, a positive effect of n‐3 fatty acid enriched protein‐dense supplements could be implicated as in cachectic patients with pancreatic cancer n‐3 FA supplementation also increased physical activity level assessed by doubly labelled water independent of muscle mass.24 Next to exercise, n‐3 FA additionally stimulate the PGC‐1α‐PPARα/δ pathway in muscle resulting not only in enhanced muscle (fat) oxidative metabolism25 and decreased muscle fatigue, but also in enhanced conversion of peripheral kynurenine into kynurenic acid, which is unable to cross the blood–brain barrier.26 Reducing plasma kynurenine protects the brain by mediating resilience to stress induced depression. Some evidence for this hypothesis was provided in this trial by the HADS depression score, which improved significantly in NUTRITION but not in PLACEBO. Based on previous research, nutritional supplementation consisted of two to three portions of 125 mL/day, which in the current trial proved to be the maximal feasible dosage. Intention‐to‐treat analysis revealed an average supplement intake of 2.1 portions/day, accompanied by significantly increased percentages of EPA and DHA, which are suggested to be useful blood biomarkers for determining adherence in clinical studies because of the linear response to its intakes.27 No changes were found in habitual dietary intake after PR (table E2), suggesting that patients in the NUTRITION group did not compensate for NS. Patients in the placebo group also did not compensate by increasing their energy intake for increased energy costs caused by the intensive exercise training, possibly implying that energy costs were compensated during the remainder of the day by an adaptive decreased number of daily steps. While the increase in FM resulting from extra caloric support may be protective in COPD patients prone to weight loss, it could be argued that attention should shift from macro‐ to targeted nutrient supplementation in weight stable COPD patients with low muscle mass. This may likely also improve compliance to nutritional support in some patients. Based on the primary outcome measure the study was negative. There may have been a difference in muscle strength between the groups that was not detected as the study is potentially underpowered. It could also be argued that the studied population was too fit, because they had a moderate level of airflow obstruction, a mean 6MWD around 500 m, and a well above recommended daily protein intake. The criterion applied for muscle wasting (FFMI‐P25) was broader than defined by the ERS statement on nutritional assessment and therapy in COPD1 but derived from the IRAD‐2 trial. This trial showed favourable effects of 3 months of home rehabilitation combining health education, oral nutritional supplements, exercise and oral testosterone on body composition and exercise tolerance compared with usual care in muscle wasted patient with chronic respiratory failure.28 Nevertheless, to explore potential deviating intervention responses following the applied criteria for muscle wasting, we performed a post‐hoc subgroup analysis in which we compared outcomes between n = 44 patients with a FFMI < 10th percentile values (according to the ERS statement) and n = 37 a FFMI 10th–25th percentile values. ANCOVA did not show differences in any of the outcomes between the groups. Although the NUTRAIN trial did not reach the targeted sample size (n = 120) and was not designed to disentangle the mechanism behind the observed dissociation between physical performance and daily physical activity, it does provide new interesting leads for further research regarding the potential of nutritional modulation in COPD beyond skeletal muscle maintenance. Furthermore, adequate clinical trials in COPD are needed to explore nutritional strategies for maintaining the effects of exercise training after a pulmonary rehabilitation program. Data of the exploratory maintenance program of the NUTRAIN trial will be reported separately. In conclusion, this RCT shows that exercise training is successful in improving lower limb muscle strength and cycle exercise performance in COPD patients with low muscle mass, moderate airflow obstruction, and a sufficient protein intake. Additional specific nutritional supplementation had beneficial effects on nutritional status and inspiratory muscle strength and positively influenced physical activity. BODY.AUTHOR CONTRIBUTIONS: Each author has made substantial contributions to the current study. The contribution of the authors to the manuscript is as follows: CvdB: acquisition, analysis, and interpretation of data, drafting the manuscript. ER, AvH, FF, EW: design of the study, interpretation of data, revising the manuscript critically for important intellectual content. AS: design of the study, interpretation of data, drafting and revising the manuscript critically for important intellectual content. All authors gave final approval of the version submitted for publication and take accountability for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work were appropriately investigated and resolved. BODY.CONFLICT OF INTEREST: E.R., F.F., and E.W. declare that they have no conflict of interest regarding the present manuscript. A.vH. is employed by Nutricia Research. C.vdB. and A.S. report grants from Netherlands Lung Foundation and Nutricia Research, during the conduct of the study. A.S. is a member of International Scientific Advisory Board of Nutricia Advanced Medical Nutrition. BODY.SUPPORTING INFORMATION: Data S1. Table E1: Nutrient composition of NUTRITION and PLACEBO products per 125 mL serving. Table E2: Habitual dietary intake.Click here for additional data file.

TITLE: An Exploratory Dose-Escalating Study Investigating the Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of Intravenous Atacicept in Patients with Systemic Lupus Erythematosus ABSTRACT: Atacicept, a recombinant fusion protein containing the extracellular, ligand-binding portion of the transmembrane activator and calcium modulator and cyclophilin-ligand interactor receptor, and the Fc portion of human immunoglobulin (Ig) G, is designed to block the activity of B-lymphocyte stimulator and a proliferation-inducing ligand, and may have utility as a treatment for B-cellmediated diseases, such as systemic lupus erythematosus (SLE). This Phase Ib study investigated the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics of intravenous (i.v.) atacicept in patients with mild-to-moderate SLE. Patients (n = 24) were randomised (5:1) to receive atacicept (single dose: 3, 9 or 18 mg/kg; or multiple dose: 2 × 9 mg/kg) or matching placebo. Patients were followed for 6 weeks after dosing (9 weeks in the 2 × 9 mg/kg cohort). Local tolerability of atacicept was comparable with that of placebo, with only mild injection-site reactions reported with atacicept. Atacicept i.v. was generally well tolerated, both systemically and locally, in patients with mild-to-moderate SLE. Atacicept displayed non-linear PK, which was predictable across doses and between single and repeat doses. The biological activity of atacicept was demonstrated by its marked effect in reducing B-cells and Ig levels in patients with SLE. This supports the utility of this therapeutic approach in the treatment of autoimmune diseases, such as SLE. BODY.INTRODUCTION: Systemic lupus erythematosus (SLE) is a chronic, autoimmune disease that primarily affects women.1 The symptoms of SLE affect many different parts of the body, including the joints, skin, blood, brain and kidneys, and its effects can be fatal.1,2 Historically, hydroxychloroquine sulphate and corticosteroids, together with varying combinations of immunosuppressants, have been the main treatments for SLE.3 These therapies have been associated with poor safety profiles and/or low response rates, leading to a high unmet therapeutic need among patients with SLE.3,4 Recent developments in genetic and immunological techniques have advanced our understanding of the basic molecular and cellular processes underlying the pathogenesis of SLE.5–7 This has led to the development of a new generation of SLE therapies designed to target specific steps in the disease process.8–13 It is hoped that these new, targeted therapies, when compared with conventional SLE treatments, will have improved safety profiles and similar or improved efficacy. SLE is characterised by the production of anti-nuclear antibodies (ANA),2 which are thought to play a central role in the disease, targeting healthy tissues throughout the body. The origin of ANA is unclear, but may involve B-cell hyperresponsiveness.6 Abnormal T-cell behaviour, complement deficiencies and abnormal cytokine function are also thought to play a role in SLE.5–7 The involvement of B-cells in autoantibody production has lead to numerous investigations into B-cell receptor signalling in patients with SLE.14 B-lymphocyte stimulator (BLyS; also known as B-cell-activating factor of the tumour necrosis factor [TNF] family [BAFF]) and a proliferation-inducing ligand (APRIL) are two related members of the TNF ligand superfamily that both regulate B-cell maturation, function and survival.15–17 Experiments in animals and humans suggest a role for these molecules in SLE. Transgenic mice engineered to express high levels of BLyS exhibit an expanded peripheral B-cell compartment, with considerably increased numbers of mature B-cells.18,19 In addition, these mice show autoimmune-like manifestations, including high levels of immunoglobulins (Igs), rheumatoid factors and anti-DNA autoantibodies, proteinuria and glomerulonephritis, characteristic of SLE-like symptoms.18,19 Furthermore, increased levels of BLyS correlate with the onset and progression of disease in these mice.18 In humans, elevated levels of APRIL, BLyS and/or BLyS/APRIL heterotrimers have been detected both in patients with SLE20,21 and in patients with other autoimmune diseases.21–24 BLyS and APRIL share signalling through the transmembrane activator and calcium modulator and cyclophilin-ligand interactor (TACI) and B-cell maturation antigen (BCMA) receptors.25,26 In addition, BLyS has a high affinity for the BAFF receptor (BAFF-R),26,27 and APRIL can bind with low affinity to heparin-sulphate proteoglycans.28,29 Atacicept (formerly known as TACI-Ig) is a recombinant fusion protein containing the extracellular, ligand-binding portion of the TACI receptor and the Fc portion of human IgG.18 Atacicept binds to BLyS and APRIL, and inhibits their effects on B-cells. Experiments in mice have demonstrated that atacicept reduces mature B-cell counts and serum antibody levels in normal mice30 and delays disease progression in a mouse model of SLE.18 Overall, this evidence suggests that atacicept has potential utility in the treatment of SLE, and a clinical development programme to investigate the use of atacicept in SLE has been initiated. A Phase Ia study demonstrated that a single subcutaneous dose of atacicept (up to 630 mg) is well tolerated in healthy volunteers.31 Single and multiple doses of subcutaneous atacicept were well tolerated in exploratory studies, where biological effects in line with the proposed mechanism of action (MoA) of atacicept were observed in patients with rheumatoid arthritis.32 Here, we describe a Phase Ib study investigating the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of intravenous (i.v.) atacicept in patients with SLE. BODY.METHODS.STUDY DESIGN: We conducted a double-blind, randomised, placebo-controlled, sequential, dose-escalating, exploratory Phase Ib study (protocol number: 25842) at three centres in Russia. The primary objective of the study was to evaluate the safety, tolerability, PK and PD of single or multiple i.v. injections of atacicept in patients with mild-to-moderate SLE. The study protocol was approved by the local independent ethics committee or institutional review board at each centre, and the study was performed according to the principles of Good Clinical Practice and the Declaration of Helsinki (2000). All patients gave written informed consent prior to entry into the study. On enrolment, patients were assigned to four sequential cohorts (Figure 1). A different atacicept dose (single or multiple i.v. injection) was investigated in each cohort. Patients within each cohort were randomised 5:1 to atacicept or matching placebo and followed for 6 weeks after dosing (9 weeks in the 2 × 9 mg/kg cohort). Prior to each dose escalation, safety and tolerability data at 2 weeks post-dose were assessed by a safety review board. The dosing regimen and dose-escalation scheme were selected based on the design and results of the Phase I trial of atacicept in healthy volunteers.31 Figure 1Patient cohorts and dose-escalation scheme. i.v., intravenous; SRB, safety review board. BODY.METHODS.PATIENTS: We recruited patients aged 18–70 years, with a body mass index <40 kg/m2 and with a proven diagnosis of mild-to-moderate SLE. Patients were required to meet at least four of the American College of Rheumatology (ACR) criteria for SLE classification33 and have a score of 0–10 on the SLE Disease Activity Index (SELENA SLEDAI).34 Patients were excluded if they had received immunosuppressive drugs (e.g. azathioprine, cyclosporine, methotrexate, cyclophosphamide, mycophenolate mofetil, leflunomide) during the 8 weeks prior to the study, prednisone >20 mg/day, hydroxychloroquine >400 mg/day, non-steroidal anti-inflammatory drugs, a change in dose regimen during the 4 weeks prior to the study or previous therapy with other biological therapies. Patients were excluded if they had severe renal disorder, neurological symptoms suggestive of significant central nervous system involvement, significant impairment of liver function, active acute infective illness (reactivation of Epstein-Barr virus), chronic serious infectious disease (e.g. tuberculosis) or opportunistic infections. Patients were also excluded if they had positive serology results for hepatitis B surface antigen, hepatitis C virus or human immunodeficiency virus, a medical history of congestive heart failure or cancer, clinically significant abnormalities in haematological tests or clinically significant serious abnormalities on chest X-ray or electrocardiogram (ECG). Women who were pregnant or breastfeeding were excluded from the study. BODY.METHODS.ASSESSMENTS.SCHEDULED VISITS: For single-dose cohorts (Cohorts 1–3), scheduled visits took place on days 2, 3, 4, 8, 15, 22 and 29, with a post-study visit on day 43. For the repeat-dose cohort (Cohort 4), scheduled visits took place on days 2, 3, 4, 8, 21, 29, 36, 43 and 57, with a post-study visit on day 64. BODY.METHODS.ASSESSMENTS.PK/PD ASSESSMENTS: In Cohorts 1–3, serum samples for PK/PD measurements were taken before dosing, 15 minutes, 30 minutes and 4 hours post-dose, and at each subsequent scheduled visit thereafter. In Cohort 4, serum samples for PK/PD measurements were taken before dosing, 30 minutes and 4 hours post-dose, and at each subsequent scheduled visit thereafter (with the exception of the visit on day 36). Patients in Cohorts 3 and 4 also returned on days 84 and 120 for PK/PD sampling. PK was assessed by measuring serum levels of free atacicept, atacicept•BLyS complex and total atacicept (defined as free atacicept + atacicept•BLyS complex) quantified using enzyme-linked immunosorbent assay. Serum was incubated with a biotin-conjugated monoclonal antibody specific for atacicept (free or total atacicept detection) or BLyS (atacicept•BLyS complex detection) and immobilised on a streptavidin-coated microplate. After washing, an atacicept-specific monoclonal antibody conjugated to horseradish peroxidase (HRP) was added. For detection of total atacicept, a BLyS-specific monoclonal antibody conjugated to HRP was also added at this stage. In all three assays, atacicept species were detected and quantified using standard chemiluminescence methods. PD was assessed by measuring serum levels of Igs (IgG, IgM, IgA) and lymphoid cell populations in peripheral blood. Igs were measured using standard methods. Four-colour immunophenotyping, using standard flow cytometry methods, was used to identify the following cell types: total T-cells (CD3+, CD45+), T-helper cells (CD8−, CD3+, CD4+, CD45+), T-cytotoxic/suppressor cells (CD4−, CD3+, CD8+, CD45+), total B-cells (CD19+), mature B-cells (CD19+, CD27−, IgD+), naïve B-cells (CD38−, CD27−, CD19+, CD20+), memory B-cells (CD38−, CD19+, CD20+, CD27+), monocytes (CD14+, CD45+) and natural killer (NK) cells (CD3−, CD45+, CD56+). Peripheral blood was collected from patients at the clinical sites and transported to a contract research organisation within 24 hours. Whole blood was incubated with appropriate dilutions of directly labelled fluorescent antibodies and red blood cells were lysed using a standard celllysing reagent. Counting beads were used to determine total T- and B-cell concentrations. Approximately 100,000 events were collected and analysed using flow cytometry analysis software. For data analysis, a lymphocyte gate was used for T- and NK-cell determinations, and a lymphocyte and monocyte gate was used for B-cell subset determinations. Levels of complement-3 (C3) and ANA, including anti-dsDNA antibodies, were measured as markers of SLE disease activity. C3 levels were measured using standard methods, and ANA were measured using the AtheNA Multi Lyte® ANA test system (Zeus Scientific Inc., Raritan, NJ, USA). Pre-dose BLyS serum concentrations were also measured during this study. BODY.METHODS.ASSESSMENTS.SAFETY AND TOLERABILITY: A physical examination, including measurement of vital signs, was conducted at screening, before and 4 hours after dosing, and at each subsequent visit. For Cohorts 1–3, standard 12-lead resting ECGs were performed at screening, before dosing on day 1 and on days 2, 15, 29 and 43. For Cohort 4, ECGs were performed at screening, before dosing on day 1, on day 2, before and 30 minutes post-dose on day 21 and on days 36 and 64. The incidence, severity and relationship to treatment of adverse events (AEs) were assessed throughout the study. The incidence of any injection-site pain, itching, tenderness, swelling, redness or bruising were assessed regularly by the investigator (any other local reactions were also noted). Pain was patient-graded using a 10-cm visual analogue scale. Concomitant medication usage was recorded throughout the study. Laboratory analyses (haematology, blood chemistry, coagulation, urinalysis) were conducted at the screening visit, prior to dosing on day 1, and on days 2, 8, 15, 22, 29 and 43 in Cohorts 1–3, and at the screening visit, prior to dosing on day 1, on days 2 and 8, before and 30 minutes post-dose on day 21, and on days 36 and 64 in Cohort 4. Vaccine immunisation status was assessed by measuring tetanus toxoid antibody titres on day 1 and at the final post-study visit (patients in Cohort 4 were also assessed on day 29). Assays for binding and neutralising antibodies to atacicept were performed on samples taken on day 1 and at the final post-study visit; patients in Cohort 4 also had an assessment 30 minutes post-dose on day 21. BODY.METHODS.STATISTICAL ANALYSES: Due to the exploratory nature of this study, sample size determination was not based on any formal statistical assumptions. Six patients were to be included at each dose level, with five receiving active medication and one receiving placebo. This number of patients was considered sufficient to allow an initial assessment of the systemic and local tolerability of atacicept and to allow a description of its PK and PD properties. All analyses were performed on the safety analysis set (all patients who received at least one dose of study medication) using an as-treated approach. Results were summarised for the combined placebo group, for atacicept recipients in each cohort and for the overall trial population. Continuous variables were tabulated using summary statistics (number of observed values, number of missing values, mean, standard deviation, median, minimum and maximum). Categorical variables were summarised using frequencies and percentages. AEs were coded using the Medical Dictionary for Regulatory Activities (MedDRA, version 8.0), and treatment-emergent AEs (those with onset between the first treatment injection and the post-study follow-up visit) were summarised by System Organ Class and Preferred Term. Concomitant medications were coded using WHO-Drug (March 2006) by ATCtext4 and ATCtext2. ATCtext2 was used in place of ATCtext4 for medications for which no ATCtext4 classification was given. Concentration—time profiles for PK data were subjected to non-compartmental analysis using WinNon-Lin software (version 5.0.1), and the resulting values were summarised statistically. BODY.RESULTS.PATIENTS: Of the 32 patients who were screened, 24 patients were enrolled in the study and randomised into four cohorts (six patients per cohort). All 24 patients completed the trial. Baseline demographic and clinical characteristics were well balanced between groups (Table 1). All patients were White, the vast majority were female (92%) and the median age was 45.5 years. Median disease duration was 6.0 years, and patients generally showed characteristics of mild-to-moderate SLE, with signs and symptoms affecting a wide range of organ systems. Overall, all patients (100%) satisfied at least four of the 11 ACR criteria for SLE; 38% of patients satisfied four ACR criteria, 42% satisfied five criteria, 13% satisfied six criteria and 8% satisfied seven criteria. Baseline SLE clinical manifestations based on the ACR criteria included: arthritis (91.7%), photosensitivity (66.7%), malar rash (58.3%), oral ulcers (16.7%), serositis (12.5%), haematological manifestations (33.3%), discoid lupus (41.7%) and renal involvement (25.0%). In line with the trial entry criteria, no patients had neurological disorders at baseline. Table 1 Baseline demographic and clinical characteristics of patients involved in the study Placebo ( n = 4) Atacicept 3 mg/kg ( n = 5) Atacicept 9 mg/kg ( n = 5) Atacicept 18 mg/kg ( n = 5) Atacicept 2 × 9 mg/kg ( n = 5) All ( n = 24) Age, years 44.0 (21–54) 54.0 (25–66) 45.0 (32–64) 41.0 (34–60) 47.0 (36–55) 45.5 (21–66) Sex, n (%) female 4 (100) 4 (80) 5 (100) 4 (80) 5 (100) 22 (92) BMI, kg/m 2 24.63 (20.7–35.2) 27.53 (22.9–39.6) 24.09 (17.7–31.2) 29.45 (24.0–36.0) 25.46 (20.1–32.8) 26.57 (17.7–39.6) Number of ACR 33 criteria met    4 1 1 3 2 2 9    5 3 4 0 1 2 10    6 0 0 1 1 1 3    7 0 0 1 1 0 2 Disease duration, years 4.5 (3–27) 7.0 (4–33) 5.0 (4–12) 14.0 (3–20) 14.0 (5–28) 6.0 (3–33) SELENA SLEDAI score 34 0.00 (0.0–4.0) 0.00 (0.0–4.0) 0.00 (0.0–1.0) 0.00 (0.0–3.0) 0.00 (0.0–4.0) 0.00 (0.0–4.0) Abbreviations: ACR: American College of Rheumatology; BMI: body mass index; SELENA SLEDAI: systemic lupus erythematosus Disease Activity Index. All data are presented as median (range, min—max) unless specified otherwise. A total of 71% of patients were receiving corticosteroids and 58% of patients were receiving hydroxychloroquine or chloroquine. No use of other immunosuppressive agents was reported during the trial. BODY.RESULTS.PATIENTS.SAFETY: Total cumulative exposure ranged from 182 to 1735 mg per patient in the atacicept groups, and the median cumulative dose reflected the assigned dose level in each cohort (3 mg/kg: 201 mg; 9 mg/kg: 612 mg; 18 mg/kg: 1440 mg; 2 × 9 mg/kg: 1278 mg). During the study, 15 treatment-emergent AEs were reported in 10 patients (Table 2). All AEs were mild (12 events) or moderate (three events) in severity. AEs were reported most frequently in the System Organ Class of infections and infestations (four events in four patients). All infection-related events were considered to be unrelated to trial medication. Of the 15 AEs that were reported, four were considered possibly or probably related to study medication: three reports of decreased white blood cell (WBC) count and one report of pyrexia. The decreased WBC counts occurred in two patients who received 2 × 9 mg/kg atacicept, both of whom had low WBC counts before dosing on study day 1. WBC counts had recovered by the end of the study. There were no infections or other AEs reported for these two patients. All three AEs reported in the placebo group occurred in the same patient. One serious event was reported, which was considered unrelated to trial medication: a subcutaneous abscess of the ear in a patient with a long-standing history of an ear cyst. The abscess resolved following surgical and antibiotic treatments. No deaths were reported, and no AEs led to treatment or study discontinuation. Table 2 Treatment-emergent adverse events reported during the study (the observation period was 6 weeks for single-dose cohorts and 9 weeks for the repeat-dose cohort) Patient Event a (events) , n Placebo ( n = 4) Atacicept 3 mg/kg ( n = 5) Atacicept 9 mg/kg ( n = 5) Atacicept 18 mg/kg ( n = 5) 2 × 9 mg/kg Atacicept ( n = 5) All ( n = 24) Total 1 (3) 3 (5) 1 (1) 1 (1) 4 (5) 10 (15) Virus respiratory tract infection 2(2) 2 (2) Subcutaneous abscess 1 (1) 1 (1) Upper respiratory tract infection 1(1) 1 (1) WBC count decreased 2 (3) 2 (3) Constipation 1 (1) 1 (1) Gastrointestinal motility disorder 1 (1) 1 (1) Gastroesophageal reflux disease 1 (1) 1 (1) Blepharitis allergic 1 (1) 1 (1) Conjunctivitis allergic 1 (1) 1 (1) Pyrexia 1 (1) 1 (1) Headache 1 (1) 1 (1) Eczema 1 (1) 1 (1) Abbreviations: MedDRA: Medical Dictionary for Regulatory Activities; WBC: white blood cell. a Events listed by MedDRA-preferred term. There were no notable changes over time or differences between groups for vital signs, ECGs or laboratory values. No patient developed antibodies to atacicept, and atacicept treatment did not affect vaccine immunisation status. BODY.RESULTS.PATIENTS.LOCAL TOLERABILITY: Of the patients who received atacicept, seven experienced local reactions: redness (four patients), tenderness (three patients), bruising (one patient) and swelling (one patient). All local reactions in the atacicept group were mild in severity. Local injection-site reactions were reported in two patients who received placebo: mild tenderness (two patients), moderate bruising (one patient) and mild redness (one patient). Local injection-site reactions in all patients resolved within 4 hours after injection. No notable difference in the incidence of local injection-site reactions was seen between atacicept dose groups. Pain scores were low in all groups: the maximum pain score was 15 (out of a possible 100) in the placebo group and 8 in the atacicept groups. In all but two cases, injection-site pain resolved within 30 minutes after injection. BODY.RESULTS.PK: Maximum free atacicept concentrations were achieved at a median of 0.25–0.5 hours post-dose in all cohorts, corresponding to the first two PK sampling points (Table 3 and Figure 2). Free and total atacicept concentration—time profiles displayed multi-phasic behaviour. This comprised at least two distribution phases, which occurred within 14 days post-dose, and a prolonged terminal phase characterised by median terminal half-lives of 642–765 hours for free atacicept (27–32 days) and 1100–2050 hours for total atacicept (46–85 days). The distribution phases appear to represent both the distribution of atacicept across tissues and binding of the drug to its ligands. Figure 2Free atacicept serum concentration versus time profiles after administration of (A) a single intravenous dose or (B) multiple intravenous doses of atacicept. Table 3 Pharmacokinetic values a for free atacicept in patients who received active treatment Atacicept 3 mg/kg ( n = 5) Atacicept 9 mg/kg ( n = 5) Atacicept 18 mg/kg ( n = 5) Atacicept 2 × 9 mg/kg first dose ( n = 5) Atacicept 2 × 9 mg/kg second dose ( n = 5) t 1/2 , h 642 (496–1000) 765 (489–880) 702 (638–1050) 102 (85.7–121) 710 (659–847) t max , h 0.50 (0.50–0.50) 0.25 (0.25–0.50) 0.50 (0.25–0.50) 0.50 (0.25–4.0) 504.25 b (504.25–504.25) C max , mg/L 38.6 (32.4–46.4) 91.7 (70.7–641) 257 (214–448) 148 (104–160) 118 (83.2–132) AUC inf , mg h/L 953 (609–1130) 1770 (1450–2940) 4880 (4200–6090) 2650 (2580–3080) 4700 (2820–5170) CL, L/h 0.282 (0.201–0.315) 0.319 (0.229–0.421) 0.287 (0.237–0.316) 0.223 (0.177–0.338) — Abbreviations: AUC inf : area under the concentration—time curve extrapolated to infinity; CL: clearance; C max : peak plasma concentrations; t max :time to peak concentrations; t 1/2 elimination half-life. a Data are presented as median (range). b Last dose administered at 504 hours. Serum levels of atacicept•BLyS complex showed a saturated (less than dose-proportional) increase across the doses studied (Figure 3). The evidence of non-linearity was weaker for free and total atacicept. The latter can be attributed to the diminishing influence of the binding non-linearity with the dose escalation. Despite the existing non-linearities, the PK of atacicept was very consistent and predictable across the doses and between single and repeat doses for all three variables. Figure 3B-lymphocyte stimulator (BLyS) • atacicept complex serum concentration versus time profiles after administration of (A) a single intravenous dose or (B) multiple intravenous doses of atacicept. The study design, involving a maximum of two doses administered over a 3 -week interval, did not permit any conclusions to be made about accumulation of atacicept in the body. BODY.RESULTS.PD: Atacicept induced an initial transient increase followed by a dose-dependent reduction in mature and total B-cells (Figure 4A and B). The greatest reductions were seen in mature B-cells 6 weeks after a single dose in the 9 mg/kg group (40% reduction from baseline) and 7 weeks after the repeated dose in the 2 × 9 mg/kg group (55% reduction from baseline). Recovery of total and mature B-cell counts from the reduction phase was not seen during the relatively short course of the study. An initial transient increase was also observed in memory and naïve B-cells, with a subsequent recovery toward baseline. No notable drug-dependent changes were observed for T-cells (total, helper or cytotoxic), NK-cells or monocytes. Figure 4Median percentage change from baseline in (A) mature B-cell counts for single doses, (B) mature B-cell counts for a repeat dose, (C) immunoglobulin M (IgM) levels for single doses and (D) IgM, IgA and IgG levels after repeat doses, after intravenous administration of atacicept. Ig levels decreased rapidly after atacicept dosing, with the greatest reductions seen for IgM (Figure 4C and D). The largest effect was seen in the 2 × 9mg/kg group, which showed a median decrease frombaseline of ∼40% for IgM 6 weeks after the last dose (33% decrease in IgA, 20% decrease in IgG). In some treatment groups, decreases in IgM and IgA were preceded by transient increases of 5–10% from baseline. Comparison of results from the 18 mg/kg and 2 × 9 mg/kg groups at 4 weeks post-dose suggested a greater response with administration of the same total dose as two injections. C3 levels remained within the normal range throughout the study for most patients. At baseline, only three of 24 patients had C3 values below the normal range (0.9–1.8 g/L): one patient in the 9 mg/kg group, one patient in the 18 mg kg group and one patient in the 2 × 9 mg/kg group. All three of these patients showed transient increases in C3 to normal levels during treatment. Anti-dsDNA antibody titres were negative at baseline for all except one patient. Therefore, no conclusions can be drawn about the effects of atacicept i.v. on autoantibody levels. BODY.DISCUSSION: We conducted a study to investigate the safety of single and multiple i.v. injections of atacicept in patients with SLE. Results showed that atacicept was well tolerated by patients with SLE, both systemically and locally, at a total dose of up to 18 mg/kg (as either a single dose or 2 × 9 mg/kg injections). Few AEs were observed and they were mild or moderate in severity. Decreased WBC counts occurred in two patients from the 2 × 9 mg/kg atacicept dose cohort. However, both patients had low WBC counts at baseline, and no significant decreases in WBC count were observed in the other patients in the study. Nevertheless, the number of patients is too small to draw any conclusions with respect to the use of multiple i.v. doses of atacicept in patients with pre-existing leucopenia. Local tolerability of atacicept was good with only transient, mild injection-site reactions reported. Overall, our results confirmed the favourable safety profile of atacicept that was observed previously with subcutaneous administration in healthy volunteers and in patients with rheumatoid arthritis.31,32 Notably, there did not appear to be any safety concerns associated with concomitant administration of atacicept and medications commonly used in the management of SLE. The PK profile of atacicept was non-linear but was predictable across doses and between single and repeat doses. Maximum free atacicept concentrations were achieved at a median of 0.25–0.5 hours post-dose. The slight delay in the maximum (relative to the instantaneous input assumption related to i.v. administration) is probably due to initial mixing into the central compartment. Treatment with atacicept resulted in a reduction of B-cells and Ig levels in patients with SLE indicating biological activity consistent with its MoA. Specifically, reductions in mature B-cell counts and decreases in Ig levels are consistent with inhibition of BLyS and APRIL activity. Overall, PD effects were most marked in the 2 × 9 mg/kg group. This group showed a 55% reduction from baseline in mature B-cells 7 weeks after the second dose. In this study, we also observed marked reductions in IgM (–40%), IgA (–33%) and IgG (–20%) in the 2 × 9 mg/kg group 6 weeks after repeat dosing. Taken together, these data show that atacicept has a potent effect on B-cells and Ig levels in patients with SLE. These findings are consistent with those of a recent study of subcutaneously administered atacicept in patients with SLE that demonstrated sustained reductions in total and mature B-cells and in Ig levels at all doses, which were particularly marked in the repeat-dose groups.35 This supports the utility of this therapeutic approach in the treatment of autoimmune diseases such as SLE. Atacicept is one of a number of agents designed specifically to target B-cells in patients with SLE.9,13 Rituximab, epratuzumab, atacicept and belimumab are all at various stages of clinical development for SLE. All of these agents bind to B-cell surface molecules, but affect B-cell activity in different ways. The monoclonal antibodies rituximab and epratuzumab bind to CD20 and CD22 cell-surface antigens, respectively, causing B-cell death,36,37 whereas atacicept and belimumab (anti-BLyS monoclonal antibodies) prevent ligands from binding to cell-surface receptors, inhibiting B-cell growth and development, and reducing Ig production.30,38 These differential MoAs are likely to result in different benefit-to-risk profiles for each agent. For example, the ability of atacicept to bind to both BLyS and APRIL may lead to a greater efficacy compared with belimumab, which binds only to BLyS.38 Safety profiles may also differ according to the mechanism by which B-cell activity is reduced. While rituximab and epratuzumab may increase the risk of AEs associated with B-cell depletion, this may not be the case for atacicept and belimumab, which reduce B-cell activity but have a less profound effect on B-cell numbers. With this in mind, it is encouraging that none of the infection-related events we report here were considered to be related to atacicept and that atacicept did not compromise vaccine immunisation status. In conclusion, i.v. atacicept was well tolerated, both systemically and locally, in patients with mild-to-moderate SLE at the doses investigated and demonstrated clear biological activity consistent with its predicted MoA. PK and PD results from this study will aid dose selection in larger studies that will further investigate the safety and efficacy of atacicept in patients with SLE.

TITLE: Real-Time fMRI Neurofeedback Training of Amygdala Activity in Patients with Major Depressive Disorder ABSTRACT.BACKGROUND: Amygdala hemodynamic responses to positive stimuli are attenuated in major depressive disorder (MDD), and normalize with remission. Real-time functional MRI neurofeedback (rtfMRI-nf) offers a non-invasive method to modulate this regional activity. We examined whether depressed participants can use rtfMRI-nf to enhance amygdala responses to positive autobiographical memories, and whether this ability alters symptom severity. ABSTRACT.METHODS: Unmedicated MDD subjects were assigned to receive rtfMRI-nf from either left amygdala (LA; experimental group, n = 14) or the horizontal segment of the intraparietal sulcus (HIPS; control group, n = 7) and instructed to contemplate happy autobiographical memories (AMs) to raise the level of a bar representing the hemodynamic signal from the target region to a target level. This 40s Happy condition alternated with 40s blocks of rest and counting backwards. A final Transfer run without neurofeedback information was included. ABSTRACT.RESULTS: Participants in the experimental group upregulated their amygdala responses during positive AM recall. Significant pre-post scan decreases in anxiety ratings and increases in happiness ratings were evident in the experimental versus control group. A whole brain analysis showed that during the transfer run, participants in the experimental group had increased activity compared to the control group in left superior temporal gyrus and temporal polar cortex, and right thalamus. ABSTRACT.CONCLUSIONS: Using rtfMRI-nf from the left amygdala during recall of positive AMs, depressed subjects were able to self-regulate their amygdala response, resulting in improved mood. Results from this proof-of-concept study suggest that rtfMRI-nf training with positive AM recall holds potential as a novel therapeutic approach in the treatment of depression. BODY.INTRODUCTION: Major Depressive Disorder (MDD) is a disabling and common medical condition[1]. Approximately two-thirds of patients who seek pharmacological and/or psychological interventions will not respond fully to treatment, and only one-half of treatment-responders achieve sustained remission[2]. Cognitive-behavioral therapy (CBT), the most commonly implemented psychological treatment for MDD, is most effective for mildly-to-moderately depressed patients[3], but is generally insufficient as monotherapy for severely ill patients[4]. Treatments available for severely ill patients who don't respond to multiple conventional treatments such as psychotherapy, pharmacotherapy, and/or a combination of the two include electroconvulsive therapy, vagus nerve stimulation, and deep brain stimulation, which are invasive and associated with significant adverse event risks[5], [6]. Therefore, substantial need exists to investigate novel therapeutic approaches for MDD that can improve the effectiveness of non-invasive treatments. Real-time functional magnetic resonance imaging (rtfMRI), in which blood oxygen-level-dependent (BOLD) fMRI data processing and display are performed concomitantly with image acquisition[7], has enabled rtfMRI neurofeedback (rtfMRI-nf) training, allowing a person to see and regulate the fMRI signal from their own brain[8]. Contrary to other biofeedback methods (such as EEG), rtfMRI-nf training results in the precise localization and modulation of relevant brain structures, allowing focal investigation of relationships between cognitive-behavioral functions and neuroplasticity changes[9], [10]. By using rtfMRI-nf, healthy individuals can learn to self-regulate brain activity in structures relevant to emotional processing including the insula, amygdala, ventrolateral prefrontal cortex (VLPFC), and anterior cingulate cortex (ACC)[11]–[15]. Emerging evidence also suggests rtfMRI-nf has clinical utility in reducing the symptoms of chronic pain[16], tinnitus[17], and Parkinson's disease[18]. Furthermore, a recent study in depressed men found the ability to up-regulate activity in various emotion-related brain regions though rtfMRI-nf was associated with clinical improvement[19]. It is important to note, however, that these clinical studies were pilot studies with relatively small samples (on the order of 5–12 patients) and replication and randomized clinical trials are needed to draw definitive conclusions regarding the clinical utility of neurofeedback procedures. The current rtfMRI-nf study targets a brain region critically involved in both emotional processing and the pathophysiology of MDD: the left amygdala (LA). Studies show amygdala BOLD activity increases in response to both positive and negative emotional stimuli in healthy humans[20]–[22]. A functional dissociation between left and right amygdala has been proposed such that the right is engaged in rapid/automatic detection of emotional stimuli, while the left is involved in detailed and elaborate stimulus evaluation[21], [23]. While abundant evidence suggests LA hemodynamic responses to negative stimuli are exaggerated in MDD[24]–[26], extant evidence further suggests MDD-associated amygdala abnormalities are "doubly dissociated" from healthy individuals by virtue of showing a greater response to negative stimuli and an attenuated response to positive stimuli [20], [27]. Furthermore, amygdala responsiveness to positive stimuli is inversely correlated with depression severity[27], and this response increases following successful antidepressant pharmacotherapy[20] or Cognitive Control Therapy[28]. These findings suggest that altered amygdala activation to positive stimuli is clinically relevant and that some antidepressant drugs and cognitive therapies may exert their therapeutic effect by normalizing this emotional processing bias[29]. The current study aimed to determine whether depressed individuals are able to use rtfMRI-nf to enhance the amygdala hemodynamic response to positive autobiographical memories, and whether this ability alters mood. Specifically, we predicted MDD subjects receiving rtfMRI-nf regarding left amygdala activity would demonstrate greater activity in this region while contemplating positive autobiographical memories (AMs) compared to those who received rtfMRI-nf from a region putatively not involved in emotional processing. Furthermore, we predicted significant improvements in mood-ratings would be evident in the experimental relative to the control rtfMRI-nf group. BODY.METHODS.SUBJECTS: The study was conducted at the Laureate Institute for Brain Research. The research protocol was approved by the Western Institutional Review Board. Human research in this study was conducted according to the principles expressed in Declaration of Helsinki. All subjects gave written informed consent to participate in the study and received financial compensation. Because it employed an experimental rather than clinical trial design, the study was not registered in a public trials database. Twenty-three right-handed, unmedicated adults ages 18–55 who met the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR)[30]_ENREF_28 criteria for MDD in a current major depressive episode participated in the study. Volunteers, recruited from the community via advertisements, underwent screening evaluations at the Laureate Institute for Brain Research, including the Structural Clinical Interview for DSM-IV disorders[31]. Exclusion criteria included current pregnancy, general MRI exclusions, serious suicidal ideation, psychosis, major medical or neurological disorders, exposure to any medication likely to influence cerebral function or blood flow within three weeks (8 weeks for fluoxetine), and meeting DSM-IV criteria for drug/alcohol abuse within the previous one year or for alcohol/drug dependence (excepting nicotine) within the lifetime. All volunteers were naïve to rtfMRI neurofeedback. BODY.METHODS.EXPERIMENTAL PARADIGM: The experimental paradigm is based on work previously published within our laboratory using healthy control subjects [14], and a task outline is depicted in Figure 1. 10.1371/journal.pone.0088785.g001Figure 1Design of the rtfMRI neurofeedback experiment. A) Regions of Interest (ROI) for the rtfMRI neurofeedback procedure. Three regions of interest (spheres of 7 mm radius) were used to assess changes in BOLD activity. These regions were the left amygdala (LA, red, centered at -21, -5, -16), right amygdala (RA, yellow, centered at 21, -5, -16), and left horizontal segment of the intraparietal sulcus (HIPS, green, centered at -42, -48, 48). ROI placements are illustrated on T1-weighted coronal (upper row) and axial (lower row) human brain sections in Talairach space[48]. Following radiological notation, the left side (L) of the brain is shown on the right, and the right side (R) of the brain on the left. B) Real-time display screen for the rtfMRI neurofeedback procedure. During the Happy condition, the word "Happy," two color bars, and a number indicating the neurofeedback signal were displayed on the screen. Participants were instructed to recall happy autobiographical memories to make themselves feel happy while trying to increase the level of the red bar representing the feedback signal from the target ROI to a given target level indicated by the fixed height of the blue bar (but not to exceed that target level). C) Protocol for the rtfMRI neurofeedback experiment. The experimental protocol consisted of seven runs each lasting 8 min 40 sec. During the Rest runs, participants were instructed to rest with their eyes open. During the Practice run, the participants were given the opportunity to become comfortable with the procedure and test out different memories. During Runs 1–3 participants underwent rtfMRI neurofeedback training consisting of alternating blocs of Rest (R, pink block), Happy (H, red block), and Count (C, green block, instructed to count backwards from 300 by a given integer), each lasting 40 sec. During the Transfer Run, participants were instructed to perform the same task as during the neurofeedback training, but no neurofeedback information (bars, numbers) was provided. Participants were informed that they would be assigned to receive neurofeedback from one of two brain regions; one region involved in emotional processing or another region that is independent of emotional processing and which may be difficult to regulate. Participants were instructed to retrieve positive AMs that potentially would help them control the level of activity in the target brain region. The strategy of positive AM retrieval was selected based on findings of amygdala activity (with other medial temporal regions) during AM retrieval[32], and is commonly reported by participants post-hoc as an effective strategy in neurofeedback studies targeting emotional processing brain regions[12], [19], [33]. Because depressed individuals are impaired at recalling specific and positive AMs[34], [35], each participant was interviewed prior to scanning to facilitate their AM recall and ensure five highly arousing and vivid, specific, and happy AMs could be evoked during rtfMRI-nf. Participants were instructed to recall those or other happy AMs while attempting to increase the hemodynamic activity in the assigned ROI to that of the blue bar representing the target level of activation, but not to exceed that level. The decision to include the instruction not to exceed the target level was based on post-scan interviews with the first few participants who indicated that they tried so hard to get their amygdala level as high as possible during the Practice Run that they felt fatigued during the next training run. Therefore, we deemed the instruction not to exceed the target level necessary in order to avoid overexertion early in the task which could result in fatigue as the task progressed and the target level increased. They were informed to maintain this strategy even if they felt it was ineffective at raising their brain activity, though they could change the positive memories utilized or the aspects of the memories focused on. Each neurofeedback run consisted of three conditions: Happy Memories, Count, and Rest. For each condition, cues were presented on the screen using both text and color icons to indicate each condition. During the Happy Memory Condition (Figure 1b), the cue "Happy" and two color bars (red, blue) were displayed on the screen. The red bar represented the actual neurofeedback signal, which was updated continuously by changing the height of the bar either upwards or downward based on the corresponding level of BOLD activity. This neurofeedback signal was also indicated by a number shown above the red bar representing the percent signal change within the target region. During this condition, participants were instructed to retrieve and contemplate positive autobiographical memories while also attempting to increase the level of the red bar to the fixed target level displayed by the blue bar. Because the Happy Memories condition required memory recall and rumination on those memories could potentially not be stopped quickly[13], [36], two control conditions were implemented to distract participants' attention from contemplating positive memories and to dampen the activation of the emotion regulation network[37]. During the Count condition, the participants were shown the cue "Count" with the specific instruction to count backwards from 300 by subtracting a specified integer. This integer was 3, 4, 6, 7, and 9 for Practice, Run 1, Run 2, Run 3, and the Transfer run respectively. During the Rest condition, participants were presented with the cue "Rest" and were asked to relax and breath regularly while looking at the display screen. No bars were displayed during the Count and Rest conditions. The rtfMRI-nf procedure consisted of seven fMRI runs each lasting 8 minutes and 40 seconds (Fig 1c); a resting run, a practice run (PR), three training runs (R1-3), a final transfer run (TR) in which no neurofeedback information was provided, and a final resting run. During the Rest runs, a resting state paradigm was employed and participants were instructed to not think of anything in particular while fixating on the display screen. During the Practice run, participants were given an opportunity to become comfortable with the neurofeedback procedure. As with the training and transfer runs, the practice run consisted of alternating blocks of Rest (5 blocks lasting 40 seconds each), Count (4 blocks lasting 40 seconds each), and Happy (4 blocks lasting 40 seconds each). For the first three Happy Memory blocks participants were instructed to recall and contemplate the positive AMs prepared prior to the task with the experimenter, and then, for the last Happy condition block, to use the one memory that elevated their mood to the greatest extent. Thus, the Practice run allowed participants (i) to accommodate to the neurofeedback task, (ii) evaluate the emotional impact of the prepared happy memories within the experimental setting, and (iii) practice switching from one memory to another during neurofeedback training. During the subsequent 3 Training runs (Runs 1-3) the same alternating 40s blocks of Rest (5 blocks), Happy Memories (4 blocks), and Count (4 blocks) were presented. Participants were encouraged to try various other happy memories if the currently chosen one did not help them raise the red bar during neurofeedback training. Because our preliminary experiments indicated that the activation level of the left amygdala could be as high as a 2% BOLD signal change, the target level of the blue bar was set to 0.50%, 1.0%, 1.5% and 2.0% for PR, R1, R2, and R3, respectively. Finally, during the Transfer Run, the participants were instructed to perform the same task as during neurofeedback training, but rtfMRI-nf information was not provided for the Happy Memory blocks and the bars were not shown. The transfer run was performed to assess the transfer of the learned control and to check whether the training effect generalized to situations where no neurofeedback was available. Prior to the rtfMRI-nf session, participants completed the 20-item Toronto Alexithymia Scale (TAS-20)[38], the Emotional Contagion Scale (EC)[39], and the Snaith-Hamilton Pleasure Scale (SHAPS)[40]. These ratings scales were selected as they examine the ability of individuals to experience emotion. As the current study trains participants to use the experience of internally generated positive emotions to regulate their amygdala, individual who have difficulty experiencing or describing positive emotions may have difficulty with the current neurofeedback task, and therefore ratings on these scales may explain important individual differences in the ability to perform the current task. Additionally, these scales were selected in order to compare our results to that of [14], which used the same neurofeedback paradigm in healthy control participants and found correlations between the ability to regulate the amygdala and ratings on these self-report measures. Clinician-administered rating scales included the 21-item Hamilton Depression Rating Scale (HDRS)[41], the Montgomery-Asberg Depression Rating Scale (MADRS)[42], and the Hamilton Anxiety Rating Scale (HARS)[43]. Both prior to and immediately following the fMRI session, participants completed the Profile of Mood States (POMS)[44], State/Trait Anxiety Scale (STAI)[45], and Visual Analog Scale (VAS). For the VAS, participants indicated along a 10 point scale (0 being not at all and 10 being extremely) how Happy, Sad, Restless, Angry, Anxious, Alert, and Drowsy they felt at the time of rating. The primary outcome measures for assessing the antidepressant effect were the POMS depression and VAS happy subscales because of their sensitivity to rapid changes in emotional state. Results obtained using other scales were considered secondary outcome measures. Preliminary results of this study have been presented in abstract form at the Annual Meeting of the Organization for Human Brain Mapping[46], and the Annual Meeting of the American College of Neuropsychopharmacology[47]. BODY.METHODS.REGION OF INTEREST PLACEMENT: The rtfMRI-nf procedure was based on an MRI based region of interest (ROI) approach. Three ROIs were defined as spheres of 7 mm radius in the stereotaxic array of Talairach and Tournoux [48] and placed, respectively, in the left amygdala (LA: -21, -5, -16), the right amygdala (RA: 21, -5, -16) and the left horizontal segment of the intraparietal sulcus (HIPS: -42, -48, 48), as illustrated in Figure 1a. The neurofeedback signal was based on fMRI activation in the left amygdala ROI for participants in the experimental group and on the fMRI activation in the HIPS ROI for participants in the control group. Feedback was not given from the right amygdala ROI, rather this ROI was used in later analyses to determine laterality effects of our rtfMRI-nf procedure within the amygdala. The experimenter (KY) assigned participants on a 2∶1 ratio to either the experimental (LA rtfMRI-nf; n = 14) or control group (HIPS) rtfMRI-nf; n = 7) under double-blind conditions. The 2∶1 experimental/control ratio is commonly used in proof-of-concept rtfMRI-nf experiments[9], [12], [33]. Experimental procedures for both groups were identical, except control participants received rtfMRI-nf from a region putatively not involved in emotion regulation[49]–[51]. Upon completion of the study procedures, participants were informed as to which condition they were assigned, and participants in the HIPS rtfMRI-nf condition were offered the opportunity to return to the lab to repeat the rtfMRI-nf experiment with the amygdala as the target ROI. The selection of a control task for rtfMRI-nf experiments is challenging, and no consensus has yet been reached as to the optimal approach. Studies utilizing out of scanner control conditions,[19] control conditions in which the neurofeedback bar remains static,[33] or no control condition (examining only within subject changes),[52], [53] run the substantial risk of false positives as control participants know they are not receiving feedback, and experimenter blinding is impossible. Therefore improvements evident in the active relative to the control group may be due to experimenter bias or the appeal of a novel, technology-based intervention and not to gaining control over the target region. Control conditions using neurofeedback from a different region are best suited to determine a) specificity of the procedure; whether feedback from the target region is necessary for enhanced control of that region and b) whether changes in mood ratings are due to feedback from the target region or due to a placebo effect. Therefore, for our rtfMRI-nf protocol, we employed a control condition in which subjects received rtfMRI-nf from the HIPS, a region implicated in number and not in emotional processing.[49]–[51], [54] BODY.METHODS.DATA ACQUISITION: MR imaging was conducted at the Laureate Institute for Brain Research using a General Electric Discovery MR750 whole-body 3 Tesla MRI scanner (GE Healthcare, USA) equipped with a custom rtfMRI system[55]. A standard 8-channel receive-only head coil array was used. A single-shot gradient-recalled EPI sequence with Sensitivity Encoding (SENSE) was employed for fMRI. The following EPI imaging parameters were used: FOV/slice = 240/2.9 mm, axial slices per volume = 34, acquisition matrix = 96×96, repetition/echo time TR/TE = 2000/30 ms, SENSE acceleration factor R = 2 in the phase encoding (anterior-posterior) direction, flip angle = 90°, sampling bandwidth = 250 kHz, number of volumes = 263. Each functional scan time lasted 8 min 40 sec. Three EPI volumes (6 sec) were added at the beginning of each fMRI run to allow the fMRI signal to reach steady state, and were excluded from data analysis. The EPI images were reconstructed into a 128×128 matrix, in which the resulting fMRI voxel volume was 1.875×1.875×2.9mm3. Additionally, simultaneous physiological pulse oximetry and respiration waveform recordings were conducted (with 50 Hz sampling) for each fMRI run. A photoplethysmograph with an infra-red emitter placed under the pad of the subject's left index finger was used for pulse oximetry, and a pneumatic respiration belt was used for respiration measurements. A T1-weighted magnetization-prepared rapid gradient-echo (MPRAGE) sequence with SENSE was used to provide an anatomical reference for the fMRI analysis. It had the following parameters: FOV = 240 mm, axial slices per slab = 128, slice thickness = 1.2 mm, image matrix = 256×256, TR/TE = 5/1.9 ms, acceleration factor R = 2, flip angle = 10°, delay time TD = 1400 ms, inversion time TI = 725 ms, sampling band-width = 31.2 kHz, scan time = 4 min 58 sec. BODY.METHODS.IMAGING ANALYSIS: ON-LINE: The image data analyses were performed using Analysis of Functional NeuroImages (AFNI, http://afni.nimh.nih.gov/). The neurofeedback was implemented using the custom real- time fMRI system utilizing the real-time features of AFNI [56] and a custom developed graphic user interface (GUI) software. For each subject we acquired a high- resolution MPRAGE image and a short (10s) EPI scan prior to the neurofeedback procedure. The MPRAGE image was transformed to the Talairach space. The target ROIs (as defined above) were defined in Talairach space. They were first transformed to the original MPRAGE space, and then to the EPI space defined by a single EPI volume from the short EPI scan (for steady state). Thus, the target ROIs were defined in the EPI space. During the rtfMRI neurofeedback experiment, all acquired EPI volumes were volume-registered to the same single EPI volume. This way, the ROI masks in the EPI space were applied to all fMRI data in real time, and no Talairach transform during real-time processing was required. The resulting ROIs in the EPI space contained approximately 140 voxels each. In our neurofeedback implementation, the AFNI real-time plug-in was used to perform volume registration of EPI images and to export mean values of fMRI signals for the three ROIs in real time. The first three volumes of each experimental run were excluded to allow the fMRI signal to reach steady state. The rtfMRI signal for each Happy Memories condition was measured as a percent signal change relative to the baseline obtained by averaging the fMRI signal for the preceding 40-sec long Rest condition block. This neurofeedback signal (percent signal change) was updated every 2 sec and displayed on the screen as the red bar. To reduce bar fluctuations due to noise in the fMRI signal, the bar height was computed at every time point as a moving average of the current and two preceding fMRI percent signal change values. BODY.METHODS.IMAGING ANALYSIS: POST-SCAN OFF-LINE: Pre-processing of single-subject fMRI data included correction of cardiorespiratory artifacts using AFNI implementation of the RETROICOR method [57]. The cardiac and respiratory waveforms recorded simultaneously during each fMRI run were used to generate the cardiac and respiratory phase time series for the RETROICOR. Further fMRI pre-processing included volume registration and slice timing correction for all EPI volumes in a given exam. Standard GLM analysis was then applied separately for each of the fMRI runs. The following regressors were included in the GLM model: two block stimulus conditions (Happy Memories, Count), six motion parameters as nuisance covariates to take into account possible artifacts caused by head motion, and five polynomial terms for modeling the baseline. The stimulus conditions for all runs consisted of 40-second-long blocks. Hemodynamic response amplitudes were estimated using the standard regressors, constructed by convolving a boxcar function (representing the block duration) with the canonical hemodynamic response function using standard AFNI parameters. The GLM β coefficients were computed for each voxel using the 3dDeconvolve AFNI program and then converted to percent signal changes for Happy versus Rest, Count versus Rest, and Happy versus Count contrasts. The resulting fMRI percent signal change maps for each run were spatially transformed to the stereotaxic array of Talairach and Tournoux [48] and re-sampled to 2×2×2 mm3 isotropic voxel size. They were subsequently used for whole-brain statistical group analyses. The voxel-wise percent signal change data were also averaged within the three ROIs (LA, RA, HIPS) and used as a performance measure. To obtain performance measures during the RE run, the GLM analysis procedure used for the neurofeedback runs (PR,R1,R2,R3,TR) was applied to the RE run. The same condition blocks (Happy, Count) were employed, even though there were obviously no such conditions during the resting run. We performed this procedure for additional verification of the results. The % BOLD activity levels for such condition blocks should be close to zero for RE runs, which was indeed the case in our analysis. In preparation for the whole-brain statistical group analysis, the spatially-normalized fMRI percent signal change maps were spatially smoothed using a Gaussian kernel with full width at half maximum (FWHM) of 5 mm. For each group, statistical activation maps (t-tests comparing percent signal change to zero activation) were computed for Happy versus Rest, Count versus Rest, and Happy versus Count contrasts during TR. A group t-test comparing Happy versus Rest examined statistical differences between groups during TR. The significance criterion for detecting activation was set at p corrected<0.05 determined using the AFNI program 3dClustSim (cluster size>30 voxels, thresholded at voxel p<0.005). We expect activation detected during TR when no neurofeedback information was provided during happy AM recall reflects the regions involved in maintaining the elevated amygdala response to positive AM recall after completing rtfMRI-nf training. BODY.METHODS.BEHAVIORAL DATA ANALYSIS: Analysis of behavioral data was performed using SYSTAT 13 (Systat Software Inc., USA). The training effect was evaluated by applying a three-way (Training [PR, R1, R2, R3, TR] x ROI [LA, RA, HIPS] x Group [Experimental, Control]) ANOVA for percent signal change. Specificity of the training effect to the LA was evaluated (within each group) by applying a two-way (Training x ROI) ANOVA for percent signal change. Follow-up t-tests were performed to characterize significant differences underlying ANOVA main effects and interactions. Monotonic properties of participants' control over brain activation across all runs were evaluated (for each ROI and Group) by using a one-way ANOVA trend analysis for repeated measures on percent signal changes with Training (RE, PR, R1, R2, R3) as a within-subjects factor. Association between average percent signal changes for LA ROI and scores on self-report measures was determined using Pearson bivariate correlations. The threshold for significance was set at p<0.05, one-tailed corrected for multiple comparisons using the Bonferroni method. A one-tailed test was selected for this proof-of-concept study because our aprioi hypotheses were directional in that we expected LA rtfMRI-nf to increase mood and amygdala BOLD activity. BODY.RESULTS.BASELINE DEMOGRAPHIC AND CLINICAL DATA: At the pre-treatment baseline evaluation (Table 1) the groups did not differ on mean age, HDRS, MADRS, or HARS scores, time since last antidepressant medication, or length of current depressive episode (ts(19)<0.84, ps>0.21). A Fisher's exact test revealed no significant difference between groups in the proportion of females, major depressive episodes experienced (1, 2, or 3+), or number of antidepressant medications previously taken (0, 1–2, or 3+) (ps>0.26). There was a difference between groups in the proportion of comorbid diagnoses (p = 0.045) such that the experimental group had more participants without a co-morbid diagnosis than the control group. When we compared pre-scan ratings (Table 2) between groups, there was no significant difference on any rating (ts(19)<1.61, ps>0.13). 10.1371/journal.pone.0088785.t001 Table 1 Participant Characteristics by Experimental Group. Experimental [n = 14] Control [n = 7] Age 38 (10) 36 (9) % Female 79% [n = 11] 100% [n = 7] Clinical Ratings – mean (SD) MADRS 27.1 (6.69) 31.4 (6.71) HDRS 19.9 (5.15) 23.9 (5.49) HARS 19.1 (5.32) 23.3 (7.74) Mean length in years (SD) of current MDE 4 (5) 5 (5) Number of Previous MDEs 1 Episode 14.3% [n = 2] 14.3% [n = 1] 2 Episodes 21.4% [n = 3] 0.00% [0] 3+ Episodes 64.3% [n = 9] 85.7% [n = 6] Comorbid Diagnoses * None 50.0% [n = 7] 0.00% [0] GAD 14.3% [n = 2] 28.6% [n = 2] Social Phobia 14.3% [n = 2] 42.9% [n = 3] OCD 7.14% [n = 1] 0.00% [0] PTSD 28.6% [n = 4] 57.1% [n = 4] Past Antidepressant Use None 42.9% [n = 6] 14.3% [n = 1] 1-2 28.6% [n = 4] 42.9% [n = 3] 3+ 28.6% [n = 4] 42.9% [n = 3] Time (in years) since last antidepressant use 2.33 (2.53) 2.83 (1.83) Numbers in () indicate one standard deviation of the mean. Numbers in [] indicate the number of participants in the reported category. Abbreviations: GAD  =  generalized anxiety disorder; HARS  =  Hamilton Anxiety Rating Scale; HRSD  =  Hamilton Rating Scale for Depression; MADRS Montgomery-Asberg Depression Rating Scale; MDE  =  major depressive episode; OCD  =  obsessive-compulsive disorder; PTSD  =  post-traumatic stress disorder. * Some participants had more than one co-morbid diagnosis 10.1371/journal.pone.0088785.t002 Table 2 Pre-Scan Mood Ratings for each experimental group. Pre-Scan Self-Ratings Experimental [n = 14] Control [n = 7] SHAPS 29.7 (5.94) 31.6 (6.73) TAS Identifying 18.6 (6.58) 21.0 (4.50) Describing 14.4 (5.15) 17.5 (4.60) Externally Orienting 18.2 (3.87) 22.0 (4.54) Total 51.2 (13.1) 60.4 (11.1) EC Positive 3.32 (0.52) 3.12 (0.47) Negative 3.32 (0.45) 3.30 (0.45) Total 3.33 (0.31) 3.23 (0.33) STAI State 44.3 (8.98) 43.1 (11.5) Trait 55.4 (8.05) 57.0 (12.6) POMS Depression 15.9 (9.05) 16.7 (13.7) Tension 6.64 (6.72) 9.29 (9.25) Anger 4.00 (5.16) 6.71 (10.3) Vigor 9.00 (6.25) 6.29 (8.04) Fatigue 13.8 (5.89) 11.1 (6.99) Confused 6.71 (3.41) 7.86 (5.40) Friendly 17.2 (7.12) 16.6 (5.03) Total Mood Disturbance 20.1 (29.0) 28.9 (47.2) VAS Happy 3.14 (1.70) 3.14 (2.11) Restless 3.57 (1.74) 5.00 (3.32) Sad 4.18 (2.80) 4.57 (2.99) Anxious 3.54 (2.69) 4.57 (3.60) Irritated 2.25 (2.75) 2.43 (2.64) Drowsy 5.29 (2.87) 2.86 (2.91) Alert 4.50 (2.77) 4.14 (2.61) Numbers in parentheses indicate one standard deviation of the mean. Abbreviations: EC  =  Emotional Contagion; POMS  =  Profile of Mood States; SHAPS  =  Snaith-Hamilton Pleasure Scale; STAI  =  State-Trait Anxiety Inventory; TAS  =  Toronto Alexithymia Scale; VAS  =  Visual Analogue Scale. BODY.RESULTS.ROI ANALYSIS: Two participants (one/group) reported falling asleep during the task and their data was removed from the analysis, leaving a final sample size of 21. Results of the neurofeedback experiment based on the ROI analysis of BOLD data appear in Figure 2. A Training (PR, R1, R2, R3, TR) x ROI (LA, RA, HIPS) x Group (Experimental, Control) repeated measures ANOVA for Happy-Rest revealed a main effect of ROI (F(2,38) = 6.00, p = 0.005), an ROI x Group interaction (F(2,38 = 3.93, p = 0.02), and a significant ROI x Training x Group interaction (F(4,60) = 2.66, p = 0.045). These results indicate that the experimental and control groups differed in neurofeedback training effects on the BOLD signal based on the specific target region. 10.1371/journal.pone.0088785.g002Figure 2Percent BOLD Signal Change for each ROI, run, and group.Mean percent BOLD signal change for the Happy – Rest condition for each experimental run for the left amygdala (LA; panels A, B, C), right amygdala (RA; panels D, E, F), and horizontal segment of the intraparietal sulcus (HIPS; panels G, H, I) for the LA rtfMRI-nf group (panels A, D, G), HIPS rtfMRI-nf group (panels B, E, H), and for the difference between the LA and HIPS rtfMRI-nf groups (C, F, I). Error bars indicate +/− one standard error of the mean. * indicates a significant difference from 0 at p<0.05. * indicates a significant difference from 0 at p<0.10. # indicates a significant difference from the experimental group at p<0.05. When the Training x ROI ANOVA was performed for groups separately, the experimental group had a significant effect of ROI (F(2,26) = 3.13, p = .025), but not Training (F(4,52) = 0.44, p = 0.39), and the ROI x Training interaction approached significance (F(4,52) = 2.24, p = 0.05). No significant main effects or interactions were found for the control group (Fs(4,24)<1.37, ps>0.14). This indicates the significant Training x Group and ROI x Group interactions were accounted for by effects on the BOLD signal in the target region of the experimental group only. Follow-up t-tests examined significant ANOVA effects. Independent sample t-tests comparing percent signal change in each ROI for each run showed a significant difference between groups in the LA during Runs 2, 3, and TR (ts(19)>2.33, ps<0.038), while there was no significant difference between groups for HIPS BOLD activity during any run (ts(19)<0.97, ps>0.18). When examining the effects for the RA, there was a significant difference between groups during Run 3 (t(19) = 2.60, p = 0.01), and a during TR (t(19) = 2.02, p = 0.035) suggesting the effects of LA neurofeedback training, while specifically targeted to LA, also influenced right amygdala function. One-sample t-tests (comparing activation to 0) assessed the significance of the BOLD change within each ROI and run. Within the LA in the experimental group there was a trend towards significance during PR (t(13) = 1.94, p = 0.05), and significant effects during R1, R2, R3 and TR (ts(13)>2.41, ps<0.02). No significant difference was found within the RA or HIPS for the experimental group (ts(13)<1.83, ps>0.024). In the control group, no significant difference was evident in any ROI or run (ts(6)<1.86, ps>0.08). These results suggest only participants in the experimental group were able to significantly elevate BOLD activity, and this effect largely was limited to the LA. Importantly, the finding that LA BOLD activity significantly increased during TR and did not differ from that in the final training run (TR v R3 (t(13) = 0.34, p = 0.37) in the experimental group suggests training effects persisted in the absence of neurofeedback. Finally, we performed a one-way ANOVA trend analysis across all runs for LA activation. The experimental group showed a significant linear trend (F(1,19) = 4.68, p = 0.024) and the control group showed a nonsignificant trend (F(1,19) = 3.84, p = 0.05). These results were attributable to a linear increase in LA BOLD activity within the experimental group but a linear decrease in LA activity in the control group as training progressed (Figure 2). Tests for quadratic and cubic trends were not significant for either group (Fs(1,19)<0.80, ps >0.39). There was no significant change for either group within the RA (Fs(1,19)<2.36, ps>0.08) or HIPS regions (Fs(1,19)<0.75, ps>0.20). BODY.RESULTS.ASSOCIATED MOOD EFFECTS: The mean changes on self-report emotional rating scales for each group are listed in Table 3. For the experimental group, significant changes were seen in our main outcome measures: POMS depression ratings decreased (t(13) = 2.12, p = 0.025), and VAS happiness ratings increased (t(13) = 3.04, p = 0.005) following LA rtfMRI-nf. The increase in happiness was significantly greater in the experimental compared to control group (t(19) = 2.33, p = 0.01), while the change in POMS depression between groups failed to reach significance (t(19) = 1.33, p = 0.10). Significant decreases were also found within the experimental group for ratings of POMS anger (t(13) = 2.42, p = 0.015), trait anxiety (STAI-Trait subscale; t(13) = 2.62, p = 0.01), state anxiety (STAI-Trait subscale; t(13) = 2.34, p = 0.02), and VAS ratings of restlessness (t(13) = 2.23, p = 0.02), anxiety (t(13) = 4.37, p<0.001), and irritability (t(13) = 2.14, p = 0.025), and the difference between the groups on state anxiety was significant t(19) = 2.58, p = 0.03). No other rating significantly changed pre-post scan (ts(13)1.85, ps>0.08). In the control group, VAS ratings of sadness decreased (t(6) = 2.57, p = 0.02), while no other rating score changed significantly pre-post scan (ts(6)<1.73, ps>0.10). 10.1371/journal.pone.0088785.t003 Table 3 Change in mood ratings (post-pre scan scores) following neurofeedback training for each experimental group. Change Post-Scan Experimental [n = 14] Control [n = 7] STAI State −2.14 (3.21)* 3.86 (7.65)** Trait a −3.00 (4.30)* −1.14 (3.39) POMS Depression −4.50 (8.48)* −2.43 (5.26) Tension −3.00 (6.06) −1.43 (5.86) Anger −2.36 (3.65)* −3.29 (5.82) Vigor −0.29 (5.14) 0.29 (8.48) Fatigue 0.57 (8.50) −0.43 (5.32) Confused −1.00 (3.98) 1.29 (4.23) Friendly 0.57 (6.32) −0.14 (5.58) Total Mood Disturbance −9.21 (27.2) −5.43 (29.0) VAS Happy 1.75 (1.16)* 0.29 (1.63)** Restless −1.04 (1.73)* −1.00 (3.37) Sad −0.93 (2.09) −1.57 (1.62)* Anxious −1.79 (1.53)* −0.57 (2.64) Irritated −1.36 (2.37)* −0.86 (1.07) Drowsy 0.11 (2,34) 0.72 (2.69) Alert −0.54 (2.53) −0.86 (2.12) Numbers in parentheses indicate one standard deviation of the mean. * indicates a significant change from pre to post-scan ratings at p<0.05. ** indicates a significant differences from the experimental group at p<0.05. Abbreviations: POMS  =  Profile of Mood States; STAI  =  State-Trait Anxiety Inventory; VAS  =  Visual Analogue Scale a One may wonder why the trait subscale of the STAI was administered both pre and post neurofeedback. While the Trait measure of anxiety is supposed to be a relatively stable measure, there is no set time period that the rating scale covers (such as the SHAPS which asks over the past week how often have you felt…) and instead asks how one “generally feels.” We wondered whether the neurofeedback procedure would alter how participants viewed themselves more generally, and indeed participants in the experimental group reported less general anxiety following the neurofeedback procedure. This could indicate they reflect more positively on themselves after the procedure and is another example of amygdala neurofeedback improving mood. However, as the test-retest reliability of the Trait measure of the STAI ranges from.65-.8645 (Spielberger CD, Gorsuch RL, R.E L (1970) Manual for the State-Trait Anxiety Inventory. Palo Alto, CA: Consulting Psychologists Press) the changes could simply be chance fluctuations in scores and replication is needed to confirm our hypothesis that how anxious one generally feels is indeed decreasing following amygdala neurofeedback Finally, we tested whether there was an association between neurofeedback performance and clinical or mood rating variables. Within the experimental group, the mean percent signal change in the LA over the three training runs was inversely correlated with both the current depressive episode duration (r = −0.63, p = 0.025; Figure 3a), and the TAS "difficulty describing feelings" score (r = −0.60, p = 0.025; Figure 3b). No outliers were present in the data (see Figure S1 in Supporting Information). While the TAS scores followed a normal distribution (Shapiro-Wilk W = 0.952, p = 0.563), the average MDE length did not (Shapiro-Wilk W = 0.739,p = 0.001). Therefore, we performed an additional correlation analysis on this data using Spearman's Rho and still found a significant correlation (ρ = −0.51, p<0.05) No significant correlations were found in the control group. 10.1371/journal.pone.0088785.g003Figure 3Relationship between left amygdala neurofeedback training effect and individual characteristics in the experimental rtfMRI-nf group.A) Correlation with length of the current major depressive episode. The training effect for the left amygdala (the average Happy – Rest percent signal change over the three training runs) was inversely correlated with the length of participants' current depressive episode. B) Correlation with difficulty describing feelings (TAS-20). The more difficulty a participant had describing their own feelings, the less average BOLD activation for the Happy-Rest contrast was observed in the left amygdala for the three training runs. BODY.RESULTS.WHOLE BRAIN ANALYSIS: Results of fMRI data analysis for TR appear in Table 4. For the Happy versus Count contrast, the experimental group showed increased BOLD activity during positive AM recall within bilateral orbitofrontal cortex, dorsomedial prefrontal cortex, superior temporal and middle temporal gyrus, left ACC, posterior cingulate cortex, parahippocampal gyrus, and amygdala (encompassing the amygdala ROI) and right VLPFC and thalamus. The experimental group had increased activity during counting compared to positive AM recall in bilateral middle frontal gyrus, and inferior parietal lobe. When we compared regional BOLD activity between the Happy versus Rest conditions between the experimental and control groups during TR, the experimental group had increased activity in left superior temporal gyrus and temporal pole, and right thalamus (Figure 4). 10.1371/journal.pone.0088785.g004Figure 4Group differences in BOLD activity during the Transfer Run.Regions A) Left Superior Temporal Gyrus B) Left Temporal Pole C) Right Thalamus and associated percent signal change graphs, are shown where groups had differential activation during Happy AM recall vs Rest during the Transfer run in which no neurofeedback was provided. Error bars indicate +/1 one standard deviation of the mean. 10.1371/journal.pone.0088785.t004 Table 4 Regional increases in BOLD activity during the Transfer Run. Area Cluster Size x, y, z t score Happy AM condition > Count L Orbitofrontal C 429 −37, 25, −10 8.16 R Orbitofrontal C 43 43, 23, 6 5.27 L DMPFC 581 −5, 51, 16 6.40 R DMPFC 41 5, 61, 22 5.56 R VLPFC 182 47, 23, 8 5.71 L Anterior Cingulate C 56 −5, 53, −2 5.39 L Posterior Cingulate C 850 −5, −53, 12 7.73 L Superior Temporal G 52 −43, −59, 18 4.76 R Superior Temporal G 371 37, 21, −22 6.16 L Middle Temporal G 167 −41, −5, −26 6.17 R Middle Temporal G 230 43, −57, −2 5.34 L Parahippocampal G 227 −25, −31, −14 5.32 L Amygdala 30 −23, −5, −15 5.17 L Thalamus 74 −3, −21. 10 5.23 Count > Happy Memories L Middle Frontal G 101 −23, −5, 52 5.09 R Middle Frontal G 69 43, 37, 26 4.55 L Inferior Parietal Lobe 769 −45, −39, 38 7.49 R Inferior Parietal Lobe 971 45, −57, 28 6.55 Experimental > Control L Superior Temporal G 140 −29, 3, −34 5.25 L Temporal Pole 71 −47, 17, −22 4.73 R Thalamus 31 11, −15, −4 5.24 Control > Experimental no significant clusters Coordinates correspond to the stereotaxic array of Talairach & Tournoux [48] . Cluster size refers to the number of contiguous voxels for which the voxel t value corresponds to p corrected <0.05. Abbreviations: C  =  cortex; DMPFC  =  dorsomedial prefrontal cortex; G  =  gyrus; L  =  left; R  =  right; VLPFC  =  ventrolateral prefrontal cortex. BODY.DISCUSSION: We investigated the feasibility of training MDD patients to regulate the hemodynamic activity their left amygdala using rtfMRI-nf and recall of positive AMs. Results demonstrate that, given appropriate direction, practice, and rtfMRI-nf information, MDD patients can enhance their amygdala BOLD activity by contemplating positive AMs within a single training session. That activity within the HIPS control region did not change over the course of the study in the experimental group suggests that feedback from the target amygdala region is necessary for enhanced control of that region and does not affect other regions not involved in emotional processing. Importantly, engaging in a single session of amygdala rtfMRI-nf (but not HIPS) training significantly improved mood. While the mental strategy of recalling positive AMs likely played a considerable part in this improved mood, neurofeedback from LA was crucial as evinced by lack of mood improvement in the control group who also recalled positive AMs while receiving rtfMRI-nf from the HIPS. Previous functional neuroimaging studies had implicated the HIPS region primarily in number processing[49]–[51], [54], along with visual attention[58], [59], and perceptual motor coordination[60], but not in emotional processing. Although the right amygdala showed a nominal increase in the experimental group and decrease in the control group across the neurofeedback trials, this effect was only significant within the last training run of the experimental group. Statistically significant self-regulation was mostly specific to the target ROI of the LA. These data appear compatible with the lack of correlation between left and right amygdala glucose metabolism[61]. Nevertheless, the left and right amygdala share direct intrahemispheric anatomical connections[62], [63], and our data suggest the hemodynamic responses to our experimental task are not completely independent. A linear trend was evident in which LA BOLD activity increased across runs in the experimental group, and decreased across runs within the control group. This suggests that patients in the experimental group were able to increase their LA BOLD response to positive stimuli, and to maintain this elevated response throughout the entire training session as well as during the transfer run in which neurofeedback was not provided. In the control group, amygdala BOLD activity was reduced over time, with no training effect evident in the HIPS. This decreased recruitment of the left amygdala over time in the control group could be related to the inability of MDD patients to maintain positive affect over time. It is well established that MDD patients suffer from anhedonia and an inability to sustain positive affect[64], [65], including an inability to sustain activity in brain regions involved in positive affect and reward[66]. That the experimental group was able to maintain this amygdala BOLD response during positive AM recall over the course of the training runs and into the transfer run further supports the clinical potential of the amygdala rtfMRI-nf procedure. While "Transfer" in the context of the current study refers to the ability to maintain the skill without feedback, it can also refer to the ability to perform the behavior within a different setting[67]. An important next step for future studies will be to examine whether training can be transferred to contexts outside of the scanner in order to further assess the clinical potential of the procedure. An alternate explanation for the linear decrease evident in the HIPS rtfMRI-nf group is that receiving rtfMRI-nf from the HIPS could have influenced participants to change their mental strategy, which could have resulted in the decreasing amygdala activation. We find this an unlikely explanation as participants were explicitly instructed not to change their mental strategy and debriefing interviews suggest they followed these instructions. Furthermore, participants were not successful in regulating their HIPS activity (percent signal change did not differ from 0 in any run), again suggesting participants maintained the strategy of positive AM recall. Notably, while both groups improved nominally on many of the self-report measures, only participants in the experimental rtfMRI-nf group showed statistically significant improvements. Participants in the amygdala rtfMRI-nf group reported significant decreases in ratings of depression, anxiety, anger, restlessness, and irritability along with significant increases in ratings of happiness, while mood ratings in the control group did not significantly change (except for a decrease in VAS sadness, conceivably due to the effects of positive AM recall). While it is conceivable that increasing the sample size in the control group would result in pre-post neurofeedback changes becoming significant, our conclusion that amygdala rtfMRI-nf improved mood is not solely based on significant within-subject changes in the experimental group and non-significant changes in the control group, but also significant improvements in anxiety and happiness ratings in the experimental relative to the control group. These results suggest that the experience of increasing LA BOLD activity during positive AM recall has therapeutic potential, beyond that of simply recalling positive AMs. While the control group did not have the experience of successful self-regulation associated with their AM recall, there was no evidence of any mood deterioration in these participants. Indeed, self-ratings of tension, anger, and irritability showed a nominal decrease in the HIPS rtfMRI-nf group that failed to reach statistical significance. Therefore, the mood effects observed in the amygdala rtfMRI-nf group are unlikely attributable to false positives due to increased frustration weakening mood effects in the HIPS rtfMRI-nf group. However, replication with a different control task or region is warranted to verify these results of improved mood in the experimental relative to control group. We believe that the results support the conclusion that LA rtfMRI-nf resulted in the improved mood, and were not due to a placebo effect. The strategy of recalling positive AMs was not effective at increasing HIPS activation (evinced by no change from 0 in HIPS activity for any run in either group), therefore, one might expect participants to become unblinded as activation of the ROI was unresponsive to strategies of recalling positive AMs. We do not believe this to be the case, however, as during debriefing interviews in which condition assignment was revealed most participants in the experimental group expressed surprise and stated that they had believed themselves to be in the control HIPS condition. While surprising at first that even patients whose average LA activity was well above 0 would think they were in the control group, we believe this finding to be related to the negative self-schemas inherent to MDD [68]. That despite performing well, participants felt they must have been performing poorly. These findings argue against a placebo effect. Furthermore, these results suggest that amygdala rtfMRI-nf can result in mood improvements without conscious knowledge of training having occurred. Not every participant in the experimental group was able to maintain an elevated amygdala response. The average LA BOLD response across the training runs (Runs 1–3) varied from +0.60% to −0.17%, and 4 participants in the experimental group had negative or near 0 amygdala BOLD responses indicating a failure to learn to regulate their amygdala. We found the amygdala training effect inversely correlated with length of the current depressive episode and with difficulty describing feelings. The correlation with depressive episode length suggests the clinical success of rtfMRI-nf of the amygdala may be dependent upon targeting patients early in the course of their depressive episode. This is consistent with previous research reporting that patients within a year of onset of their current depressive episode are more likely to respond to treatment than those whose episodes were of longer duration[69]. Indeed, patients in the current study who were within the first two years of onset of their current depressive episode were more likely to be able to regulate their amygdala activity via rtfMRI-nf. The negative correlation between LA modulation and difficulty describing feelings is similar to that found in healthy controls engaging in the same paradigm[14]. However, an inverse correlation between amygdala modulation and difficulty identifying feelings (on the TAS) was reported within the healthy sample, while the current study found the correlation to exist within the describing feelings TAS subscale. These two TAS subscales are positively correlated and may load on a common factor related to emotional insight[70]. The observed correlation between insight and ability to regulate suggests certain MDD patients may be better suited to rtfMRI-nf treatment, as is the case for other psychological treatments for depression [71], [72]. Our results are partially consistent with those of the only study to date to examine the clinical potential of rtfMRI-nf training in MDD[19]. This work reported reduced depressive symptoms following a different rtfMRI-nf procedure, however, only clinician-administered ratings of depression improved following neurofeedback, and only following multiple sessions. In contrast, we found immediate improvements in self-reported mood ratings following a single neurofeedback session. The results of [19] must be interpreted with caution, however, as the control condition was performed outside of the fMRI environment, raising the question as to whether clinical improvement following neurofeedback were in fact due to learning to up-regulate brain regions involved with positive emotions or instead due to the appeal of a novel, technology-based intervention. Indeed, group differences in self-report measures (such as POMS) did not differ between experimental and control groups while non-blinded clinician administered ratings differed significantly, further suggesting that experimenter bias and placebo effects may have driven their results. Furthermore, the target neurofeedback regions used in [19] were adjusted, and could change, for each participant and run. While patients were able to increase BOLD activity in the target regions over time, interpretation and replication of these results is difficult as the BOLD response from run-to-run could include activity from several different regions. These different and changing regions makes it difficult to conclude that symptom improvement was not due to the general experience of gaining control over brain activity rather than gaining control over the specific regions modulated. The results from the current rtfMRI-nf protocol, in which the target region selection for neurofeedback was based on existing knowledge of emotional processing and psychiatric disorders, allows for easier interpretation of results and application for treatment designs. The whole-brain voxel-wise analysis showed the ability to maintain elevated amygdala activity during positive AM recall following rtfMRI-nf training engaged a prefrontal-temporal cortical-limbic network implicated in emotion processing and AM recall[73], [74]. Many of these regions share extensive anatomical and functional connections with the amygdala and are recruited during emotional learning[73] and in the modulation of emotional processes[75]. These regions also form part of the core network recruited during AM recall[74], and showed increased hemodynamic activity in healthy males as they performed the same task[14], as well as increased functional connectivity following neurofeedback using the same procedure[76]. This pattern suggests that rtfMRI-nf from the amygdala is not dependent on a signal brain region, but upon a network. Future studies investigating rtfMRI-nf may benefit from using a network as opposed to a signal ROI. The superior temporal gyrus, temporal pole and thalamus were more active in the experimental group than the control group during TR, indicating these regions are critically involved in maintaining the elevated amygdala response to positive AMs following training but in the absence of neurofeedback. Increased thalamic activity may reflect increased processing or relay of information between frontal and limbic regions recruited during the task[77], or increased arousal[78], which may be needed to maintain an elevated amygdala response during AM recall. Superior temporal and temporopolar regions are involved in emotional processing and social cognition[79]–[81], and are less active in MDD versus healthy individuals[82]–[84]. Therefore, the increased activity in these regions in MDD patients during LA but not HIPS rtfMRI-nf suggests the rtfMRI-nf procedure effectively recruits other regions important in emotional regulation which show abnormal BOLD responses in MDD, further suggesting potential for rtfMRI-nf in MDD treatment. Some limitations of our study design merit comment. In this proof-of-concept study, the relatively small sample size, unmatched clinical characteristic of the experimental and control groups, and few male participants may limit the generalizability of our findings. While the control group appeared nominally more depressed than the experimental group, no differences reached significance with regard to clinical characteristics (except comorbidity), and ratings on the HDRS and MADRS for both groups were in the severely depressed range, further indicating comparable symptom severity. Nevertheless, future studies should include larger, more diverse, and better matched samples of MDD participants, which will enable better characterization of populations this treatment might best be suited to. Additionally, there was a relatively large inter-participant variability of the results, as mentioned above. While differences in the length of current depressive episode and ability to describe feelings partly accounted for this variability, other individual factors (e.g., learning ability, attention, motivation, fatigue) likely contributed as well. Repeating the rtfMRI-nf session multiple times would likely increase the training effect. Additionally, other means of enhancing training such as adjusting the target level of activation based upon individual performance should be considered in future studies. Furthermore, the current study included a single rtfMRI-nf training session, therefore we were unable to determine whether effects on clinician administered ratings (which usually cover a several day period) were evident or how long observed improvements on self-report mood ratings last, allowing us to conclude only that the amygdala neurofeedback procedure has short term benefits on mood. Additional sessions and ratings will allow us to further determine the potential of this rtfMRI-nf paradigm as an MDD intervention. Finally, randomization was not employed in the current study. Randomized blinded clinical trials are now needed to determine whether rtfMRI-nf might become a useful addition to current therapies for depression. BODY.DISCUSSION.CONCLUSIONS: In conclusion, our study demonstrates that MDD patients can self-regulate their amygdala activation using positive AM recall while receiving rtfMRI-nf. In contrast to the HIPS rtfMRI-nf, the feedback provided from the left amygdala resulted in a significant BOLD signal increase during rtfMRI-nf training, which persisted during TR in which no neurofeedback was provided. This training resulted in significant improvements in short-term state-dependent mood ratings, including increased ratings of happiness and decreased ratings of depression and anxiety only in the experimental group. In the experimental group, the training effect was inversely correlated with the length of the current depressive episode as well as with difficulty describing feelings, suggesting important individual differences are related to the ability to regulate the amygdala in response to rtfMRI-nf training. Further investigating characteristics associated with successful amygdala modulation and rtfMRI-nf related improvements of mood, along with studies aimed at improving and extending the neurofeedback effects, could ultimately allow this rtfMRI-nf procedure to be translated into a non-invasive MDD treatment. BODY.SUPPORTING INFORMATION: Figure S1 Box plots for the experimental group for a) average amygdala percent signal change over the 3 training runs (R1–R3) b) length of the current major depressive episode and c) the difficulty describing feelings subscale of the Toronto Alexithymia Scale. (DOCX)Click here for additional data file.

TITLE: The soluble guanylate cyclase activator cinaciguat prevents cardiac dysfunction in a rat model of type-1 diabetes mellitus ABSTRACT.BACKGROUND: Diabetes mellitus (DM) leads to the development of diabetic cardiomyopathy, which is associated with altered nitric oxide (NO)—soluble guanylate cyclase (sGC)—cyclic guanosine monophosphate (cGMP) signalling. Cardioprotective effects of elevated intracellular cGMP-levels have been described in different heart diseases. In the current study we aimed at investigating the effects of pharmacological activation of sGC in diabetic cardiomyopathy. ABSTRACT.METHODS: Type-1 DM was induced in rats by streptozotocin. Animals were treated either with the sGC activator cinaciguat (10 mg/kg/day) or with placebo orally for 8 weeks. Left ventricular (LV) pressure–volume (P–V) analysis was used to assess cardiac performance. Additionally, gene expression (qRT-PCR) and protein expression analysis (western blot) were performed. Cardiac structure, markers of fibrotic remodelling and DNA damage were examined by histology, immunohistochemistry and TUNEL assay, respectively. ABSTRACT.RESULTS: DM was associated with deteriorated cGMP signalling in the myocardium (elevated phosphodiesterase-5 expression, lower cGMP-level and impaired PKG activity). Cardiomyocyte hypertrophy, fibrotic remodelling and DNA fragmentation were present in DM that was associated with impaired LV contractility (preload recruitable stroke work (PRSW): 49.5 ± 3.3 vs. 83.0 ± 5.5 mmHg, P < 0.05) and diastolic function (time constant of LV pressure decay (Tau): 17.3 ± 0.8 vs. 10.3 ± 0.3 ms, P < 0.05). Cinaciguat treatment effectively prevented DM related molecular, histological alterations and significantly improved systolic (PRSW: 66.8 ± 3.6 mmHg) and diastolic (Tau: 14.9 ± 0.6 ms) function. ABSTRACT.CONCLUSIONS: Cinaciguat prevented structural, molecular alterations and improved cardiac performance of the diabetic heart. Pharmacological activation of sGC might represent a new therapy approach for diabetic cardiomyopathy. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12933-015-0309-x) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: Diabetes mellitus (DM) is associated with cardiovascular complications, such as myocardial infarction, chronic heart failure or vascular diseases. It acts as an independent risk factor for coronary atherosclerosis and ischemic heart disease, however, the altered metabolic state—due to elevated glucose levels—has a direct impact on cardiac structure and function independently of coronary artery disease. Thus DM leads to the development of a special disease entity, termed diabetic cardiomyopathy [1]. Although, both systolic and diastolic dysfunction [2–5] as well as several key morphological and cellular/subcellular features (including myocardial fibrotic remodelling, cardiomyocyte hypertrophy, nitro-oxidative stress, inflammation and apoptosis) have been described in diabetic cardiomyopathy, the exact mechanisms in the pathophysiology are still unknown [1, 6–9]. In the cardiovascular system, under physiological conditions, nitric oxide (NO) is produced by the endothelial nitric oxide synthase (eNOS) in endothelial cells and diffuses into target cells, such as vascular smooth muscle cells or cardiomyocytes. It activates its intracellular receptor, the soluble guanylate cyclase enzyme (sGC) which results in the rapid formation of the second messenger cyclic guanosine monophosphate (cGMP). cGMP activates the cGMP-dependent protein kinase (PKG), which mediates most of its physiological effects, such as vasodilatation or inhibition of platelet aggregation [10]. The NO–sGC–cGMP–PKG signalling pathway has been described to get disturbed in DM through several mechanisms, such as increased formation of reactive nitrogen (RNS) and oxygen species (ROS) (nitro-oxidative stress), eNOS uncoupling and decreased NO bioavailability, increased expression of the cGMP-degrading enzyme phosphodiesterase (PDE)-5 [1]. Our research group and others demonstrated protective effects of enhanced cGMP-signalling via pharmacological inhibition of PDE-5 in several cardiovascular diseases [11–15] and in DM in particular [3, 16]. Nitro-oxidative stress directly deteriorates the structure of sGC resulting in a heme-deficient, NO-insensitive and inactive form of the enzyme [17]. Therefore, drugs that are able to activate the NO-insensitive form of sGC thus reactivating it might have cardioprotective effects in various pathological conditions through the enhancement of the impaired cGMP-signalling. The sGC activator cinaciguat (BAY 58-2667) has been reported to bind to the heme pocket of NO-insensitive sGC [17] thus increasing its cGMP-producing activity and augmenting cGMP-PKG signalling. Recent studies described its beneficial effects in experimental myocardial infarction [18], myocardial ischemia/reperfusion injury [19, 20], endothelial dysfunction induced by nitro-oxidative stress [21], vascular neointima formation [22] or in prevention of cardiomyocyte hypertrophy [23]. The safety and tolerability of cinaciguat have been assessed by phase-I human clinical trials [24]. Although cinaciguat has demonstrated efficacy in a proof-of-concept study in patients with acute decompensated heart failure [25], the phase-IIb clinical studies for the same indication has been terminated prematurely due to the high number of adverse events (mainly a significant drop of blood pressure) during acute intravenous application [26, 27]. The investigators concluded that rather a chronic, long term oral administration of the compound could improve endothelial and preserve myocardial function in heart failure patients [28]. Based upon the concept of cardioprotection by enhanced cGMP-signalling via chronic pharmacological activation of the sGC enzyme, we investigated in the present study whether long-term administration of the sGC-activator cinaciguat can preserve left ventricular (LV) systolic and diastolic function and prevent DM-associated myocardial alterations in the rat model of streptozotocin (STZ)-induced experimental type-1 DM. BODY.METHODS.ANIMALS: The investigation conformed to the EU Directive 2010/63/EU guidelines and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996) and was reviewed and approved by the appropriate institutional and national ethical committees (permission number: 22.1/1162/3/2010). All animals received humane care. 8-week-old male Sprague–Dawley rats (n = 46; body weight (BW): 225–250 g) (Charles River, Sulzfeld, Germany) were housed individually in a room (constant temperature, 12/12 h light/dark cycles) and received standard laboratory rat diet and water ad libitum. BODY.METHODS.INDUCTION OF DM: Type-1 DM was induced with a single i.p. injection of STZ (60 mg/kg) in our rats as described previously [3]. STZ was freshly dissolved in citrate buffer (0.1 M). Control animals received only the buffer. 72 h after the injection a drop of blood was collected from the tail vein and blood glucose concentration was determined by using a digital blood glucose meter (Accu-Chek® Sensor, Roche, Mannheim, Germany). Animals with a random blood glucose level >15 mmol/l were considered to be diabetic and were included into the study. BODY.METHODS.EXPERIMENTAL GROUPS, CHRONIC TREATMENT PROTOCOL: After confirmation of DM, rats were randomised into four groups: vehicle-treated control (Co; n = 12), cinaciguat-treated control (CoCin; n = 12), vehicle-treated diabetic (DiabCo; n = 12) and cinaciguat-treated diabetic (DiabCin; n = 10) groups. Animals were treated for 8 weeks with 0.5 % methylcellulose (Co, DiabCo) vehicle or with the sGC activator cinaciguat in suspension p.o. (CoCin, DiabCin; 10 mg/kg/day), starting immeadiately after DM confirmation. Water bottles were filled every morning with the same amount of fresh tap water and daily water intake was measured. Animal cages were handled with care and were not moved after water bottle replacement to prevent spilling of water from the bottles. Body weight of the animals were recorded every 2 days and the dose of cinaciguat was adjusted accordingly. BODY.METHODS.HEMODYNAMIC MEASUREMENTS: 24–28 h after the last administration of cinaciguat/vehicle, LV pressure–volume (P–V) analyses were carried out using a pressure-conductance microcatheter system (MPVS-Ultra, Millar Instruments, Houston, TX, USA) to evaluate LV performance. Animals were anesthetised with a mixture of xylazine (3 mg/kg) and ketamin (100 mg/kg) i.p. [3, 29]. Mean arterial blood pressure (MAP), heart rate (HR), maximal LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), maximal slope of systolic pressure increment (dP/dtmax) and diastolic pressure decrement (dP/dtmin), time constant of LV pressure decay (Tau), ejection fraction (EF), stroke work (SW) and cardiac output (CO) were calculated. The slope (Ees) of the LV end-systolic P–V relationships (ESPVR; according to the parabolic curvilinear model) and preload recruitable stroke work (PRSW) were calculated as load-independent indexes of LV contractility while the slope of the LV end-diastolic P–V relationship (EDPVR) was calculated as reliable index of LV diastolic stiffness. For detailed hemodynamic description see Additional file 1. BODY.METHODS.BIOCHEMICAL MEASUREMENTS: A drop of blood was collected from the tail vein and blood glucose concentration was determined using a digital blood glucose meter (Accu-Chek® Sensor, Roche). At the end of the hemodynamic measurements blood was collected from the inferior caval vein, plasma samples were prepared and stored at −80 °C. Plasma cGMP levels were determined by enzyme immunoassay (EIA) using a commercial kit (Amersham cGMP EIA Biotrak System, GE Healthcare, Buckinghamshire, UK). BODY.METHODS.MYOCARDIAL MRNA ANALYSIS: We performed quantitative real-time polymerase chain reaction (qRT-PCR) experiments as described previously [30]. Frozen LV samples were homogenised, total RNA was isolated. Reverse transcription was performed and cDNA samples were amplified on the StepOnePlusTM Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using TaqMan® Universal PCR MasterMix and TaqMan® Gene Expression Assays (Applied Biosystems) for the following targets: atrial natriuretic factor (ANF), α myosin heavy chain (MHC) and β-MHC (ratio of β/α-MHC expression was assessed as pathological cardiomyocyte hypertrophy marker), antiapoptotic mediator B cell CLL/lymphoma-2 (Bcl-2), proaptotic mediator Bcl2-associated X protein (BAX), eNOS, mediators of cardiac remodelling such as collagen 1a1 (Col1), 3a1 (Col3) and fibronectin, matrix metallopeptidase (MMP)-2 and MMP-9 and their endogenous inhibitors tissue inhibitor of MMP (TIMP)-1 and TIMP-2, members of different antioxidant systems like catalase, thioredoxin-1, gluthatione-reductase, superoxide dismutase (SOD)-2 and heat shock 70 kD protein 1A (HSP70a1). Gene expression data were normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The mRNA expression levels were calculated using the CT comparative method (2−ΔCT) and adjusted to a positive calibrator. For detailed description see Additional file 1. BODY.METHODS.IMMUNOBLOT ANALYSIS: LV tissue samples were homogenised, protein concentration was determined and equal amounts of protein were separated via gel-electrophoresis. Proteins were transferred to nitrocellulose membranes. After blocking, membranes were incubated with primary antibodies against various targets: eNOS (1:1000, SC-654, SantaCruz Biotechnology, Santa Cruz, CA, USA), sGC β1 (1:1000, NB100-91798, Novus Biologicals, Cambridge, UK), PDE-5 (1:2000, ALX-210-099, Enzo Life Sciences, Farmingdale, NY, USA), PKG (1:2000, ADI-KAP-PK005-F, Enzo Life Sciences), vasodilator-stimulated phosphoprotein (VASP) and phospho-VASP (1:1000, 3112 and 1:2000, 3114, Cell Signaling, Danvers, MA, USA) as marker of PKG activity, profibrotic mediator transforming growth factor (TGF)-β1 (1:250, SC-146, SantaCruz Biotechnology), MMP-2 (1:5000, NB200-193, Novus Biologicals) and MMP-9 (1:1000, SC-6840, SantaCruz Biotechnology). After washing, membranes were incubated in horseradish peroxidase-conjugated secondary antibody. Immunoblots were developed by enhanced chemiluminescence detection. Protein band densities were quantified using GeneTools software (Syngene, Frederick, MD, USA). After adjusting protein band densities to GAPDH (1:10000, MAB374, Millipore, Billerica, MA, USA), band density values were normalised to the average value of the Co group for statistical analysis. Detailed description of immunoblot analysis is available as Additional file 1. BODY.METHODS.HISTOLOGY, IMMUNOHISTOCHEMISTRY: LV sections were stained with hematoxylin-eosin (H&E) and Masson's trichrome (MT) to examine histopathological characteristics and interstitial myocardial fibrotic remodelling, respectively. To evaluate cardiomyocyte hypertrophy, transverse transnuclear widths (cardiomyocyte diameter) were measured of 100 longitudinally oriented, mono-nucleated cardiomyocytes on H&E stained LV sections cut on the same plane. Immunohistochemistry for the fibrosis marker fibronectin (Sigma-Aldrich, Budapest, Hungary), for the profibrotic mediator TGF-β1 (Santa Cruz Biotechnology), for the sGC derived second messenger cGMP (AbD Serotec, Düsseldorf, Germany) and for the nitro-oxidative stress marker nitrotyrosine (NT) (Millipore) were performed and analysed. Histological and immunohistochemical analyses were performed by two blinded observers. For detailed description see Additional file 1. BODY.METHODS.TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE DUTP NICK END LABELING (TUNEL) ASSAY: TUNEL assay was performed to detect DNA strand breaks using a commercial kit (DeadEndTM Colorimetric TUNEL System, Promega, Mannheim, Germany). TUNEL positive cell nuclei were counted in 20 randomly selected fields of each section by two blinded observers. Data were normalised to the mean value of the Co group for statistical analysis. BODY.METHODS.DRUGS: The sGC activator cinaciguat (BAY 58-2667; 4-({(4-carboxybutyl)[2-(2-{[4-(2-phenylethyl)benzyl]oxy}phenyl)ethyl]amino}methyl)benzoic acid) was provided by Bayer HealthCare (Wuppertal, Germany) for oral application. Cinaciguat was suspended in 0.5 % methylcellulose solution. STZ was purchased from Sigma-Aldrich (Taufkirchen, Germany). BODY.METHODS.STATISTICS: Data are presented as means and standard errors of the mean (SEM). After testing normal distribution of the data, two-factorial analysis of variance (ANOVA) (with diabetes and drug treatment as factors) was carried out. A P value of <0.05 was used as a criterion of significance. Where F value of diabetes × treatment interaction reached the level of significance Tukey HSD post hoc testing was performed to evaluate differences between the groups. Data that did not show normal distribution were transformed logarithmically before performing two-factorial ANOVA. BODY.RESULTS.BODY WEIGHT, HEART WEIGHT (HW) AND GLUCOSE LEVELS: All animals survived the study period and reached the end-point of the investigation. HW and BW significantly decreased in both diabetic groups while HW to BW ratio increased in the diabetic groups (Table 1). When compared with controls, DM led to significantly increased blood glucose levels and daily water intake. Cinaciguat treatment in diabetic rats did not influence blood glucose levels, but led to attenuated water intake (Table 1). Time-course of body weight changes is available as Additional file 2: Figure S1.Table 1Basic characteristics of study groupsCharacteristicCoCoCinDiabCoDiabCinPdiabetes Ptreatment Pinteraction Blood glucose (mmol/l)5.8 ± 0.16.2 ± 0.130.8 ± 0.5*29.3 ± 1.4*<0.0010.4600.209Water intake (ml/gBW/day)0.078 ± 0.0020.090 ± 0.0020.787 ± 0.006*0.597 ± 0.013*† <0.001<0.001<0.001Heart weight (g)1.20 ± 0.061.24 ± 0.070.91 ± 0.04*0.84 ± 0.03*<0.0010.8050.278Body weight (g)480.7 ± 17.6477.8 ± 20.9293.5 ± 11.1*247.6 ± 14.8*<0.0010.1500.202Heart weight/body weight0.249 ± 0.0080.259 ± 0.0080.311 ± 0.009*0.348 ± 0.015*<0.0010.0260.196The values of blood glucose, daily water intake, heart weight, body weight (BW) and heart weight to body weight ratio are shown of the study groups—vehicle-treated controls (Co), cinaciguat-treated controls (CoCin), vehicle-treated diabetic (DiabCo) and cinaciguat-treated diabetic (DiabCin) groups. Values are mean ± SEM of 10–12 experiments per group* P < 0.05 vs. Co †P < 0.05 vs. DiabCo (Tukey HSD test) BODY.RESULTS.EFFECTS OF CINACIGUAT ON PLASMA AND MYOCARDIAL CGMP LEVELS IN DM: Cinaciguat treatment had no effect on plasma cGMP levels in control animals (Fig. 1a), however it resulted in a pronounced increase of plasma cGMP in DM (Fig. 1a). According to cGMP immunohistochemistry, the cGMP content of LV myocardium was significantly lower in DM than in controls (Fig. 1b, c). However, chronic treatment with cinaciguat restored cGMP to the control level (Fig. 1b, c).Fig. 1Effect of diabetes mellitus (DM) and cinaciguat treatment on plasma and myocardial cyclic guanosine monophosphate (cGMP) content. a Result of plasma cGMP enzyme immunoassay. b Quantification of cGMP (P value of diabetes × treatment interaction in the two-factorial ANOVA, Pinteraction = 0.025) immunohistochemistry. c Representative images of cGMP immunohistochemistry. Magnification: ×400, Scale bar 40 μm. Groups: vehicle-treated control (Co), cinaciguat-treated control (CoCin), vehicle-treated diabetic (DiabCo) and cinaciguat-treated diabetic (DiabCin). Bar graphs represent mean ± SEM of 9–11 experiments per group. *P < 0.05 vs. Co, †P < 0.05 vs. DiabCo (Tukey HSD test) BODY.RESULTS.EFFECTS OF DIABETES MELLITUS AND CINACIGUAT TREATMENT ON MYOCARDIAL NITRO-OXIDATIVE STRESS: DM was associated with increased NT immunoreactivity in LV myocardium referring to pronounced nitro-oxidative stress which was significantly alleviated by cinaciguat treatment (Fig. 2a, b). Increased expression of HSP70a1, catalase, gluthatione-reductase and thioredoxin-1 was found in DM (Fig. 2c). HSP70a1 and gluthatione-reductase mRNA expression in the DiabCin group remained on the level of healthy controls (Fig. 2c). SOD-2 expression did not show any difference among the groups (Fig. 2c).Fig. 2Cinaciguat treatment alleviates diabetes mellitus related oxidative stress. a Representative images of nitrotyrosine (NT) immunohistochemistry. Magnification: ×200, Scale bar 40 μm. b Quantification of NT immunohistochemistry (P value of diabetes × treatment interaction in the two-factorial ANOVA, Pinteraction <0.001) (n = 9–11/group). (c) Relative mRNA expression of catalase, gluthatione-reductase (Pinteraction = 0.046), heat shock 70 kD protein 1A (HSP70a1) (Pinteraction = 0.021), superoxide dismutase (SOD)-2 and thioredoxin-1 (n = 5–6/group). Groups: vehicle-treated control (Co), cinaciguat-treated control (CoCin), vehicle-treated diabetic (DiabCo) and cinaciguat-treated diabetic (DiabCin). Bar graphs represent mean ± SEM. *P < 0.05 vs. Co, †P < 0.05 vs. DiabCo (Tukey HSD test) BODY.RESULTS.CINACIGUAT PROTECTS AGAINST DM RELATED ALTERATION OF THE NO-SGC-CGMP-PKG SIGNALLING: Protein expression of eNOS and sGC β1 did not differ between healthy and diabetic rats (Fig. 3a) while eNOS gene expression was significantly lower in both diabetic groups (Fig. 3b). We detected elevated PDE-5 and PKG protein expression in the DiabCo group (Fig. 3a) whereas the p-VASP/VASP ratio (marker of PKG activity) was significantly reduced, showing severe deterioration of PKG signalling in DM (Fig. 3a). Application of cinaciguat in diabetic animals significantly reduced the expression of PDE-5, markedly increased PKG activity (as indicated by elevated p-VASP/VASP ratio) (Fig. 3a), while the expression of PKG did not differ between the two diabetic groups (Fig. 3a).Fig. 3The effect of diabetes mellitus and cinaciguat on myocardial NO-sGC-cGMP-PKG signalling. a Relative protein expression and representative immunoblot bands of endothelial nitric oxide synthase (eNOS), soluble guanylate cyclase β1 (sGC β1), phosphodiesterase 5A (PDE-5) (P value of diabetes × treatment interaction in the two-factorial ANOVA, Pinteraction = 0.024), protein kinase G (PKG), vasodilator-stimulated phosphoprotein (VASP) to phospho-VASP (p-VASP) ratio (Pinteraction = 0.003). b Relative gene expression of eNOS (Pinteraction = 0.039). Groups: vehicle-treated control (Co), cinaciguat-treated control (CoCin), vehicle-treated diabetic (DiabCo) and cinaciguat-treated diabetic (DiabCin). Bar graphs represent mean ± SEM of 5–6 experiments per group.*P < 0.05 vs. Co, †P < 0.05 vs. DiabCo (Tukey HSD test) BODY.RESULTS.CINACIGUAT TREATMENT PROTECTS AGAINST DM RELATED FIBROTIC REMODELLING OF THE MYOCARDIUM: DM was associated with dysregulation of the MMP system indicated by markedly increased MMP-9/TIMP-1 and reduced MMP-2/TIMP-2 gene expression ratios (Fig. 4a). These alterations were attenuated in the DiabCin group (Fig. 4a). Although fibronectin expression remained unchanged, Col1 and Col3 expression levels were significantly lower in both diabetic groups (Fig. 4a). The profibrotic TGF-β1 showed increased expression in the diabetic animals, which was significantly ameliorated by cinaciguat (Fig. 4c). Expression of MMP-9 showed a twofold increase in DM, while MMP-2 remained unchanged (Fig. 4c). Cinaciguat did not significantly alter these parameters (Fig. 4c). We found severe interstitial fibrosis of diabetic myocardium indicated by increased MT staining (Fig. 4c). Additionally, elevated immunoreactivity was observed against the profibrotic mediator TGF-β1 and fibrosis marker fibronectin in the diabetic heart (Fig. 4c). Application of cinaciguat reduced MT staining intensity of diabetic myocardium (Fig. 4c) while TGF-β1 immunoreactivity strongly tended to decrease (P = 0.051) (Fig. 4c).Fig. 4Effects of diabetes mellitus and cinaciguat on myocardial fibrotic remodelling. a Relative gene expression values of matrix metallopeptidase (MMP)-9 (P value of diabetes × treatment interaction in the two-factorial ANOVA, Pinteraction = 0.038), tissue inhibitor of MMP (TIMP)-1, MMP-9/TIMP-1 ratio (Pinteraction < 0.001), MMP-2, TIMP-2, MMP-2/TIMP-2 ratio, collagen 1a1 (Col1), 3a1 (Col3) and fibronectin (n = 5–6/group). b Representative images of Masson's trichrome (MT) staining and representative immunohistochemical images of profibrotic mediator transforming growth factor (TGF)–β1 and fibrosis marker fibronectin. Quantification of MT staining (Pinteraction = 0.002), TGF-β1 (Pinteraction = 0.029) and fibronectin immunohistochemistry. Magnification: ×400, Scale bar 40 μm. (n = 9–11/group) c Relative protein expression and representative immunoblot bands of TGF-β1 (Pinteraction = 0.049), MMP-2 and MMP-9. (n = 5–6/group) Groups: vehicle-treated control (Co), cinaciguat-treated control (CoCin), vehicle-treated diabetic (DiabCo) and cinaciguat-treated diabetic (DiabCin). Bar graphs represent mean ± SEM. *P < 0.05 vs. Co, †P < 0.05 vs. DiabCo (Tukey HSD test) BODY.RESULTS.DM RELATED MYOCARDIUM HYPERTROPHY AND APOPTOSIS IS ALLEVIATED BY CINACIGUAT: Myocardial mRNA expression data from LV myocardium of diabetic rats showed a marked increase of ANF and the β-MHC/α-MHC ratio (Fig. 5a). Treatment with cinaciguat caused a significant decrease of ANF expression in DM (Fig. 5a) while β-MHC/α-MHC ratio showed slight decrease (Fig. 5a). When compared with controls, increased cardiomyocyte width was observed in the DiabCo group indicative of cardiomyocyte hypertrophy. This increase was completely prevented by cinaciguat (Fig. 5b, c). The mRNA expression levels of proapoptotic BAX and antiapoptotic Bcl-2 did not differ among the study groups resulting in unchanged BAX/Bcl-2 ratio (Fig. 5a). DM was associated with increased TUNEL positivity in LV myocardium referring to pronounced DNA fragmentation (Fig. 5b, c). TUNEL positivity was effectively reduced in the DiabCin group (Fig. 5b, c).Fig. 5Effects of diabetes mellitus and cinaciguat on myocardial hypertrophy and apoptosis. a Relative mRNA expression of pathological hypertrophy markers atrial natriuretic factor (ANF) (P value of diabetes × treatment interaction in the two-factorial ANOVA, Pinteraction = 0.036), β myosin heavy chain (MHC) to α-MHC ratio and the apoptosis marker Bcl2-associated X protein (BAX) to B-cell CLL/lymphoma 2 (Bcl-2) ratio (n = 5–6/group). b Mean cardiomyocyte diameter (Pinteraction = 0.002) (as marker of cardiomyocyte hypertrophy) and quantification of TUNEL-positive cardiomyocyte nuclei (Pinteraction = 0.009). (n = 9–11/group). c Representative images of hematoxylin—eosin stained sections and TUNEL assay of the left ventricle. Magnification: ×400, Scale bar 40 μm. Groups: vehicle-treated control (Co), cinaciguat-treated control (CoCin), vehicle-treated diabetic (DiabCo) and cinaciguat-treated diabetic (DiabCin). Bar graphs represent mean ± SEM. *P < 0.05 vs. Co, †P < 0.05 vs. DiabCo (Tukey HSD test) BODY.RESULTS.IN VIVO CARDIAC FUNCTION IS IMPROVED BY CINACIGUAT IN DM: In comparison with non-diabetic controls DiabCo group showed remarkably reduced MAP, LVSP, EF, SW, dP/dtmax and impaired dP/dtmin values, while LVEDP and Tau increased, indicating LV systolic and diastolic dysfunction (Table 2). HR significantly decreased in the diabetic groups while CO was not significantly different among the study groups (Table 2). MAP, LVSP, SW, dP/dtmax and dP/dtmin remained unchanged in the DiabCin group, however drug treatment markedly improved LVEDP and Tau in DM (Table 2). As a result of cinaciguat treatment EF tended towards improvement (P = 0.054) in DM (Table 2).Table 2Basic hemodynamic data of the study groupsVariableCoCoCinDiabCoDiabCinPdiabetes Ptreatment Pinteraction HR (beats/min)231 ± 11246 ± 12208 ± 8*204 ± 10*0.0030.6080.351MAP (mmHg)80.0 ± 2.081.0 ± 3.563.7 ± 2.5*64.4 ± 3.1*<0.0010.8450.969LVSP (mmHg)99.5 ± 2.6103.5 ± 2.185.5 ± 1.3*82.3 ± 2.3*<0.0010.8580.102SW (mmHg μl)14,561 ± 106013,293 ± 9489789 ± 592*12,032 ± 10670.0030.6110.072CO (μl/min)42,347 ± 247241,306 ± 280433,360 ± 216238,605 ± 42880.0570.4840.297EF (%)70.42 ± 2.5068.17 ± 2.7058.09 ± 2.54*68.02 ± 2.560.0210.1460.024dP/dtmax (mmHg/s)6539 ± 2406804 ± 1884933 ± 207*4785 ± 230*<0.0010.7910.350dP/dtmin (mmHg/s)−6135 ± 362−6570 ± 446−3883 ± 133*−3723 ± 248*<0.0010.6790.374LVEDP (mmHg)7.0 ± 0.67.2 ± 0.49.7 ± 0.7*6.8 ± 0.3†0.0260.0820.034Tau (Weiss; ms)10.3 ± 0.310.1 ± 0.317.3 ± 0.8*14.9 ± 0.6*†<0.0010.0160.054Heart rate (HR), mean arterial pressure (MAP), maximal left ventricular (LV) systolic pressure (LVSP), stroke work (SW), cardiac output (CO), ejection fraction (EF), maximal slope of systolic pressure increment (dP/dtmax) and diastolic pressure decrement (dP/dtmin), LV end-diastolic pressure (LVEDP) and time constant of LV pressure decay (Tau) are shown. Groups: vehicle-treated controls (Co), cinaciguat-treated controls (CoCin), vehicle-treated diabetic (DiabCo) and cinaciguat-treated diabetic (DiabCin) animals. Values are mean ± SEM of 9–11 experiments per group* P < 0.05 vs. Co †P < 0.05 vs. DiabCo (Tukey HSD test) The values of load-independent, P–V-loop derived contractility indexes (Ees, PRSW) were significantly reduced in diabetic animals indicating severe contractile dysfunction (Fig. 6a, b) Treatment with cinaciguat led to a significant increase in PRSW (Fig. 6b) while Ees showed a strong tendency towards improvement in DM (P = 0.092) (Fig. 6b).Fig. 6Effect of cinaciguat on left ventricular (LV) contractility and cardiac stiffness in diabetes mellitus. a Representative pressure–volume (P–V) loops, b preload recruitable stroke work (PRSW) (P value of diabetes × treatment interaction in the two-factorial ANOVA, Pinteraction = 0.003), slope (Ees) of LV end-systolic P–V relationship (Pinteraction = 0.006) and the slope of LV end-diastolic P–V relationship (EDPVR) (Pinteraction = 0.004) are presented in vehicle-treated control (Co), cinaciguat-treated control (CoCin), vehicle-treated diabetic (DiabCo) and cinaciguat-treated diabetic (DiabCin) groups. Bar graphs represent mean ± SEM of 9–11 experiments per group. *P < 0.05 vs. Co, †P < 0.05 vs. DiabCo (Tukey HSD test) In comparison with Co group the slope of EDPVR tended to increase (P = 0.063) in diabetic animals, which was significantly reduced by cinaciguat (Fig. 6b). Cinaciguat had no hemodynamic effects in non-diabetic rats. BODY.DISCUSSION: In the current study we investigated the effects of the sGC activator cinaciguat on DM related cardiac alterations in an experimental type-1 diabetic animal model. We found that chronic application of cinaciguat did not affect blood glucose levels, however it effectively raised cGMP levels and restored cGMP-PKG signalling in the myocardium. These molecular changes were associated with attenuated cardiomyocyte hypertrophy, less severe fibrotic remodelling and markedly improved systolic and diastolic cardiac performance. Rat model of STZ induced type-1 DM is widely used in research studies aiming at the characterisation of diabetic cardiomyopathy and its possible new therapeutic approaches. STZ is taken up via glucose transporter 2 to the pancreatic beta cells which ensures its selective toxicity for beta cells and the ability to induce insulin-dependent DM. In the present study increased blood glucose and daily water intake levels indicated obvious evidence of pronounced DM in STZ-treated rats which was concordant with earlier data [31]. The most important cause in DM that presumably leads to severe complications, including diabetic cardiomyopathy, is hyperglycemia. The imbalance in glucose homeostasis triggers numerous mechanisms and signalling pathways which result in cardiovascular dysfunction. Despite the fact, that many studies investigated its pathological background so far, the exact pathomechanisms of diabetic cardiomyopathy are still poorly understood. It is known that hyperglycemia increases production of ROS and RNS and subsequent upregulation of endogenous antioxidant systems and overexpression of heat shock proteins [32] through several mechanisms [1]. As a marker of excessive nitro-oxidative stress [33] a significantly higher NT positivity was shown in the diabetic myocardium which might have contributed to the observed induction of endogenous antioxidant systems (see thiorexodin-1, catalase or gluthatione-reductase gene expression levels) and to a remarkable heat shock response (increased HSP70a1 level). Pronounced nitro-oxidative stress can lead to the oxidation of the sGC enzyme which causes the loss of its heme prosthetic group and results in a NO-insensitive (inactive) form of sGC. Additionally, an imbalance of the different types of phosphodiesterases was presented in the failing heart which might have contributed to the rapid degradation of intracellular cGMP [34]. Decreased cGMP levels and subsequent impairment of NO-cGMP-PKG signalling is a well-known feature occuring in the diabetic myocardium. This observation was confirmed by our results showing reduced myocardial cGMP content in diabetic rats while sGC β1 and eNOS protein expression remained unchanged. Moreover, NO-cGMP-PKG signalling showed a severe deterioration in the diabetic group indicated by a reduced gene expression level of eNOS, increased PDE-5 protein levels (in accordance with previous data [35]) and significantly reduced phosphorylation of the PKG target VASP (lower p-VASP/VASP ratio, an index of PKG activity [23]). However, elevated PKG protein expression was observed which might reflect an ineffective compensatory mechanism in the LV. The presumable loss of the widely described cGMP related cytoprotection was associated with a four times higher extent of DNA damage in DM compared with controls (indicated by the TUNEL reaction). The observed discrepancy between impairment of myocardial cGMP-signalling and unchanged plasma cGMP levels in DiabCo vs. Co animals might be the consequence of diabetes-associated increase in ANF levels and the subsequent activation of natriuretic peptide receptors and particulate guanylate cyclase (pGC) in organs other than the heart, as plasma cGMP is seen as an overspill of intracellular cGMP from various tissues [36]. The sGC activator cinaciguat has been described to exert disease specific effects since it preferentially activates the oxidised, inactive form of the enzyme (that is present mainly in diseased conditions) thus restores its cGMP producing ability [17] in various pathological conditions associated with excessive nitro-oxidative stress [19, 21]. With the application of cinaciguat we achieved massive cytoprotective effects in DM, that was confirmed by lower NT staining (indicative of attenuated nitro-oxidative stress), normalised expression of endogenous antioxidant and heat shock protein systems as well as lower extent of DNA breaks (less severe TUNEL positivity). The mechanism by which cinaciguat decreased nitro-oxidative stress remains unclear and needs further investigations. A possible explanation might be the cGMP-induced down-regulation of NADPH-oxidases, as proposed by a recent experimental work [37]. By reducing oxidative damage to the myocardium with cinaciguat, pathologically high levels of PDE-5 enzyme reversed to the level of control animals, which might have contributed to the accumulation of cGMP in the diabetic heart and plasma, too. The augmented cGMP production consequently reinforced the impaired PKG activity (proven by markedly increased myocardial p-VASP/VASP ratio) that further contributed to the cardioprotective effects of cinaciguat in DM. Pathological remodelling is a well-known phenomenon in the diabetic myocardium, however there are several remaining questions regarding the underlying mechanisms to be clarified. A number of key pathophysiological features such as cardiac hypertrophy, fibrotic remodelling, myocardial tissue injury with DNA fragmentation are lying in the background of diabetic cardiomyopathy [1]. In our diabetic rat model pathological cardiomyocyte hypertrophy was developed demonstrated by increased cardiomyocyte diameter, increased ANF gene expression values and a shift in the β/α-MHC expression ratio. Chronic pharmacological activation of sGC showed antihypertrophic effects not only on the histological level (decreased cardiomyocyte diameter) but also in the molecular environment (significantly reduced ANF gene expression, strong tendency towards reversed shift in β/α-MHC ratio). Our results are concordant with data from a recent publication [23] demonstrating antihypertrophic and antifibrotic effects of cinaciguat in cultured cells. Fibrotic remodelling of the diabetic myocardium is strongly associated with dysregulation of MMPs [1], increased fibroblast proliferation [38], decreased Col1 and Col3 mRNA expression [39] and TGF-β1 signalling [40]. In accordance with previous literature data we found an upregulation of the profibrotic mediator TGF-β1, downregulation of Col1 and Col3 mRNA levels, intense MT and fibronectin staining (as fibrosis markers) and increased DNA fragmentation in diabetic cardiomyopathy. Data from our experiments revealed that treatment with the sGC activator cinaciguat effectively prevented pathological changes in the myocardium by improving MMP dysregulation (reverting altered MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios), by decreasing myocardial level of TGF-β1 and preventing fibrotic changes, as well as repressing myocardial TUNEL positivity. Interestingly, we did not find any differences in BAX/Bcl-2 ratio among the study groups which suggest that classical apoptotic pathways are not involved in the observed beneficial effects. A possible explanation might be a cross-talk between sGC-cGMP axis and TGF-β-dependent extracellular signal-regulated kinase signalling (as reported by Beyer et al. [41]), however this hypothesis requires further investigations. Several clinical and experimental studies have focused on the investigation of cardiac function in DM. Both systolic and diastolic dysfunctions have been described in type-1 diabetes in the literature using different non-invasive and invasive methods [2, 31]. We found similar alterations in our current study regarding DM related cardiac dysfunction. We observed a significant reduction of MAP (probably as a consequence of diabetic polyuria [42]) in DM compared with controls that was not affected by chronic cinaciguat treatment. Whether cinaciguat administration acutely affects MAP in diabetic rats, needs further non-invasive investigations. DiabCo group showed significantly deteriorated systolic function (as reflected by decreased LVSP, SW, EF and dP/dtmax). EF and dP/dtmax are widely used to assess contractile function, however they are known to be affected by pre- and afterload [29]. Therefore, in the present study we determined P–V-loop derived, pre- and afterload independent contractility parameters (Ees, PRSW) that are more informative on intrinsic myocardial contractile function [29]. The specific indexes of LV contractility showed severe systolic dysfunction in the DiabCo group. As a result of sGC activation a remarkable improvement of systolic function has been achieved in diabetic animals: a significant increase of PRSW clearly reflects improved LV contractility, whereas EF and SW were restored to the levels of healthy controls. When investigating diastolic function we found significantly deteriorated LV active relaxation (as shown by impaired dP/dtmin and prolonged Tau) and markedly elevated LV diastolic stiffness (indicated by higher LVEDP and strong tendency towards increased EDPVR) in DM compared with controls. Cinaciguat effectively improved not only active relaxation (by remarkably shortening Tau) but also diastolic stiffness (by reverting LVEDP and EDPVR values). Study limitations: Results in the current study are limited to young, male, type-1 diabetic rats. Rats did not receive any glucose-lowering medication. Acute effects of pharmacological activation of sGC on hemodynamics were not investigated. A possible explanation of cinaciguat's beneficial effects in DM could be the improvement of cardiac microvascular perfusion, however that needs further investigations. BODY.CONCLUSIONS: To our knowledge, this is the first study reporting the effects of pharmacological sGC activation on diabetic cardiomyopathy and DM related myocardial dysfunction. We confirmed that the sGC activator cinaciguat improves pathological features of diabetic cardiomyopathy without affecting blood glucose levels. Molecular mechanisms underlying the advantagous effects of sGC activation include the upregulation of NO-sGC-cGMP-PKG axis which might crosstalk with antioxidant, antihypertrophic and antifibrotic cascades hence preventing cardiac complications of diabetic metabolism and improving DM related cardiac dysfunction. Based on the data presented here pharmacological sGC activation might be a potential therapeutic approach to improve cardiovascular dysfunction in DM.

TITLE: Assessment of Unconscious Decision Aids Applied to Complex Patient-Centered Medical Decisions ABSTRACT.BACKGROUND: To improve patient health, recent research urges for medical decision aids that are designed to enhance the effectiveness of specific medically related decisions. Many such decisions involve complex information, and decision aids that independently use deliberative (analytical and slower) or intuitive (more affective and automatic) cognitive processes for such decisions result in suboptimal decisions. Unconscious thought can arguably use both intuitive and deliberative (slow and analytic) processes, and this combination may further benefit complex patient (or practitioner) decisions as medical decision aids. Indeed, mounting research demonstrates that individuals render better decisions generally if they are distracted from thinking consciously about complex information after it is presented (but can think unconsciously), relative to thinking about that information consciously or not at all. ABSTRACT.OBJECTIVE: The current research tested whether the benefits of unconscious thought processes can be replicated using an Internet platform for a patient medical decision involving complex information. This research also explored the possibility that judgments reported after a period of unconscious thought are actually the result of a short period of conscious deliberation occurring during the decision report phase. ABSTRACT.METHODS: A total of 173 participants in a Web-based experiment received information about four medical treatments, the best (worst) associated with mostly positive (negative) side-effects/attributes and the others with equal positive-negative ratios. Next, participants were either distracted for 3 minutes (unconscious thought), instructed to think about the information for 3 minutes (conscious thought), or moved directly to the decision task (immediate decision). Finally, participants reported their choice of, and attitudes toward, the treatments while experiencing high, low, or no cognitive load, which varied their ability to think consciously while reporting judgments. Cognitive load was manipulated by having participants memorize semi-random (high), line structured (low), or no dot patterns and recall these intermittently with their decision reports. Overall then, participants were randomly assigned to the conditions of a 3 (thought condition) by 3 (cognitive-load level) between-subjects design. ABSTRACT.RESULTS: A logistic regression analysis indicated that the odds of participants choosing the best treatment were 2.25 times higher in the unconscious-thought condition compared to the immediate-decision condition (b=.81, Wald=4.32, P=.04, 95% CI 1.048-4.836), and 2.39 times greater compared to the conscious-thought condition (b=.87, Wald=4.87, P=.027, 95% CI 1.103-5.186). No difference was observed between the conscious-thought condition compared to the immediate-decision condition, and cognitive load manipulations did not affect choices or alter the above finding. ABSTRACT.CONCLUSIONS: This research demonstrates a plausible benefit of unconscious thinking as a decision aid for complex medical decisions, and represents the first use of unconscious thought processes as a patient-centered medical decision aid. Further, the quality of decisions reached unconsciously does not appear to be affected by the amount of cognitive load participants experienced. BODY.INTRODUCTION.BACKGROUND: To improve or maintain patient health and well-being, it is of course important that patients and/or health-care providers make the best (or at least beneficial) decisions regarding treatment options, behaviors, diagnoses, test options, and so forth. Complicating this, however, these individuals must commonly consider very complex information to make medical decisions. Thus, growing research has investigated decision aids that might benefit patients and health-care providers as they consider complex medical information [1]. Decision aids are generally used to engage both patients and practitioners in the decision process, and allow patients to understand the potential risks and benefits of a given medical choice. Additionally, decision aids often incorporate value clarification exercises to help patients consider the personal values they place on potential risks and benefits. For example, patients may go through a list of values, select the five most important ones, and bear those in mind while making a medical decision. De Vries et al [1] suggest that decision aids can make use of deliberative processes (requiring intentional and analytical thinking) or intuitive processes (which are more affective, unconscious, or automatic) to consider decision-relevant information. Although decision aids that make use of deliberative processes are more common, both types of processes have strengths and weaknesses for medical decisions. Regarding this, De Vries et al warn that decision aid developers "should be aware that the current common practice to encourage patients to extensively analyze available choice options, typically immediately after information exposure, lacks solid theoretical and empirical grounding...and may even have some harmful side effects to preference construction processes" (p. 159 [1]) and suggest that optimal decision aids would take advantage of the complementarity of the two systems [1]. Consistent with this later suggestion, the current paper explores the possibility that unconscious-thinking processes [2]—which theoretically incorporate intuitive processes as well as more time-demanding and analytic unconscious deliberation processes [3-5]—can provide patients with a valuable decision aid for complex medical information. According to Dijksterhuis and Nordgren [2], unconscious thought is the "object-relevant or task-relevant cognitive or affective thought processes that occur while conscious attention is directed elsewhere" (p. 96 [2]), whereas conscious thought involves these same processes, but within conscious awareness. They suggest that unconscious thinking processes make use of vast mental resources, whereas conscious thinking processes rely on limited resources such as working and short-term memory. Thus, they argue, individuals often arrive at better decisions from complex information when they process information unconsciously (while conscious thinking processes are distracted from the relevant decision task). Much research supports this possibility [2,6-11], and generally follows a paradigm established by Dijksterhuis [6]. In this paradigm, individuals receive much information about 3-4 targets (eg, roommates). Further, one target is associated with mostly positive attributes, one with mostly negative attributes, and the other(s) with a balance of positive and negative attributes. Next, participants report their decisions regarding, or preferences for, targets either immediately (allowing for minimal conscious or unconscious thinking), after 3 minutes in which they think about the presented information (ie, think consciously), or after 3 minutes in which they engage in an unrelated task that distracts them from thinking consciously about the presented information (but can still think unconsciously). Typical results demonstrate an "Unconscious Thought Effect" (UTE) such that participants in the "unconscious thought" condition arrive at better decisions (prefer the best over the worst target) relative to participants in the immediate-decision condition. As well, participants in conscious-thought conditions often arrive at decisions comparable to those in the immediate-decision conditions. Since Dijkterhuis's first article reporting the UTE [6], much research has either replicated the effect or called the effect into question. A recent Bayesian meta-analysis of 16 studies conducted by Newell and Rakow [12] does not support the existence of the UTE, and a study by Huizenga and colleagues [13] provides additional evidence against the merits of unconscious thought processes. Conversely, a meta-analysis of 92 studies conducted by Stick et al [14] suggest that the UTE is modest but reliable, and research by Creswell and colleagues [15] provides strong fMRI (functional magnetic resonance imaging) evidence consistent with the UTE. Creswell and colleagues addressed important critiques of the UTE by providing physiological evidence that the UTE relies on specific neural reactivation to occur, and that conscious and unconscious thought processes recruit non-overlapping neural regions [15]. Nevertheless, when considering unconscious thought as a potential decision aid, developers ought to consider the present paper as part of a growing literature that deserves a thorough review before justifying any reforms. The meta-analysis conducted by Strick et al [14] demonstrated that the UTE is stronger when the presented information is complex, the goal to make a decision is emphasized and formulated in a holistic fashion, and the decision task is ecologically valid. Thus, unconscious thought may be particularly suited to aid sound medical decisions; medical decisions are typically complex (eg, involving a large number of trade-offs between length and quality of life [1]), patients and health-care providers are generally motivated to find the best course of treatment, and the outcomes of such decisions bear real-life consequences that ensure a level of ecological validity. In fact, an experiment by DeVries and colleagues investigated the UTE in a health context, and demonstrated that in-training clinical-psychologists (graduate students) achieve more accurate psychiatric diagnoses following a period of unconscious versus conscious thinking. However, the present research sought to investigate the potential benefit of unconscious thinking as a decision aid for the broad population of patients (and is the first to our knowledge to do so), without specific health or medical training. This is noteworthy because experts within a given decision domain (eg, training clinicians) demonstrate the UTE more than non-experts [16] (eg, patients and the lay-public generally). Thus, demonstrating this effect on medically related decisions—relative to a "non-thinking" control group—even among a general sample could reveal that unconscious thought is a useful decision aid for making complex medical decisions that affect patient health and well-being. Further, the reported research addresses a potential methodological criticism of past unconscious-thought research: participants could theoretically think consciously while reporting their decisions (ie, during the decision phase of the experiment), even following a period of distraction. If true, the UTE might actually result from conscious thinking processes. We explore this possibility by manipulating the amount of cognitive load participants experience during the decision phase of the experiment. This is a relevant and ecologically valid manipulation given the cognitively demanding context of many medical environments, and is novel within the unconscious-thought literature. BODY.INTRODUCTION.CURRENT AIMS, EXPERIMENT OVERVIEW, AND HYPOTHESES: The aim of the current research was to test whether patient-centered decisions regarding complex treatment options are better following a period of unconscious thought relative to immediate decisions, indicating unconscious thought can be a beneficial decision aid. For this initial investigation, a Web-based sample of participants received a cover story entailing "their" recent hospital admission and diagnosis. Next, all participants received side-effect/attribute information for four potential treatments, one of which was the best, one the worst, and two of which were in the middle. Following this, participants were randomly assigned to a thought condition in which they completed a distraction task for 3 minutes (unconscious), deliberated for 3 minutes (conscious), or were given no time (immediate decision), before reporting their judgments about the treatments. Finally, participants rendered their judgments while under a high, low, or no cognitive load. Thus, overall, participants were randomly assigned to the conditions of a 3 (thought condition) by 3 (cognitive-load level) between-subjects design. Participant's choice of treatment, and attitude ratings of each treatment, were recorded. The primary hypothesis was that we would observe a UTE such that participants in the unconscious-thought condition (but not participants in the conscious-thought condition) would choose the best treatment relative to the immediate-decision condition (control group). This same effect was predicted for participants' treatment attitudes, although this measure is less critical than investigating actual treatment choices; the choices patients and health practitioners make tend to be more consequential to health outcomes than their attitudes toward various treatments. Additionally, we predicted that participants in the unconscious-thought condition would choose (and form more favorable attitudes toward) the best treatment relative to the conscious-thought condition. Although this is not critical to demonstrating the UTE per se, this prediction is consistent with much of the unconscious-thought literature [14], and speaks directly to the possibility that unconscious thought may be an effective decision aid relative to purely deliberative decision aids. Further, we propose two competing exploratory hypotheses regarding the effect of cognitive load during the decision phase. First, if participants in the unconscious-thought condition actually generate their decisions consciously during the decision phase, then their decisions should become worse as cognitive load increases, and the UTE should only manifest under no- and low-load conditions. Second, if participants in the unconscious-thought condition truly generate their decisions unconsciously, then their decisions should be comparable across load conditions, and the UTE should occur unaffected by cognitive load. BODY.METHODS.PARTICIPANTS: A total of 173 Amazon Mechanical Turk workers participated in this Web-based experiment. The Institutional Review Board of the Montana State University approved all procedures in advance. Participants were compensated with US $0.50, and (retroactively) an additional $1.00 bonus for choosing the best treatment option. Participants were 87 males (50.3%) and 86 females (49.7%) with ages ranging from 18 to 73 years (mean 28.38, SD 8.18). One participant reported that he/she was 2 years old (we assumed this was a "typo" and the individual intended to report an age in the twenties); excluding this participant from the analyses had no effect on the results. In total, 135 participants categorized their ethnicity as White/Caucasian (78.0%), 12 as Black/African American (6.9%), 11 as Asian (6.4%), 3 as Native American (1.7%), and 12 as "other" (6.9%). Further, we analyzed these participant characteristics independently as a function of the independent variables, and found no significant effects (all Ps>.15). Thus, our random assignment procedure succeeded at distributing participants evenly across the experiment conditions. BODY.METHODS.MATERIALS: Treatment side-effects/attributes were first pretested using a separate sample of 52 Amazon Mechanical Turk workers. Participants from this sample rated each of 75 side-effects/attributes on valence and importance. Specifically, participants were asked to "Rate the following side effect in terms of how positive/negative it is", then were randomly shown one of the 75 side-effects/attributes and responded on a 9-point scale ranging from 1 (very negative) to 9 (very positive). Following, participants were shown the same side-effect/attribute and asked to "Rate the following side effect in terms of how important it is" on a 9-point scale ranging from 1 (very unimportant) to 9 (very important). Of note, positive side effects/attributes in the present design were independent of the intended treatment effects (to cure the patient), but were considered to be positive or beneficial. A total of 35 (17 positive and 18 negative) side-effects/attributes were selected as stimuli for the current experiment (see Multimedia Appendix 1 for selected side-effects/attributes and pre-test ratings). We chose side-effects/attributes with moderate pre-test ratings on both valence and importance dimensions to ensure that one or a few side-effects/attributes would not dominate choices, thereby oversimplifying the decision process. Using these pre-test data, we then assessed the actual quality of each treatment that was used in the main experiment by weighting the valance of treatment side-effects/attributes by importance. This follows logically from research conducted by Bos and colleagues [5], which demonstrates that unconscious thought makes use of valance and importance information in a logical way (approximately weighting valance by importance) to make sound decisions. For example, a highly positive side-effect/attribute of low importance can affect decisions less than a mildly positive side-effect/attribute of high importance. Thus, for each treatment, we first multiplied the valance rating by the importance rating for each positive side-effect/attribute, then summed these products. We did this again for each negative side-effect/attribute, independently for each treatment. Next, for each treatment, we subtracted the resulting sum for the negative side-effects/attributes from the sum for the positive side-effects/attributes. This created a "quality rating" for each treatment (more positive numbers indicate higher quality), which was analyzed using contrasts within a repeated-measure ANOVA (analysis of variance). These contrasts confirmed that the best treatment was viewed as having better quality (mean 297.75, SD 139.32) than the two balanced treatments (mean 128.92, SD 110.67 and mean 137.48, SD 117.76; F 1,51=366.95, P<.001), and the worst treatment was viewed as having lower quality (mean=−48.71, SD 88.73) than the two balanced treatments (F 1,51=308.51, P<.001). Further, the two balanced treatments were rated comparably, F<1. BODY.METHODS.PROCEDURE.INTERNET SAMPLE AND PLATFORM: Participants were recruited through Amazon Mechanical Turk (a Web-based crowdsourcing marketplace), and redirected to Qualtrics (a survey website) to complete the experiment. Amazon Mechanical Turk users were eligible to participate if they resided in the United States, had completed over 100 remunerated tasks (known as Amazon Mechanical Turk HITS), and had an approval rating over 90% (meaning that 90% of tasks completed by users where deemed worthy of remuneration by previous employers). Data collection started on April 30, 2014 and finished on May 9, 2014; participants had the option of leaving the experiment at any time but were unable to return to previous pages. A completeness check was automatically recorded by Qualtrics (dependent upon viewing the last page of the survey), and the completion rate was 59.6% (173/290). Incomplete surveys were not included in the final analyses. Amazon Mechanical Turk's account registration system was used to prevent multiple entries (a given account could only complete the experiment once). The average time of survey completion was 13.5 minutes; no atypical timestamp surveys were excluded. BODY.METHODS.PROCEDURE.EXPERIMENT FLOW: Participants were first presented with a consent form, completed a demographic questionnaire, and then read the following scenario: Please imagine yourself as a recently admitted patient at a hospital. The doctors have diagnosed you with a Campylobacter infection. They then present you with different treatment options which all have a large number of positive and negative side effects. Since all of the treatments will treat the Campylobacter infection, the only basis for comparison are their associated side effects. Also, given the progression of the infection, a decision must be taken in the next few minutes. This part of the experiment is concerned with the way in which we form an impression on the basis of a number of attributes. In a few moments you will be presented with four treatments along with side effects that each of the treatments possess. Please read these sentences carefully, study each one until the next appears. Later, we will ask you a series of questions concerning the impressions that you have formed of the four different treatments. Following, participants were told that choosing the best treatment option would grant them a $1 bonus. The possibility for bonus remuneration helped ensure that participants were motivated to choose the best treatment. Subsequently, each participant was sequentially shown 48 side-effects/attributes in random order. Each side-effect/attribute was presented for 4 seconds and attributed to one of four treatment options. Overall, one option was best (8 positive and 4 negative side-effects/attributes), one was worst (4 positive and 8 negative side-effects/attributes), and the other two were balanced (6 positive and 6 negative side-effects/attributes). This type of stimuli presentation was used to ensure decision complexity following past research methods [6-8]. Next, participants were randomly assigned to either an unconscious-thought, conscious-thought, or immediate-decision condition. Finally, participants were randomly assigned to render their treatment choices and attitudes while experiencing high, low, or no cognitive load (details for all manipulations provided below). Last, participants were debriefed, thanked for their participation, and given a code to redeem compensation through Amazon Mechanical Turk. BODY.METHODS.INDEPENDENT VARIABLES.THOUGHT CONDITION: After participants received all of the side-effect/attribute information, they were randomly assigned to one of three thought conditions. Participants in the unconscious-thought (or distraction) condition were instructed to complete as many anagrams as they could within 3 minutes, and were presented with a list of 36 anagrams. This task is commonly used in unconscious-thought experiments to consume and distract conscious thought, yet allows unconscious thought to continue processing decision-relevant information. Participants in the conscious-thought condition read the following instructions: "For the next 3 minutes, consider the four different treatments and the side effects you read about. Think about which treatment is the best and/or which treatment you like the most. Try to only think of the treatments and which treatment you might personally prefer." Thus, these participants were specifically instructed to think consciously about the side-effect/attribute information, and had time to do so. Finally, participants in the immediate-decision condition directly moved on to the judgment task, and had insufficient time to think consciously or unconsciously about the side-effect/attribute information. BODY.METHODS.INDEPENDENT VARIABLES.COGNITIVE LOAD: Cognitive load was manipulated using 4 x 4 matrices with 4 dots presented within 16 possible locations. The manipulation stimuli were modeled after Haymen et al [17], who demonstrated their effectiveness in producing high or low cognitive load. High cognitive-load manipulations consisted of a semi-random scatter, whereas the low cognitive-load manipulation consisted of a 4 dot line (see Multimedia Appendix 2 for actual stimuli). Participants were instructed to memorize the exact pattern and warned that they would later be asked to reproduce it. Ultimately, participants reported judgments about the treatments interspersed with the load manipulations. Specifically, the decision phase entailed a repeated sequence of events: pattern exposure, treatment choice, pattern recall, new pattern exposure, treatment evaluations, pattern recall (see Figure 1). Participants in the no cognitive-load condition were only presented with the judgment tasks. We reasoned that the semi-random dot patterns were difficult to memorize and that participants in the high cognitive-load condition would not be able to consciously process the side-effects/attributes information and rehearse the dot pattern as they were reporting their choice/attitude ratings. Conversely, the linear dot patterns (low cognitive load) would require little active cognition to maintain in working memory and therefore could allow participants to engage in conscious deliberation. Figure 1Flow of the decision phase. The first decision task consisted of the choice variable while the second decision task consisted of the attitude rating. The high cognitive load is displayed for illustrative purposes; the actual pattern depended on cognitive load condition. BODY.METHODS.DEPENDENT MEASURES.CHOICE: Participants were asked to make two main treatment judgments. First, participants were instructed to: "Choose a treatment" and could click on one of the four treatment options. This is the primary dependent measure, as this choice directly affects patient health outcomes and experienced side-effects. For this measure, participants who chose the best treatment option were scored "1", whereas participants who chose any of the other options were scored "0". BODY.METHODS.DEPENDENT MEASURES.ATTITUDE MEASURE: Next, participants separately rated each of the four treatments (eg, "Your impression of Treatment A was...") on scales ranging from −25 (very negative) to 25 (very positive). Following previous research in the unconscious-thought literature, participants' rating for the worst treatment was subtracted from their rating for the best treatment, resulting in an attitude preference measure. Higher numbers on this measure indicate a more positive rating for the best over the worst option. BODY.RESULTS.MANIPULATION CHECK: On average, participants in the low cognitive-load condition correctly recalled significantly more dots (mean 6.91, SE 0.29) than participants in the high cognitive load condition (mean 5.73, SE 0.25; t 110=−3.031, P=.003). This worse recall in the high versus low cognitive-load conditions supports the idea that the pattern task was more cognitively demanding than in the former, and is consistent with the results and interpretation of Heyman et al [17]. BODY.RESULTS.DEPENDENT VARIABLES.CHOICE: We conducted a hierarchical logistic regression to predict participants' choice for the best treatment, entering Thought Condition as a predictor in the first step (given this was a theoretically important variable), Cognitive-Load Condition as an additional predictor in the second step (given it was exploratory), and the interaction between these two factors as yet an additional predictor in the third step. As hypothesized, the first step revealed a main effect of thought condition; this predictor demonstrated a significant improvement over the constant-only model (χ 2 2=6.39, P=.04, Nagelkerke R 2=.05), and this single-predictor model fit the data well (for the Hosmer and Lemeshow test, P=.81). Further, there was no main effect of cognitive-load condition, nor a Thought Condition by Cognitive-Load Condition interaction. Adding Cognitive-Load in the second step revealed no improvement in the model (χ 2 2=1.54, P=.46), nor did adding the interaction term in the third step (χ 2 4=3.83, P=.43). Given this, we interpreted the main effect of thought condition based on the first step of the analysis. Demonstrating a UTE, the odds of participants choosing the best treatment were 2.25 times higher in the unconscious-thought condition compared to the immediate-decision condition (b=.81, Wald=4.32, P=.04, 95% CI 1.048-4.836), and 2.39 times greater in the unconscious-thought condition compared to the conscious-thought condition (b=.87, Wald=4.87, P=.027, 95% CI 1.103-5.186). Further, the odds of choosing the best treatment were comparable across the conscious-thought and immediate-decision conditions (b=−.06, Wald=0.22, P=.88; for the constant, b=−.88, Wald=9.31, P=.002). Overall, thought condition was the only significant predictor of choice, and participants in the unconscious-thought condition demonstrated a higher probability of making the correct choice versus the control condition (see Table 1 for choice contingency table). Further, participants overall found the decision task quite complex, given 112 of 173 participants (64.7%) chose an incorrect treatment option. This is important given UTEs tend to manifest in complex decision tasks. We also explored choice as a function of cognitive-load condition separately for each thought condition. None of these analyses demonstrated a significant effect of cognitive load (all X 2< 2.56, all Ps>.05).Of note, if correct decisions arising from unconscious thought actually result from conscious thought during the decision phase, then higher levels of load should result in worsening decisions within the unconscious thought conditions. But, if anything, load resulted in better choices in the unconscious thought conditions (see Table 1). Nonetheless, so as to fully assess the interaction of cognitive load and thought conditions, we also reanalyzed our data after combining the immediate and conscious-thought conditions (unconscious thought vs others), and after combining the low- and high-load conditions (no load vs load). Again, this analysis revealed an effect of thought condition (b=.84, Wald=6.34, P=.012, 95% CI 1.205-4.463), and again the effect of load condition and the interaction of thought and load were not significant (all Ps>.28). Further, we found the power of our experiment to be .73 for this simplified interaction. Thus, our experiment had reasonable power to detect an effect of load on the UTE. Table 1 Contingency table for choice broken down by thought condition and cognitive-load condition (n=173). Thought Condition Cognitive Load Choice Totals Correct Incorrect Unconscious No Load 10 13 23 Low Load 10 8 18 High Load 8 9 17 Total 28 (48.3%) 30 (51.7%) 58 Immediate No Load 3 9 12 Low Load 4 20 24 High Load 10 12 22 Total 17 (29.3%) 41 (70.7%) 58 Conscious No Load 7 19 26 Low Load 4 10 14 High Load 5 12 17 Total 16 (28.1%) 41 (71.9%) 57 BODY.RESULTS.DEPENDENT VARIABLES.ATTITUDE MEASURE: The attitude measure was analyzed using a 3 (thought condition) by 3 (cognitive load condition) between-subjects ANOVA. This analysis revealed no significant effects (all Fs<1.42), and no planned comparisons were significant (ts<1). BODY.DISCUSSION.PRINCIPAL FINDINGS: In this experiment, participants who were distracted for 3 minutes after receiving treatment information—and thus had the opportunity to think unconsciously—were significantly more likely to choose the best treatment option relative to participants who made their choices immediately following the information (or thought consciously). To our knowledge, this is the first replication of the UTE using a patient-oriented medically related decision task. Further, no such advantage was observed for participants who consciously thought about the information for 3 minutes after receiving treatment information. These trends were mirrored in participants' attitudes toward the best versus worst treatment, although not significantly. In the field, however, the choices patients and health practitioners make are more consequential to health outcomes than their attitudes toward various treatments. The choice results are strongly in line with a growing body of evidence demonstrating that individuals are more likely to make the best decisions when they think unconsciously, provided the decision task is complex, they are motivated to be accurate, and the task has ecological validity. These conditions were met in the current research: the task was complex and most participants chose an incorrect treatment, participants were motivated to choose the correct treatment (with a US $1 incentive), and the task was constructed to represent a real-life medical decision (albeit, with fictitious treatment information and a fictitious medical condition). Further, these conditions are clearly met in many medical contexts, as treatment information is often complex and all parties involved are motivated to arrive at correct treatment choice. Thus, unconscious thinking processes may greatly aid decision making within many medical contexts. The present research also explored the possibility that UTEs are not actually the result of unconscious thinking that occurs while people are distracted, but of conscious processes that occur while people solidify and report a judgment. And, to our knowledge, this is the first experiment to investigate this potential alternative account for the UTE. To test this possibility, we varied the cognitive load participants experienced while they reported their choice and attitude judgments, and this manipulation was successful. If the UTE actually results from conscious thinking at the time of judgment, then participants in the unconscious-thought conditions should do worse if they experience high load (compared to low or no load) while reporting their judgments. However, the cognitive-load manipulation had no effect on either of the dependent measures, nor did it interact with thought condition. This overall null effect of cognitive load suggests the UTE does not result from conscious processing at the time of judgment, and judgments are accessed with negligible effort during the decision phase. BODY.DISCUSSION.LIMITATIONS AND FUTURE DIRECTIONS: The reported results indicate that unconscious thought may serve as a beneficial decision aid for patients facing complex medical decisions. But, of course, this initial investigation has limitations and we encourage further research into unconscious thinking in medical contexts before advocating any decision-making reform. Foremost, the tested decision task involved an imaginary scenario and not a personal health event. Still, participants were motivated to arrive at the correct decision and the greater motivation real patients likely experience should theoretically enhance the UTE [5]. Related, the stimuli used in this experiment were fictitious, although designed to appear medically relevant. Future research should employ real information as the basis of decisions and focus on adapting the unconscious thought paradigm to real-life examples such as the trade-offs between length and quality of life faced by older or terminally ill populations or the time-sensitive medical decisions that one may face in an emergency room. Furthermore, the side-effect/attribute information was presented randomly rather than organized by treatment as would normally happen. This was important to create the decision difficulty needed to verify the results stemmed from unconscious, and not conscious, processes in this initial investigation. However, given the present results, future research could further test the benefit of unconscious thought as a decision aid under more realistic informational settings. Finally, we tested our predictions using a geographically dispersed Web-based sample, not with participants in a controlled environment, and cannot ensure strict instruction compliance. An exact laboratory replication could easily address this limitation. At the same time, however, the present results demonstrate the UTE for medical decisions using a Web-based sample, and suggest that a Web-based platform could be used to create decision aids that foster unconscious thinking. Ultimately, it will be critical to demonstrate the UTE in actual medical contexts with participants facing real decisions for themselves or others. As of now, the present research reveals a plausible benefit of unconscious thought as an aid for patients' medical decisions and future research will have to confirm that benefit in commonplace settings, with real information, and with vested decision-makers. The present claim that unconscious judgments come to awareness relatively independently of cognitive load entails several limitations. First, the cognitive load manipulation used in the present experiment was comprised of visuospatial dot patterns whereas the decision task was primarily verbal. To the extent that visuospatial and verbal processing may employ different cognitive resources, it is possible that the present cognitive load manipulation did not interfere with the decision task enough to adequately test our hypotheses. Although visuospatial stimuli were preferred in the present study to help ensure instruction compliance (reporting random dot patterns requires memory processes because they are difficult to reproduce or write down), future studies should make use of verbal cognitive-load manipulations to address this issue. Additionally, participants were instructed to memorize dot patterns and report their judgments intermittently, but were not instructed to focus primarily on one task or the other. As such, participants had the opportunity to neglect the cognitive task so as to minimize its impact on their performance on the decision task. This eventuality may account for the difference in mean recall observed between the high and low cognitive-load conditions (of note, however, this difference replicates Heyman et al, who interpreted these differences as indicating greater task difficulty rather than decreased task compliance). Future studies could address this issue by specifically stating the primary and secondary task in the instructions. Finally, a baseline assessment of performance on the cognitive load task could also be implemented to assess the extent to which it is affected by the decision task. As ours is the first experiment to explore the influence of cognitive load during the decision phase on UTEs, we strongly encourage future research to address these issues to better couch our current findings. BODY.DISCUSSION.CONCLUSION AND IMPLICATIONS: Patients and health practitioners alike commonly consider a vast amount of information to reach optimal medical decisions. Unfortunately, considerable evidence now indicates that conscious processes can be ill-equipped to integrate complex information, at least without aids (eg, notes, computers, etc). Quite simply, people cannot consciously retain and process vast amounts of information, and thus often form poor decisions via conscious processes. But, according to Dijksterhuis and Nordgren [2], unconscious thinking can process vast amounts of information with just a little time (eg, 3 minutes), and thus somewhat counterintuitively, individuals often come to better decisions when they are distracted from consciously considering decision-relevant information. The current research demonstrates this can also be true for patient-centered medical decisions. A hypothetical (and relatively long-term) implication of the current research lies in the type of media used to test the UTE. Unconscious thought research readily utilizes computer and research software platforms, and a Web-based platform was used presently. Demonstrating the UTE in this fashion may constitute evidence that unconscious-thinking decision aids and value clarification exercises may be integrated with Web and mobile technologies within health care realms (given that current limitations are addressed of course). For example, a Web or mobile phone app may in some instances present the relevant information to a medical decision maker, provide a timed and cognitively consuming distraction, then solicit a decision. That is, it is conceivable that a Web or mobile app could model the stages used in the current research for real medical-decision tasks in a way that fosters unconscious thought, and thus better decisions. Further, the merits of unconscious thought should not be limited to treatment side-effects/attributes. Personal values could (theoretically) also be processed unconsciously. This implication is particularly relevant because unconscious thought can process decisional factors that are difficult to articulate or too numerous to maintain in conscious awareness [1-3]. Patients may therefore be able to use an "unconscious thought mobile app" as a beneficial value clarification exercise and successfully incorporate numerous personal values in their decision. Finally, unconscious thinking may instill further benefits in medical contexts. First, other research demonstrates that individuals are more satisfied with the choices they make via unconscious thinking [7]. Thus, patients might experience more satisfaction with a treatment, and thus better adhere to it, if they chose that treatment following unconscious thought. Second, we investigated mock-patient decisions, but health practitioners might experience the greatest benefit from unconscious thought for complex medical decisions. For instance, research by Dijksterhuis and colleagues [8] showed that participants with more (vs little) expertise in a domain reach higher quality judgments after a period of unconscious thought. Thus, medical experts may realize the most advantage for choosing optimal treatments or generating accurate diagnoses in the face of complex and numerous symptoms, complications, side-effects, and risks, and some research already supports this possibility [16]. Given this, exploring the UTE in medical decision making for health care providers and patients has the potential to greatly and broadly enhance patient health and well-being.

TITLE: Thiamine as a metabolic resuscitator in septic shock: a randomized, double-blind, placebo-controlled, pilot trial ABSTRACT: BODY.INTRODUCTION: The objective was to determine whether the administration of thiamine mitigates elevated lactate levels in patients with septic shock. Thiamine is essential for aerobic metabolism and we have found that thiamine levels are low and inversely correlated with lactate levels in patients with sepsis. BODY.METHODS: We performed a randomized, double-blind, placebo-controlled, two-center trial from January 2010 to October 2014. We enrolled patients with septic shock, elevated lactate (≥3 mmol/l) and no obvious competing cause of lactate elevation. Patients received thiamine 200 mg or placebo i.v. twice/day for 7 days. The primary outcome was lactate levels at 24 hours. Secondary outcomes included the SOFA score at 24 hours and mortality. Lactate levels at 24 hours were compared between groups using the Wilcoxon rank-sum test and categorical variables were compared using the Fisher's exact test. Lactate values at 24 hours, for those who died before 24 hours, were imputed according to a predefined plan. We performed a preplanned analysis in those with baseline thiamine deficiency (≤7 nmol/l). BODY.RESULTS: We enrolled 88 patients; 43 received thiamine and 45 placebo. Baseline characteristics were similar between groups. We found no overall statistical significant difference in 24-hour lactate levels between thiamine and placebo groups (2.5 (IQR: 1.5 to 3.4) vs. 2.6 (IQR: 1.6 to 5.1), P = 0.40). Fewer patients in the thiamine group had lactate levels >4 mmol/l at 24 hours (21% vs. 38%, P = 0.10) and this was statistically significant if only evaluating survivors at 24 hours (7% vs. 33%, P = 0.03), although our preplanned analysis was to impute data. We found no difference in 24-hour SOFA score or mortality. A total of 28 (35%) patients were thiamine deficient. Of the deficient patients, those receiving thiamine had statistically significant lower lactate levels at 24 hours (2.1 (IQR: 1.4 to 2.5) vs. 3.1 (IQR: 1.9 to 8.3), P = 0.03) and more patients in the placebo group had a lactate >4 mmol/l (38% vs. 7%, P = 0.07). Mortality in the thiamine and placebo groups was 13% and 46%, respectively (P = 0.10). BODY.CONCLUSION: Thiamine deficiency is prevalent in septic shock. Thiamine did not decrease overall median lactate levels at 24 hours. In the patients with thiamine deficiency, there were statistically significant lower lactate levels at 24 hours in the thiamine group and a large, although nonsignificant, difference in mortality.

TITLE: Phase II randomized study of PM01183 versus topotecan in patients with platinum-resistant/refractory advanced ovarian cancer ABSTRACT.BACKGROUND: PM01183 is a new compound that blocks active transcription, produces DNA breaks and apoptosis, and affects the inflammatory microenvironment. PM01183 showed strong antitumor activity in preclinical models of cisplatin-resistant epithelial ovarian cancer. ABSTRACT.PATIENTS AND METHODS: Patients with platinum-resistant/refractory ovarian cancer were included in a two-stage, controlled, randomized (in a second stage), multicenter, phase II study. Primary endpoint was overall response rate (ORR) by RECIST and/or GCIG criteria. The exploratory first stage (n = 22) confirmed the activity of PM01183 as a single agent at 7.0 mg flat dose every 3 weeks (q3wk). The second stage (n = 59) was randomized and controlled with topotecan on days 1–5 q3wk or weekly (every 4 weeks, q4wk). ABSTRACT.RESULTS: ORR was 23% (95% CI, 13%–37%) for 52 PM01183-treated patients. Median duration of response was 4.6 months (95% CI, 2.5–6.9 months), and 23% (95% CI, 0%–51%) of responses lasted 6 months or more. Ten of the 12 confirmed responses were reported for 33 patients with platinum-resistant disease [ORR = 30% (95% CI, 16%–49%)]; for the 29 patients treated with topotecan in the second stage, no responses were found. Median PFS for all PM01183-treated patients was 4.0 months (95% CI, 2.7–5.6 months), and 5.0 months (95% CI, 2.7–6.9 months) for patients with platinum-resistant disease. Grade 3/4 neutropenia in 85% of patients; febrile neutropenia in 21% and fatigue (grade 3 in 35%) were the principal safety findings for PM01183. ABSTRACT.CONCLUSION: PM01183 is an active drug in platinum-resistant/refractory ovarian cancer and warrants further development. The highest activity was observed in platinum-resistant disease. Its safety profile indicates the dose should be adjusted to body surface area (mg/m2). ABSTRACT.TRIAL CODE: EudraCT 2011-002172-16. BODY.INTRODUCTION: PM01183 (lurbinectedin) is a synthetic tetrahydroisoquinoline that is a selective inhibitor of active transcription. The mechanism involve the irreversible stalling of elongating RNA polymerase II (Pol II) on the DNA template and its specific degradation by the ubiquitin/proteasome machinery, with subsequent DNA breaks and apoptosis [1]. Furthermore, PM01183 affects the inflammatory microenvironment, with selective apoptotic-inducing effect on tumor-associated macrophages, and specific inhibition of inflammatory cytokines production [2]. Strong preclinical antitumor activity was observed in cisplatin-resistant epithelial ovarian cancer models [3]. This two-stage phase II trial evaluated PM01183 activity in terms of overall response rate (ORR) in patients with platinum-resistant or platinum-refractory ovarian cancer. BODY.MATERIALS AND METHODS: The study was conducted at nine sites following the ICH GCP guidelines, and approved by the respective Research Ethics Committees. Written informed consent was obtained from all patients. BODY.MATERIALS AND METHODS.ELIGIBILITY CRITERIA: Selection criteria included: age ≥18 years; histologically/cytologically confirmed epithelial ovarian, fallopian tube or primary peritoneal cancer; advanced measurable (per RECIST v.1.1) or non-measurable disease [per Gynecologic Cancer InterGroup (GCIG) criteria]; platinum-resistant (disease relapse or progression <6 months after last platinum-containing chemotherapy) or platinum-refractory (disease that did not respond during last platinum-containing chemotherapy) disease; <3 prior lines of cytotoxic-containing chemotherapy; Eastern Cooperative Oncology Group performance status (ECOG PS) score ≤2; recovery from previous toxicities; and adequate bone marrow, hepatic, renal, and metabolic function. Criteria for exclusion were: prior treatment with topotecan; brain metastases or leptomeningeal involvement; pregnancy or lactation; prior pelvic irradiation; concomitant conditions such as unstable angina, myocardial infarction, congestive heart failure, generalized edemas or grade ≥3 ascites, immune deficiency; active uncontrolled infection, myopathy. BODY.MATERIALS AND METHODS.STUDY DESIGN: This was a two-stage phase II clinical trial (Figure 1). First stage explored the activity of PM01183 7.0 mg flat dose (FD) every 3 weeks (q3wk) in platinum-resistant/refractory advanced ovarian cancer. Second stage was randomized and controlled. Control arm initially consisted of standard pegylated liposomal doxorubicin (PLD) every 4 weeks (q4wk) or standard i.v. topotecan daily times five q3wk [4]. However, due to a worldwide shortage of PLD, PLD was replaced with topotecan, including a weekly schedule, based on research suggesting better tolerability for weekly topotecan compared with standard regimen [5]. Patients in the second stage were assigned to each treatment arm, PM01183 or topotecan, by strata (platinum refractory versus resistant disease) [6] random lists (random permuted blocks method). Figure 1.CONSORT flow diagram. BODY.MATERIALS AND METHODS.STUDY TREATMENT: PM01183 was administered as a 7.0 mg FD 1-h intravenous (i.v.) infusion every 3 weeks (q3wk) in both stages [7]. Topotecan was administered as a 30-min i.v. infusion: the regimen, standard (days 1–5 q3wk) or weekly (days 1, 8, and 15 q4wk), was decided by the investigators. Topotecan starting dose was adjusted according to creatinine clearance and ECOG PS: 1.50–0.75 mg/m2 days 1–5 (standard regimen; referred to as 'daily') or 4.0–2.4 mg/m2 (weekly regimen). All patients received antiemetic prophylaxis. Granulocyte colony-stimulating factors could be added for secondary prevention of neutropenia. During the second stage, patients in the control arm with disease progression could crossover to the experimental arm. BODY.MATERIALS AND METHODS.STUDY ASSESSMENTS: Patients were assessed at baseline, and on day 1 of each new treatment cycle. Laboratory values were tested again on days 8 and 15 (cycle 1) and on day 10 (subsequent cycles). Febrile neutropenia and grade 4 neutropenia/thrombocytopenia were reassessed daily until recovery to grade ≤3 or fever resolution. Antitumor activity was evaluated every 6 weeks (±1 week) using RECIST v.1.1 [8] (measurable disease) and GCIG criteria (non-measurable disease). Laboratory abnormalities and adverse events (AEs) were graded per the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) v.4.03. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: The primary objective was to test PM01183 activity; therefore, sample size was calculated based on PM1183 response rate. If enough activity was observed in the exploratory stage I (interim analysis with 18 evaluable patients), then stage II had to include additional patients treated with PM10183 and the same number of patients for the control arm. A two-stage design to test the null hypothesis (H0) that ≤8% patients responded to PM01183 according to RECIST v.1.1 and/or GCIG criteria (P ≤ 0.08) versus the alternative hypothesis (H1) that ≥25% (P ≥ 0.25) was used (whole data, including stages I and II of this study). The type I error (alpha) was 0.025 (one-sided) and the type II error (beta) was 0.1; hence, statistical power was 90%. At the end of the study, if at least 8 of the 48 evaluable patients treated with PM01183 (18 in the first stage and 30 in the second stage) were responders, then PM01183 treatment should to be considered for further study. All tests were two-sided (significance level at 0.05). For the evaluation of the main primary endpoint (ORR), the exact binomial estimator including its 95% confidence interval was used. Time-to-event variables were calculated following the Kaplan–Meier method. BODY.RESULTS.PATIENT CHARACTERISTICS: Eighty-one patients were treated between December 2011 and March 2013: 52 received PM01183 (22 in the first stage; 30 in the second stage) (Figure 1) and 29 topotecan (15 crossed over to PM01183). Most patients had ovarian cancer, serous histology, poorly differentiated carcinoma, and platinum-resistant disease (Table 1). Table 1Baseline characteristicsAll PM01183 (n=52)Randomized, controlled second stagePM01183 (n=30)Topotecan (n=29)n (%)n (%)n (%)Median age (range) (years)59 (35–81)60 (35–81)61 (35–80) ≥708 (15%)7 (23%)11 (38%)ECOG PS 026 (50%)17 (57%)11 (38%) 124 (46%)12 (40%)14 (48%) 22 (4%)1 (3%)4 (14%)Disease evaluation RECIST (measurable disease)43 (83%)26 (87%)22 (76%) GCIG (non-measurable disease)9 (17%)4 (13%)7 (24%)Primary tumor site Ovarian44 (85%)27 (90%)23 (79%) Peritoneal7 (14%)2 (7%)4 (14%) Fallopian tube1 (2%)1 (3%)2 (7%)Most common histology type Papillary serous38 (73%)22 (73%)19 (66%) Endometrioid4 (8%)1 (3%)2 (7%) Undifferentiated carcinoma4 (8%)3 (10%)2 (7%) Clear cell1 (2%)-3 (10%)Histology grade Well differentiated8 (15%)4 (13%)- Moderately differentiated9 (17%)6 (20%)8 (28%) Poorly differentiateda24 (46%)14 (47%)9 (31%)Median time from first diagnosis (range) (months)19 (3–109)19 (3–109)14 (4–81)Disease stage (inclusion) III20 (39%)15 (50%)16 (55%) IV32 (62%)15 (50%)13 (45%)Ascites11 (21%)7 (23%)10 (35%)Most common sites of current disease Peritoneum35 (67%)21 (70%)23 (79%) Lymph node24 (46%)14 (47%)17 (59%) Liver17 (33%)8 (27%)5 (17%)Prior radical surgery38 (73%)24 (80%)24 (83%)Prior chemotherapy linesMedian2 (1–3)2 (1–3)2 (1–3) 118 (35%)9 (30%)13 (45%) 226 (50%)14 (47%)12 (41%) 38 (15%)7 (23%)4 (14%)Platinum status Refractory19 (37%)13 (43%)13 (45%) Resistant33 (63%)17 (57%)16 (55%)Platinum resistance Primary resistance30 (58%)17 (57%)18 (62%) Secondary resistance22 (42%)13 (43%)11 (38%)Platinum-free interval median (range) (months)3.6 (0.1–6.0)3.3 (0.1–5.9)2.3 (0–5.9)Prior angiogenesis inhibitor13 (25%)5 (17%)11 (38%) Prior bevacizumab6 (12%)3 (10%)9 (31%)No patients were previously treated with poly ADP ribose polymerase inhibitors.One patient in the second stage PM01183 arm was previously treated with immunotherapy.aIncludes undifferentiated. Unknown grades are not shown.ECOG PS, Eastern Cooperative Oncology Group performance status; GCIG, Gynecological Cancer Intergroup criteria; RECIST, Response Evaluation Criteria in Solid Tumors. BODY.RESULTS.TREATMENT: In the experimental arm (first/second stage, n = 52), 310 PM01183 cycles were administered (median of five cycles per patient). In the control arm (n = 29), 21 patients (72%) were treated with 53 cycles of weekly topotecan (median of two cycles per patient). Eight patients (28%) were treated with 28 cycles of daily topotecan (median of three cycles per patient). Eighteen PM01183-treated patients (36% of patients with more than one cycle) had dose reduced in a total of 23 cycles (9% of cycles); 15 dose reductions (6% of cycles) in 14 patients (28%) were due to hematological toxicities (febrile neutropenia, n = 5) and eight due non-hematological toxicity (mostly fatigue). Treatment-related fatigue resulted in three treatment discontinuations. Hematological toxicity was the reason for treatment discontinuation in two patients treated with PM01183 (one due to grade 3 febrile neutropenia and one due to grade 4 neutropenia/thrombocytopenia). Increased transaminases led to one dose reduction and one cycle delay. BODY.RESULTS.EFFICACY: Twelve (seven in the first stage, five in the second stage) confirmed responses were observed in the 52 patients treated with PM01183: one CR (per RECIST) and 11 PRs (nine per RECIST; two per GCIG criteria). ORR for PM01183 was 23% (95% CI, 13%–37%) (Table 2). Median duration of response was 4.6 months (95% CI, 2.5–6.9 months) and 23% (95% CI, 0%–51%) of responses lasted ≥6 months. Median PFS was 4.0 months (95% CI, 2.7–5.6 months) (Figure 2A). Median OS was 10.6 months (95% CI, 9.5–18.1 months). Table 2Overall response and clinical benefit (RECIST/GCIG) in all patientsAll PM01183 (n =52)Randomized, controlled second stagePM01183 (n =30)Topotecan (n =29)an (%)n (%)n (%)Overall response rate (RECIST/GCIG)23%17%-(95% CI)(13%–37%)(6%–35%)(0%–12%)PM01183 versus topotecan, P =0.0033Complete response1 (2%)1 (3%)-Partial response11 (21%)4 (13%)-Stable disease26 (50%)15 (50%)15 (52%)Progressive disease13 (25%)9 (30%)14 (48%)Clinical benefit (CR+PR+SD≥4 months)22 (42%)12 (40%)4 (14%)(95% CI)(29%–57%)(23%–59%)(4%–32%)aWeekly (n = 21): 4.0–2.4 mg/m2 days 1, 8, and 15 q4wk; standard (n = 8): 1.50–0.75 mg/m2 days 1–5 q3wk.CI, confidence interval; CR, complete response; GCIG, Gynecologic Cancer Intergroup criteria; PR, partial response; RECIST, Response Evaluation Criteria in Solid Tumors; SD, stable disease. Figure 2.Progression-free survival in (A) all patients; (B) patients with platinum-resistant disease. Nine responders (75%) had an ECOG PS score of 0 (none scored 2) and all were free of ascites at baseline; most were <65 years old; ten of 12 responders (83%) had platinum-resistant disease (seven had primary resistance). Two patients had BRCA mutation. Median number of prior advanced chemotherapy lines was one (range, 1–2). Median progression-free interval (PFI) was 4.5 months (range, 0.9–5.95 months). Seven responders (58%) had non-platinum-based therapy as last prior line. All but one received six or more PM01183 treatment cycles. Antitumor activity was observed in three of six patients who had previously received bevacizumab. No responses (either by RECIST or GCIG criteria) were found in the 29 patients treated with topotecan. In the second randomized stage, median PFS was significantly longer with PM01183: 3.9 months (95% CI, 2.5–5.7 months) versus 2.0 months (95% CI, 1.4–2.8 months) with topotecan (P = 0.0067). Median OS was 9.7 months (95% CI, 7.7–19.3 months) with PM01183, and 8.5 months (95% CI, 3.3–15.6 months) with topotecan (P = 0.2871). Noteworthy antitumor activity was observed with PM01183 in platinum-resistant disease (supplementary Table S1, available at Annals of Oncology online): ORR was 30% (95% CI, 16%–49%) (10 of 33 patients treated in both study stages), with median PFS of 5.0 months (95% CI, 2.7–6.9 months) (5.7 months in the second stage versus 1.7 months in the topotecan-treated patients with platinum-resistant disease) and median OS of 13.5 months (95% CI, 9.7–22.0 months) (15.6 months in the second stage versus 8.7 months in the topotecan-treated patients). Median PFS was significantly longer with PM01183 versus topotecan for patients with platinum-resistant disease, both in the study as a whole (P = 0.0017) and in the second stage alone (P = 0.0043) (Figure 2B). According to GCIG criteria,13 of 37 patients with tumor marker values at least two times the ULN at baseline had a >50% CA-125 decrease during PM01183 treatment; seven were subsequently confirmed (response rate of 19%; 95% CI, 8%–35%); and five responses were also confirmed response by RECIST. Fifteen patients from the control arm (52%) crossed over to PM01183 after disease progression; PM01183 antitumor activity was observed in two patients (ORR = 13%), consisting of one PR per RECIST (SD with weekly topotecan) and one PR per GCIG criteria (PD as best response with weekly topotecan); both had platinum-resistant disease and one had BRCA mutation; one had been previously treated with bevacizumab. BODY.RESULTS.TOXICITY: Myelosuppression was the most frequent AE associated with PM01183 (Table 3). The most common severe hematological abnormalities were grade 3/4 neutropenia (85%; grade 4: 64%) and grade 3/4 thrombocytopenia (33%; grade 4: 23%). There were 11 patients (21%) with febrile neutropenia meaning that primary granulocyte-colony stimulating factor (G-CSF) prophylaxis would be required according to current guidelines [9]. G-CSFs (prophylactic or therapeutic) were administered in 20 (39%) of PM01183-treated patients. Transfusions were given to 19 (37%): eight received platelets (15%, all after grade 4 thrombocytopenia) and 11 (21%) RBCs. Fourteen patients (27%) received erythropoietin. Table 3Grade 3/4 laboratory abnormalities and treatment-related (or relationship unknown) adverse events (or in ≥10% of patients)PM01183 (n =52)Topotecan (n =29)aNCI-CTCAE gradeNCI-CTCAE grade12341234n (%)n (%)n (%)n (%)n (%)n (%)n (%)n (%)Hematological laboratory abnormalitiesAnemia14 (27%)18 (35%)19 (37%)-5 (17%)12 (41%)11 (38%)-Leukopenia-12 (23%)19 (37%)15 (29%)6 (21%)8 (28%)9 (31%)2 (7%)Lymphopenia13 (25%)24 (46%8 (15%)5 (10%)6 (21%)11 (38%)5 (17%)1 (3%)Neutropenia1 (2%)1 (2%)11 (21%)33 (64%)5 (17%)7 (24%)3 (10%)8 (28%)Thrombocytopenia16 (31%)4 (8%)5 (10%)12 (23%)11 (38%)4 (14%)4 (14%)3 (10%)Biochemical laboratory abnormalitiesALP increase25 (48%)4 (8%)2 (4%)-13 (48%)4 (15%)--ALT increase23 (44%)13 (25%)8 (15%)1 (2%)14 (48%)1 (3%)--AST increase29 (56%)2 (4%)6 (12%)-8 (28%)1 (3%)--Creatinine increase40 (77%)6 (12%)2 (4%)-23 (79%)2 (7%)--GGT increase16 (31%)14 (27%)12 (23%)1 (2%)10 (37%)7 (26%)3 (11%)-Treatment-related adverse eventsAbdominal pain5 (10%)1 (2%)---1 (3%)--Alopecia4 (8%)---2 (7%)3 (10%)--Constipation11 (21%)4 (8%)--1 (3%)1 (3%)--Decreased appetite7 (14%)1 (2%)2 (4%)-2 (7%)1 (3%)1 (3%)-Diarrhea4 (8%)3 (6%)--5 (17%)2 (7%)--Fatigue10 (19%)12 (23%)18 (35%)-5 (17%)8 (28%)--Febrile neutropenia--8 (15%)3 (6%)--3 (10%)-Gastrointestinal toxicity--1 (2%)-----Nausea14 (27%)15 (29%)6 (12%)-6 (21%)5 (17%)--Rhabdomyolysis--1 (2%)-----Vomiting15 (29%)8 (15%)5 (10%)-4 (14%)--aWeekly (n = 21): 4.0–2.4 mg/m2 days 1, 8, and 15 q4wk; standard (n = 8): 1.50–0.75 mg/m2 days 1–5 q3wk.ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CPK, creatine transferase; NCI-CTCAE, National Cancer Institute Common Terminology Criteria for Adverse Events. Fatigue was the most frequent non-hematological treatment-related AE reported among PM01183-treated patients (all grades, 77%) and was also the most common severe non-hematological toxicity (grade 3, 35%; no grade 4 AEs other than febrile neutropenia were observed). Nausea was common (67%; grade 3 in 12%) as was vomiting (54%; grade 3 in 10%). There was one event of grade 3 rhabdomyolysis in cycle 2 reported as related to PM01183; the patient's dose was not reduced as required by protocol despite grade 4 neutropenia, grade 3 transaminases increase, and fatigue in cycle 1. Topotecan safety profile agreed with that previously reported for both the daily and weekly regimens. Grade 3/4 neutropenia occurred in 38% of patients (grade 4 in 28%; 88% in the daily regimen compared with 5% in the weekly); and grade 3/4 thrombocytopenia in 24% of patients (grade 4 in 10%, all in the daily regimen). There were three cases of febrile neutropenia (10% overall); all of them in the daily regimen (38% of patients). G-CSF use (prophylactic or therapeutic) was required in four (14%) patients (all four following the daily regimen; 50% of patients treated with daily topotecan required G-CSFs). Non-hematological topotecan-related AEs included mild/moderate fatigue (45%), nausea (38%), vomiting (28%), and diarrhea (24%). Apart from febrile neutropenia, only decreased appetite (3%) reached grade 3 in the control arm; all grade 3 AEs occurred with the daily regimen. No remarkable changes were seen in PM01183 safety profile in the fifteen patients who crossed over. Most AEs remained the same or slightly improved after crossover. Laboratory values did not vary greatly before and after crossover: many remained the same and noticeable shifts were in line with PM01183's known toxicities. There were no treatment-related deaths in the study. BODY.DISCUSSION: The primary endpoint of this phase II study was met: PM01183 was active in patients with platinum-resistant/refractory ovarian cancer. Twelve (of a total of 52 treated patients) responded with a median duration of response of 4.6 months. The poor prognosis of the patient population in this phase II trial needs to be borne in mind: 37% of patients had platinum-refractory disease, and 35% had previously received one prior chemotherapy line after confirmation of platinum resistant/refractory disease. The activity was not influenced by prior angiogenesis-inhibitor treatment; three of six patients with previous bevacizumab had confirmed response. Six of eighteen (33%) patients who received PM01183 as second-line after their disease became platinum-resistant/refractory had confirmed response. This response rate is superior to the 8.1% reported by Griffiths et al. [10] who evaluated the efficacy of subsequent lines of treatment in 274 patients with platinum-resistant/refractory disease. PM01183 antitumor activity was especially noteworthy in patients with platinum-resistant disease (n = 33; both stages) with ORR = 30%, median PFS = 5.0 months, and median OS = 13.5 months. More modest activity was observed in patients with platinum-refractory disease: ORR = 10.5%; median OS = 9.3 months. In platinum-resistant disease, ORR and PFS with PM01183 were statistically significant with respect to topotecan in the second stage; two patients with platinum-resistant disease treated initially with topotecan achieved PR after crossing over to PM01183 treatment (one had a BRCA germline mutation). In the randomized second stage, antitumor activity with topotecan was lower with respect to PM01183: ORR = 0%, median PFS = 2.0 months, and median OS = 8.5 months. Recent studies in ovarian cancer have also shown a poor activity with topotecan. ORR (RECIST and/or GCIG) was 3.3% and median PFS was 2.1 months (95% CI, 1.9–3.3 months) in the phase III AURELIA trial (most patients treated with the weekly regimen) [11, 12]. No responses (RECIST) and median PFS of 2.6 months (95% CI, 2.1–4.3 months) were found in the phase III PENELOPE trial (n = 24 patients treated with topotecan 1.25 mg/m2 days 1–5 q3wk) [13]. The incidence of severe hematological abnormalities found here was higher than that observed among the 15 patients (47% women) with advanced solid tumors treated at 7.0 mg FD in the first-in-human study, where 53% and 7% of patients had grade 3/4 neutropenia and grade 3/4 thrombocytopenia, respectively, and without febrile neutropenia [7]. Ongoing PK/PD modeling of pooled phase I/II data has shown some covariates [ascites, albumin, aprepitant, body surface area (BSA)] that might affect neutropenia and thrombocytopenia (unpublished data). Four of six patients who received aprepitant concomitantly with PM01183 developed severe (and in some cases prolonged) pancytopenia. Aprepitant is an inhibitor of cytochrome P-450 isoenzyme 3A4 (CYP3A4) which metabolizes PM01183, and those patients had PM01183 clearance (CL) reduced and AUC increased. Currently, aprepitant is forbidden in all clinical trials with PM01183. Preliminary PK analysis showed that PM01183 CL was unaffected by BSA, but a recent analysis of pooled phase II data showed grade 3/4 neutropenia and thrombocytopenia more frequent in patients with lower BSA [14]. BSA-based dosing (mg/m2) should now be adopted and the recommended dose reduced for future studies to improve safety. In conclusion, PM01183 is an active drug in platinum-resistant/refractory ovarian cancer and warrants further clinical development, particularly in platinum-resistant disease, where its activity was remarkable. Currently, PM01183 is being tested in a pivotal randomized trial (CORAIL study, NCT02421588) in patients with platinum-resistant ovarian cancer [15]. BODY.SUPPLEMENTARY MATERIAL: Supplementary DataClick here for additional data file.

TITLE: Efficacy and Blood Plasmalogen Changes by Oral Administration of Plasmalogen in Patients with Mild Alzheimer's Disease and Mild Cognitive Impairment: A Multicenter, Randomized, Double-blind, Placebo-controlled Trial ABSTRACT.BACKGROUND: Plasmalogens (Pls) reportedly decreased in postmortem brain and in the blood of patients with Alzheimer's disease (AD). Recently we showed that intraperitoneal administration of Pls improved cognitive function in experimental animals. In the present trial, we tested the efficacy of oral administration of scallop-derived purified Pls with respect to cognitive function and blood Pls changes in patients with mild AD and mild cognitive impairment (MCI). ABSTRACT.METHODS: The study was a multicenter, randomized, double-blind, placebo-controlled trial of 24 weeks. Participants were 328 patients aged 60 to 85 years who had 20 to 27 points in Mini Mental State Examination-Japanese (MMSE-J) score and five or less points in Geriatric Depression Scale-Short Version-Japanese (GDS-S-J). They were randomized to receive either 1 mg/day of Pls purified from scallop or placebo. The patients and study physicians were masked to the assignment. The primary outcome was MMSE-J. The secondary outcomes included Wechsler Memory Scale-Revised (WMS-R), GDS-S-J and concentration of phosphatidyl ethanolamine plasmalogens (PlsPE) in erythrocyte membrane and plasma. This trial is registered with the University Hospital Medical Information Network, number UMIN000014945. ABSTRACT.FINDINGS: Of 328 patients enrolled, 276 patients completed the trial (140 in the treatment group and 136 in the placebo group). In an intention-to-treat analysis including both mild AD (20 ≤ MMSE-J ≤ 23) and MCI (24 ≤ MMSE-J ≤ 27), no significant difference was shown between the treatment and placebo groups in the primary and secondary outcomes, with no severe adverse events in either group. In mild AD patients, WMS-R improved significantly in the treatment group, and the between group difference was nearly significant (P = 0.067). In a subgroup analysis of mild AD patients, WMS-R significantly improved among females and those aged below 77 years in the treatment group, and the between-group differences were statistically significant in females (P = 0.017) and in those aged below 77 years (P = 0.029). Patients with mild AD showed a significantly greater decrease in plasma PlsPE in the placebo group than in the treatment group. ABSTRACT.INTERPRETATION: Oral administration of scallop-derived purified Pls may improve cognitive functions of mild AD. ABSTRACT.FUNDING: The Japanese Plasmalogen Society. BODY.INTRODUCTION: 1 Alzheimer's disease (AD) is an age-related neurodegenerative disorder that has increased in prevalence as people live longer. It is estimated that the prevalence of AD may reach > 74 million worldwide by 2030 (World Alzheimer Report, 2015). The cause and mechanism of AD is not fully elucidated, while progressive deposition of amyloid-β and Tau protein is considered to be a neuropathological hallmark of AD. On the other hand, the close connection between plasmalogens (Pls) and AD has been indicated by the observations of decreased phosphatidyl ethanolamine Pls (PlsPE) in the affected brain regions of AD patients, such as the hippocampus and frontal cortex (Ginsberg et al., 1995, Guan et al., 1999, Han et al., 2001). Decreased levels of PlsPE in the blood and cerebrospinal fluid of AD patients have been reported (Goodenowe et al., 2007, Wood et al., 2010, Wood et al., 2015, Oma et al., 2012, Yamashita et al., 2015). However, it is not clear whether the decrease of Pls in brain tissue and in plasma is the cause of the disease or merely a result of the disease. Recent studies of animal models of AD by our group indicated that intraperitoneal administration of purified Pls improved cognitive function (Katafuchi et al., 2012, Hossain et al., 2013, Hossain et al., 2016). Pls are a special class of glycerophospholipids characterized by a vinyl ether bond at the sn-1 position of glycerol backbone, and they are sometimes called plasmenyl phospholipid or alkenyl acryl phospholipid. They are found in almost all mammalian tissues and constitute about 18–20% of the total phospholipids in cell membranes. Predominant Pls in mammalian tissues are PlsPE and choline plasmalogen. PlsPE is much more abundant than choline plasmalogen except in heart and skeletal muscle. It is reported that Pls are abundant in the brain, retina, leukocytes (immune cells), sperm, heart, and skeletal muscle in mammals. This characteristic distribution of Pls indicates the importance of Pls in mammals (Farooqui and Horrocks, 2001, Braverman and Moser, 2012). Pls are not only a structural component of animal cell membranes and reservoir for secondary messengers, but may also be involved in membrane fusion, ion transport, and cholesterol efflux, and act as antioxidants in cell membranes. Recently, the inhibitory effects of Pls on γ-secretase activity have been reported (Onodera et al., 2015). This study aimed to assess whether oral administration of Pls extracted from scallops would improve the cognitive function in patients with mild AD and mild cognitive impairment (MCI). BODY.METHODS: 2 BODY.METHODS.STUDY DESIGN AND PARTICIPANTS: 2.1 This study was a multicenter, randomized, double-blind, placebo-controlled trial to evaluate the memory-improving efficacy of scallop-derived Pls in patients with mild AD and MCI. The study period consisted of a 24-week administration period and a four-week post-treatment period without administration (28 weeks in total). Patients were those who had 20 to 27 points of Mini Mental State Examination Japanese Version (MMSE-J) scores, i.e., patients with mild AD (20 ≤ MMSE-J ≤ 23) or those with MCI (24 ≤ MMSE-J ≤ 27) (Solfrizzi et al., 2004). All patients were required to meet the criteria for mild AD or MCI set out in the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-V). Patients were confirmed to have no arteriosclerotic dementia with MRI or CT scans performed within the previous six months. Patients were also ensured to have five or less points of Geriatric Depression Scale Short Version in Japanese (GDS-S-J) in order to exclude depressive pseudodementia. Patients taking anti-Alzheimer drugs had no change in the regimen during the previous three months. All caregivers were required to accompany patients to all visits throughout the study, and provided information on patients' daily life and health status. Patients were excluded if they were allergic to scallops, the raw materials for test substance. Written informed consent was obtained from either patients or their caregivers. The study protocol was approved by the Institutional Review Boards of Fukuoka University Hospital (Fukuoka), Nihonbashi Sakura Clinic (Tokyo), and BOOCS Clinic Fukuoka (Fukuoka). The study was implemented in compliance with the Declaration of Helsinki. BODY.METHODS.RANDOMIZATION AND MASKING: 2.2 A computer-generated random allocation list was created by an expert at CAC Croit Corporation (Tokyo) based on the blocked randomization method with each block consisting of two placebo allocations and two Pls allocations. Each study site was provided with test substance kits numbered according to the allocation list, which were handed directly to patients. At some study sites without cold storage space, patients received test kits by courier every month. Thus enrolled patients were randomly assigned to receive either 1.0 mg Pls/day or placebo. Test substance was jelly-like substance. Active substance and placebo were identical in appearance and taste. Patients, caregivers, study physicians, and clinical staff were masked to treatment allocation throughout the study period. BODY.METHODS.PROCEDURES: 2.3 Enrolled patients were provided with the test substance at baseline visit or within one week after the baseline visit, and were instructed to take it orally twice/day for 24 weeks. They received one month's extra test substance in case they missed the next scheduled visit. To confirm compliance, they were requested to return unconsumed test substance at their next visit. They were furthermore urged not to change the regimen during the study period as far as possible. If a patient changed his or her drug use, we terminated his or her observation at the point. We recorded any complications and adverse events reported by patients at each visit. The primary outcome measure was MMSE-J. The secondary outcome measures included Wechsler Memory Scale-Revised (WMS-R), GDS-S-J, plasma PlsPE levels, and relative concentration of PlsPE in erythrocyte membrane, namely the percentage of Pls to the total phospholipids in erythrocyte membrane. Cognitive function was assessed at baseline, and at weeks 12, 24, and 28. Blood was drawn from patients in the fasting state at baseline, and at weeks 8, 16, 20, 24, and 28 for measuring erythrocyte PlsPE and plasma PlsPE. PlsPE measurement was performed using the previously reported method (Mawatari et al., 2007, Mawatari et al., 2016). Safety assessment was conducted by recording adverse events and performing a physical examination and biochemical blood tests such as liver function, renal function, blood sugar, and lipid levels at each visit. BODY.METHODS.STATISTICAL ANALYSIS: 2.4 To determine the sample size, we assumed that the MMSE-J score would improve by 5% in the placebo group and by 10% in the Pls treatment with SD of 15% in each. With a statistical power of 0.80 and a one-sided significance level of 0.01, the required sample size was estimated to be 181 in each group. The target number of enrollments was decided to be 200 in each with allowance for some extent of dropout. The between-treatment difference was assessed by unpaired t-test, and the within-group change from the baseline was evaluated by paired t-test. In terms of after-treatment outcome of the cognitive function, the mean change from the baseline and 95% confidence interval (CI) were presented. Analyses were done with Stata version 13 (StataCorp, College Station, TX). The trial is registered at the University Hospital Medical Information Network as ID UMIN000014945. BODY.METHODS.ROLE OF THE FUNDING SOURCE: 2.5 B&S Corporation Co. Ltd. (Tokyo), one of the donors to The Japanese Plasmalogen Society, was involved in provision of Pls test substance and placebo and delivery to study sites, but it had no involvement in study design and planning, investigator training, data analysis, interpretation, and writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. All co-investigators also had full access to the data. BODY.RESULTS: 3 BODY.RESULTS.STUDY PARTICIPANTS: 3.1 A total of 328 patients were enrolled at 25 hospitals or clinics in Kyushu, Kanto, and Kansai regions in Japan from November 15, 2014 to October 8, 2015. They were randomly assigned to either of the two treatment groups (166 to Pls group and 162 to placebo group). Of the 328 enrolled patients, 285 continued to participate in the study for 12 weeks, and 276 completed the 24 week study period. Those who completed the study included 11 patients (4.0%) who failed to use 80% or more of the provided test substance. Fig. 1 shows the trial profile. No significant difference was noted between the groups with respect to age, MMSE-J, GDS-S-J, WMS-R (0 min), WMS-R (30 min), erythrocyte PlsPE, and plasma PlsPE, at baseline, although the male-to-female ratio was significantly lower in the placebo group (Table 1). BODY.RESULTS.CLINICAL EFFICACY: 3.2 BODY.RESULTS.CLINICAL EFFICACY.PRIMARY ANALYSIS: 3.2.1 In an intention-to-treat analysis, there was no significant difference between the treatment and placebo groups in the primary and secondary outcomes of cognitive function. The MMSE-J score showed a nearly significant improvement in the treatment group while no such improvement was observed in the placebo group, resulting no statistically significant between-group difference. The WMS-R (0 min) and WMS-R (30 min) score each showed a significant improvement in both groups, but there was no statistically significant between-group difference. Erythrocyte PlsPE increased and plasma PlsPE decreased to different degrees after treatment in both Pls and placebo groups. There was no statistically significant between-group difference at any points of time, while the decrease in plasma PlsPE seemed greater in the placebo group (Table 2). BODY.RESULTS.CLINICAL EFFICACY.SUBGROUP ANALYSIS: 3.2.2 We examined the change in cognitive function in mild AD patients and MCI patients separately. No notable change was seen in MMSE-J in either group. WMS-R (0 min) and WMS-R (30 min) improved statistically significantly in the treatment group, and the improvement seemed greater in the Pls treatment group with respect to the 0 min value (P = 0.067) and the 30-min value (P = 0.078) (Table 3). BODY.RESULTS.CLINICAL EFFICACY.SUBGROUP ANALYSIS BY GENDER AND AGE: 3.2.3 Furthermore, we analyzed the cognitive function in patients with mild AD patients by gender and age (Table 4). Mild AD patients were divided into two groups based on the median value, 77 years or younger and 78 years or older. In the patients aged 77 years or younger, WMS-R (30 min) increased statistically significantly in the treatment group, showing a significant between-group difference (P = 0.029). In female patients with mild AD, WMS-R (30 min) improved significantly in the treatment group, with a significant between-group difference (P = 0.017). There was no notable between-group difference in the analysis of MCI patients by gender and age. BODY.RESULTS.CLINICAL EFFICACY.CHANGES IN BLOOD PLASMALOGENS: 3.2.4 The changes in blood PlsPE levels among mild AD patients were examined (Table 5). Plasma PlsPE decreased in the placebo group, showing a significant between-group difference (P = 0.016). There was a statistically significant elevation of erythrocyte PlsPE at week 16 in the treatment group and at week 24 in placebo. However, the changes were not significantly different between the two groups. BODY.RESULTS.CLINICAL SAFETY: 3.3 Table 6 summarizes adverse events. There was no notable difference in the occurrence of adverse events between the two groups. BODY.DISCUSSION: 4 To our knowledge, this is the unprecedented trial that addressed efficacy on cognitive function and blood Pls changes by oral administration of Pls in patients with mild AD and MCI. The present results showed no significant difference in the primary outcome (MMSE-J score) between the treatment and placebo groups. However, among patients with mild AD, WMS-R score significantly improved in the treatment group, and the improvement seemed greater than in the placebo group. In a subgroup analysis of mild AD patients, improvement of WMS-R was more evident in female patients and in patients below 77 years. These results indicate that oral administration of scallop-derived purified Pls may be effective to improve memory function of patients with mild AD. This study did not find the efficacy of Pls in mild AD patients over 78 years or male AD patients. The reason for the lack of efficacy in these aged patients may be ascribed to age-related irreversible degenerative changes in the brain (Kou et al., 2011). It is unknown why the efficacy was evident in females but not in males. The present study did not find the efficacy of Pls in MCI patients. It should be noted that WMS-R improved significantly even at end of the study (week 24) in the placebo group as well. These findings suggest that MCI patients may have exhibited a stronger placebo effect than AD patients. MCI patients may retain higher brain functions such as "expectation" and "hope". AD patients are seemingly less affected by placebo effects because their cognitive function declines and the above-mentioned higher brain function diminishes more markedly than that of MCI patients. This line of conjecture indicates that the mental status of people can change Pls concentration in brain tissue. Our previous studies showed that Pls in erythrocyte membrane significantly increased with orally ingested Pls, and another study also revealed that plasma PlsPE increased shortly after being ingested orally (Mawatari et al., 2012, Nishimukai et al., 2003). However, in the present study, only 1.0 mg/day of purified Pls showed an effect on memory function of mild AD patients. The physiological mechanism of this effect with such a small amount of Pls by oral administration is unclear. One hypothesis is that Pls may work through some receptors like hormones. Lipid rafts of cell membrane are considered to be associated with cell signaling (Hossain et al., 2016, New and Wong, 2007). There are reports that lipid rafts are rich in PlsPE (Pike et al., 2002). G-protein coupled receptors (GPCR) are also localized to lipid rafts and caveolae (Chini and Parenti, 2004). These may indicate the possibility that Pls work as a ligand of GPCR. Concentration of PlsPE in human plasma is about 100 μmol/l, but Pls in plasma may circulate as lipoproteins. Pls in the lipoproteins themselves are not likely to work as a ligand of receptors. However, it is possible that free Pls, which are derived from oral administration, even in a small amount, may work as a ligand of some receptors at intestinal cells before becoming lipoproteins. It is well known that there is close communication between the intestine and brain through neural, endocrine, and immune pathways (Yarandi et al., 2016). On the other hand, there have been many reports that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) relate closely to brain functions (Grimm et al., 2016, Hopperton et al., 2016, Ren et al., 2017). Scallop-derived Pls used in the present study presumably contained relatively large amounts of DHA and EPA (Kraffe et al., 2004, Hanuš et al., 2009). It is possible that these omega-3 polyunsaturated fatty acids of Pls may be effective for improvement of cognitive function in mild AD. Some studies suggest that DHA in the form of phospholipids passes through blood-brain barrier approximately ten times more efficiently than in the form of free fatty acid (Lagarde et al., 2001, Lagarde et al., 2015), although other studies report that a diet high in omega-3 fatty acids such as DHA and EPA resulted in negligible beneficial effect on cognition in AD patients (Quinn et al., 2010, Phillips et al., 2015). Regarding the change of blood Pls levels after treatment, there were significant differences between the treatment and placebo groups in mild AD patients. Plasma PlsPE in the placebo group decreased significantly after treatment, whereas that of the treatment group remained unchanged. Some studies suggest that statins, a class of cholesterol-lowering drugs, raise blood level of PlsPE (Meikle et al., 2015). In the present study, 23 (23.5%) of 98 patients with mild AD used statins during the study period. However, there were still significant differences in the changes of plasma PlsPE between the treatment and placebo groups except for statin-users. Therefore, it is unlikely that statins protected the decrease of Plasma PlsPE in the treatment group. These results might indicate that Pls production in peroxisome was concurrently reduced with the progression of Alzheimer's disease during the 24-week treatment period, and suggest that orally administered Pls may contribute to maintain the production of Pls in peroxisome. The present study has several problems and limitations. The study duration may be too short to detect the efficacy of Pls in MCI patients with MMSE-J score over 24, whose condition was ill-defined and may have little differed from that of healthy individuals. To resolve this problem, we should conduct additional research to ascertain whether it is possible to delay progression from MCI to AD in a long-term follow-up of MCI patients in the treatment group. Further randomized controlled trials for moderate and severe AD patients are also required. BODY.FUNDING SOURCES: The study was funded by The Japanese Plasmalogen Society (Pls2014-01) (Fukuoka, Japan) as an investigator-initiated trial. BODY.CONFLICTS OF INTEREST: TF and SM have applied for patents on method for manufacturing ether phospholipid (patent application number: PCT/JP2015/63617, PCT/JP2015/63740). TY, TA, YT, CW, and SK declare that they have no conflicts of interest. BODY.AUTHOR CONTRIBUTIONS: TF was responsible for central management of the trial including literature search, study design, data collection, data interpretation and writing of the manuscript. TY, TA, and YT contributed to study design, recruitment and research monitoring. CW contributed to clinical management, data collection and writing of the manuscript. SM contributed to implementation of the study with laboratory measurements and writing of the manuscript. SK did study design, statistical analysis of the data and writing of the manuscript. All authors reviewed the manuscript and approved the final manuscript.

TITLE: Pain relief after ambulatory hand surgery: A comparison between dexmedetomidine and clonidine as adjuvant in axillary brachial plexus block: A prospective, double-blinded, randomized controlled study ABSTRACT.BACKGROUND:: For ages various adjuvants have been tried to prolong axillary brachial plexus block. We compared the effect of adding dexmedetomidine versus clonidine to ropivacaine for axillary brachial plexus blockade. The primary endpoints were the onset and duration of sensory and motor block and duration of analgesia. ABSTRACT.MATERIALS AND METHODS:: A total of 90 patients (20-40 years) posted for ambulatory elective hand surgery under axillary brachial plexus block were divided into two equal groups (groups ropivacaine dexmedetomidine [RD] and ropivacaine clonidine [RC]) in a randomized, double-blind fashion. In group RD (n = 45) 30 ml 0.5% ropivacaine + 100 μg of dexmedetomidine and group RC (n = 45) 30 ml 0.5% ropivacaine + 75 μg clonidine were administered in axillary plexus block. Sensory and motor block onset times and block durations, time to first analgesic use, total analgesic need, postoperative visual analog scale (VAS), hemodynamics and side-effects were recorded for each patient. ABSTRACT.RESULTS:: Though with similar demographic profile in both groups, sensory and motor block in group RD (P < 0.05) was earlier than group RC. Sensory and motor block duration and time to first analgesic use were significantly longer and the total need for rescue analgesics was lower in group RD (P < 0.05) than group RC. Postoperative VAS value at 18 h were significantly lower in group RD (P < 0.05). Intraoperative hemodynamics were insignificantly lower in group RD (P < 0.05) without any appreciable side-effects. ABSTRACT.CONCLUSION:: It can be concluded that adding dexmedetomidine to axillary plexus block increases the sensory and motor block duration and time to first analgesic use, and decreases total analgesic use with no side-effects. BODY.INTRODUCTION: Pain frequently hampers implementation of ambulatory surgery in spite of so many analgesic drugs and regimens.[1] Postoperative pain has a negative impact on patient's early mobilization and discharge as well as it causes unanticipated hospital admission particularly in a day care setting.[2] Peripheral nerve block as an anesthetic technique plays an important role in modern regional anesthesia. The most important prerequisites for the use of peripheral regional anesthesia in daily clinical practice are success rate and safety. Upper limb surgeries especially hand surgeries below the elbow joint; commonly performed as an outpatient procedure; are mostly performed under peripheral blocks such as the axillary brachial plexus block.[3] Peripheral nerve blocks not only provide intraoperative anesthesia, but also extend analgesia in the postoperative period without major systemic side-effects by minimizing stress response and using minimal anesthetic drugs.[4] Ropivacaine is an amino-amide local anesthetic that blocks the peripheral afferents acting on voltage dependent Na+ channels. It is less cardiac and central nervous system toxic than other long acting local anesthetics like bupivacaine.[5] During hand surgeries local anesthetics alone for axillary brachial plexus block provide good operative conditions, but have shorter duration of postoperative analgesia. So various adjuvants like opioids,[6] clonidine,[7] neostigmine, dexamethasone,[8] midazolam,[9] etc., were added to local anaesthetics in axillary brachial plexus block to achieve quick, dense and prolonged block, but the results are either inconclusive or associated with side-effects. Clonidine is a selective α2 adrenergic agonist with some α1 agonist property. In clinical studies, the addition of clonidine to local anesthetic solutions improved peripheral nerve blocks by reducing the onset time, improving the efficacy of the block during surgery and extending postoperative analgesia.[1011] Dexmedetomidine is highly selective (8 times more selective than clonidine),[12] specific and potent α2 -adrenergic agonist having analgesic, sedative, antihypertensive and anesthetic sparing effects when used in systemic route.[13] Adding dexmedetomidine to local anesthetics during peripheral nerve blockade and regional anesthesia procedures may also prove efficacious for the surgical patients.[1415] The aim and objective of this study was to compare the analgesic efficacy of dexmedetomidine and clonidine as adjuvant to ropivacaine for axillary brachial plexus blockade in the in the first 20 h postoperative period of a day-care hand surgery. The severity of pain was recorded using visual analog scale (VAS) with choice options ranging from 0 (no pain) to 10 (worst possible pain). BODY.MATERIALS AND METHODS: After obtaining permission from Institutional Ethics Committee, written informed consent was taken. Totally 90 adult patients were randomly allocated to two equal groups (n = 45 in each group) using computer generated random number list. American Society of Anesthesiologists (ASA) physical status I and II, aged between 25 and 50 years of both sexes undergoing elective surgeries of hand under axillary brachial plexus block were enrolled in the study. Patients in group RC received 30 ml of 0.5% ropivacaine + 75 μg of clonidine for axillary block. Group RD received 30 ml 0.5% ropivacaine + 100 μg of dexmedetomidine for the same block. Adjuvants were made 1 ml in both the groups for blinding purpose after mixing it with normal saline. BODY.MATERIALS AND METHODS.EXCLUSION CRITERIA: Patient refusal, any known hypersensitivity or contraindication to ropivacaine, clonidine, dexmedetomidine; pregnancy, lactating mothers, hepatic, renal or cardiopulmonary abnormality, alcoholism, diabetes, long-term analgesic therapy, bleeding diathesis, local skin site infections were excluded from the study. Patients having history of significant neurological, psychiatric or neuromuscular disorders were also excluded. As we were dealing with day care surgery patients having no assistance in home and dwelling at more than 10 km from our institution were also excluded from this study. In the preoperative assessment, the patients were enquired about any history of drug allergy, previous operations or prolonged drug treatment. General examination, systemic examinations and assessment of the airway were done. Preoperative fasting of minimum 6 h was ensured before the operation in all day care cases. All patients received premedication of tablet alprazolam 0.5 mg orally the night before surgery as per preanesthetic check-up direction to allay anxiety, apprehension and for sound sleep. The patients also received tablet ranitidine 150 mg in the previous night and in the morning of operation with sips of water. All patients were clinically examined in the preoperative period, when whole procedure was explained. 10 cm VAS (0: No pain and 10: Worst pain imaginable) was also explained during preoperative visit. All patients are investigated for hemoglobin %, total leukocyte count, differential leukocyte count, erythrocyte sedimentation rate, platelet count, blood sugar, blood urea, serum creatinine and liver function tests. A 12 lead electrocardiography (ECG) and chest X-ray were also taken. On entering the patient in the operative room standard intraoperative monitors like ECG, pulse oximeter, non-invasive blood pressure were attached, and baseline parameter were recorded. Philips IntelliVue MP20 monitor used for this purpose. Intravenous (i.v.) infusion of Ringers' lactate started and oxygen given at 3 L/min via face mask. All patients received injection midazolam 0.04 mg/kg before procedure. After proper explanation of technique and positioning, axillary block was performed in the supine position with the upper arm abducted at 90° and the elbow flexed at 90°. The area was shaved the day before and disinfected. The axillary artery was palpated in the proximal part of the axilla, and a skin wheal was injected using 1 ml of lignocaine 2%. A nerve stimulator (Stimuplex Kanule A 50, B Braun, Melsungen, Germany) was used to identify the plexus. The position of the needle was judged adequate when an output current of <0.5 mA still elicited a slight distal motor response. With intermittent negative aspiration, the total 31 ml volume was injected into the perivascular area. All the blocks were performed by the same anesthetist unaware of the constituent of the drug and allotment of the group and similarly resident doctors keeping records of different parameters were also unaware of group allotment. Thus blinding was properly maintained. Sensory and motor blockade were assessed every 2 min after completion of injection till 30 min and then every 30 min after the end of surgery till first 12 h, thereafter hourly until the block had completely worn off. Sensory blockade of each nerve was assessed by pinprick. Onset time of motor blockade was defined as the time interval between the end of local anesthetic injection and paresis in all of the nerve distributions. The duration of sensory block was defined as the time interval between the onset of sensory block and the first postoperative pain. The duration of motor block was defined as the time interval between the onset of motor block and complete recovery of motor functions. After 30 min, if the block was considered to be adequate, surgery commenced. Heart rate, noninvasive blood pressure, respiratory rate, SpO2, ECG and pain VAS were recorded at 1st, 2nd, 4th, 6th, 9th, 12th, 16th, 18th and 20th postoperative h. Injection diclofenac sodium (75 mg intramuscular [IM]) was given as rescue analgesia if the pain VAS >3. First postoperative analgesia request time, total diclofenac used in first 20 h were recorded. All data will be collected by an observer who is unaware of patients' group assignment. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Sample size was estimated using first rescue analgesic requirement among two groups as the main primary variable. The average duration in each group was 750 min and to detect a difference of 10% (i.e., 75 min), at the P < 0.05 level, with a probability of detecting a difference this large, if it exists, of 80% (1−beta = 0.80). On the basis of previous study assuming within group standard deviation of 150 min and we needed to study at least 44 patients per group to be able to reject the null hypothesis that the population means of the groups are equal with probability (power) 0.80. Raw data were entered into a MS Excel spreadsheet and analyzed using standard statistical software Statistical Package for the Social Sciences (SPSS) version 18.0 (SPSS Inc., Chicago, Illinois, USA). Categorical variables were analyzed using the Pearson's Chi-square test. Normally distributed continuous variables were analyzed using the independent sample t-test and P < 0.05 was considered as statistically significant. BODY.RESULTS AND ANALYSIS: We recruited 45 subjects per group, more than the calculated sample size. There were no dropouts. 45 patients in the ropivacaine dexmedetomidine group (RD) and 45 in the ropivacaine clonidine group (RC) were eligible for effectiveness analysis. The age, body weight, sex distribution, ASA status and duration of surgery, tourniquet time, and anesthesia time in the two groups were found to be comparable [Table 1]. Indications for different hand surgeries were also similar and has no clinical significance (P > 0.05) [Table 2]. Onset of both sensory and motor block were earlier in RD group than group RC [Table 3] and they were clinically significant (P < 0.05). Table 3 also shows that sensory and motor block durations are significantly greater in the group receiving dexmedetomidine (RD) (P < 0.05) than clonidine group (RC). Table 1 Comparison of demographic data between the two study groups Table 2 Indications of upper limb orthopedic surgery for two groups Table 3 Onset and duration time for sensory and motor block The mean time from block placement to first request for pain medication, that is, the duration of analgesia was 821.11 min in the dexmedetomidine group, but 768.24 min in the clonidine group. This difference (about 52.87 min) was highly significant (P < 0.006) statistically as well as clinically [Table 4 and Figure 1]. Table 4 Rescue analgesic requirement in postoperative period (time and amount of IM diclofenac sodium injections) Figure 1Duration of sensory and motor block Table 4 and Figure 2 shows that group RD required less amount of diclofenac sodium injection as rescue analgesics than patients in group RC in first 20 h of postoperative period, and the difference is statistically highly significant (P < 0.01). Figure 2Number of intramuscular diclofenac injection as rescue analgesic in first 24 h postoperative period Figure 3 shows that VAS score was of much higher value in group RC than RD group. Again group RD suffered from bradycardia, which was statistically higher (P < 0.05) than group RC. Other side-effects were quiet comparable (P > 0.05) among two groups [Table 5]. Figure 3Comparison of visual analog scale score among group ropivacaine dexmedetomidine and group ropivacaine clonidine Table 5 Comparison of side-effects BODY.DISCUSSION: Day care surgery has proven over the years as the best method to reduce the burden on the health care resources, as well as the achievement of extreme patient satisfaction.[16] In developing countries, most of the patients avoid bearing expenses of prolonged hospital stay. At the same time, infrastructure in these countries is not organized uniformly to smoothly deliver the day care procedures. In the present day scenario, pain is the most common medical cause of delayed recovery and discharge after ambulatory surgery and a frequent cause of unplanned admission and subsequently delayed return to work.[17] Axillary brachial plexus blocks are performed at the level of the brachial plexus cords for forearm and hand surgeries.[18] Here, almost the entire sensory, motor and sympathetic innervations of the forearm and hand are carried within axillary plexus sheath just as three nerve structures (cords), confined to a very small surface area. Consequently, typical features of this block include rapid onset, predictable and dense anesthesia along with its high success rate.[19] Local anesthetics alone for axillary brachial plexus block provide good operative conditions but have a shorter duration of postoperative analgesia. Hence, various drugs have been used as an adjuvant with local anesthetics in axillary plexus block to achieve quick, dense and prolonged block, but the results are either inconclusive or associated with side-effects.[678920] Dexmedetomidine; a highly selective, α2 -adrenergic agonist; has analgesic, sedative, anesthetic sparing effects when used in the systemic route.[13] Use of dexmedetomidine as an adjuvant mixed with local anesthetics has been performed with neuraxial anesthesia in both adult and pediatric patients.[2122] Mixing dexmedetomidine as adjuvant with local anesthetics during peripheral nerve and nerve plexus blockade has recently been practiced by anesthesiologists.[2324] In this prospective, randomized, double-blinded trial we had compared the effect of 100 μg of dexmedetomidine and 75 μg clonidine as an adjuvant to 30 ml 0.50% ropivacaine in axillary brachial plexus block, on the onset time and duration of sensory and motor block as well as on the postoperative rescue analgesic (injection diclofenac sodium) requirement for the patients undergoing ambulatory elective hand surgery. The demographic profile, between two groups, which was statistically insignificant (P > 0.05) of our patients was quite similar to other research investigations and provided us the uniform platform to evenly compare the results obtained.[25] From [Table 2] it is quite evident that indications of surgical procedures were almost similar in both the groups and had no statistical significance. The onset time of sensory block (10.92 ± 4.23 min in RD group vs. 12.83 ± 3.18 min in RC group) was significantly earlier in dexmedetomidine groups (P = 0.017) [Table 3]. These findings correlate with the studies of Ammar and Mahmoud,[26] Kaygusuz et al.[27] Significantly earlier onset of motor block (17.11 ± 2.8 min in RD group vs. 18.55 ± 3.2 min in group RC) was observed in RD group (P = 0.025). Similarly Ammar and Mahmoud,[26] in their study found that motor block onset was hastened by the use of dexmedetomidine adjuvant in brachial plexus block. Again Kanazi et al.[28] found both clonidine and dexmedetomidine hastens motor block in spinal anesthesia with bupivacaine. Again in a study conducted by Marhofer et al.[14] in 36 volunteers it has been found that dexmedetomidine as adjuvant though produced early onset of motor block, sensory block was not different from control group or i.v. group. In our study, the duration of sensory block (810.34 ± 110.4 min in group RD vs. 730.25 ± 134.30 min in group RC) was significantly longer in the dexmedetomidine group than in the clonidine group (P < 0.002). The duration of motor block (610.44 ± 95.23 min in RD group vs. 565.44 ± 68.23 min in RC group) was also significantly longer in the dexmedetomidine group than in the clonidine group (P < 0.011). These findings lend support to the observations of various earlier studies by Marhofer et al.,[14] Esmaoglu et al.,[23] Ammar and Mahmoud.[26] In our study, mean duration of the sensory block (analgesia) and motor block in the dexmedetomidine plus ropivacaine group were 810.34 min and 730.25 min, respectively. While the mean duration of analgesia and motor block in the dexmedetomidine plus bupivacaine group were 2.99 h and 2.59 h respectively, in the study conducted by Ammar and Mahmoud.[26] Again the median duration of sensory and motor block in the dexmedetomidine plus levobupivacaine group in infraclavicular brachial plexus block were 14.78 h and 12.88 h respectively, in the study by Esmaoglu et al.[23] In our study, patients of RD group required significantly less amount of diclofenac sodium injection in first 20 h of postoperative period than the patients RC group (P < 0.05). This finding correlates with the studies of Kaygusuz et al.[27] Kaygusuz et al. found that 11 patients of levobupivacaine group required 75 mg IM injection of diclofenac sodium as rescue analgesic whereas dexmedetomidine plus levobupivacaine group required nothing and the result was also statistically significant.[27] Reduced requirement of rescue analgesic in the dexmedetomidine group during first 20 h of postoperative period is because of prolonged duration of sensory block. Again Ammar and Mahmoud[26] also experienced statistically much less amount (4.9 mg vs. 13.6 mg) of i.v. morphine administration as rescue analgesic in bupivacaine, dexmedetomidine group while comparing with plain bupivacaine group in infraclavicular brachial plexus block. In a study comparing intra- and post-operative analgesic effect of epidural clonidine and dexmedetomidine, Bajwa et al. found that dexmedetomidine produced longer postoperative analgesia than clonidine (316.64 vs. 296.72 min).[29] In group RD, bradycardia was observed in four patients, and all of these patients were managed with atropine. There was no such episode of bradycardia in group RC. Side-effects-including pneumothorax, Horner syndrome though noted in both the groups, but the difference was not statistically insignificant (P > 0.05). Esmaoglu et al.[23] also found significant bradycardia in dexmedetomidine plus levobupivacaine group than levobupivacaine alone. However, they found significant hypotension with dexmedetomidine group, which was absent in our study. Bajwa et al. found that dry mouth was main side-effects among two groups.[29] Ropivacaine, dexmedetomidine and clonidine dose was chosen as per the recommendation in the textbook as well as experience of our previous researchers.[2630313233] While writing this discussion, we have found the reference of lowest possible volume (10 ml) and concentration (0.375%) of ropivacaine for postoperative analgesia by Iwata et al.[34] However we had used a higher concentration and much higher volume for intra- as well as post-operative analgesia. However, the great drawback of our study was that we had not taken the equipotent dose of two above-mentioned α2 agonists due to nonavailability of proper pharmaceutical reference relating to dose equivalence. A control group was not included in our study because we regarded it as unethical to withhold any adjuvant in these patients for prolongation of postoperative pain management particularly when being posted for ambulatory surgery. We do conclude that during day care hand surgery, addition of 100 mcg dexmedetomidine is more effective than 75 mcg clonidine; regarding early onset of sensory and motor blockade, prolongation of block duration, reducing the requirement of rescue analgesic in postoperative period; when added to ropivacaine 0.50% solution in axillary brachial plexus block without any appreciable side-effect. BODY.DISCUSSION.FINANCIAL SUPPORT AND SPONSORSHIP: Nil. BODY.DISCUSSION.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: The Effect of Zinc Supplementationon the Symptoms of Gastroesophageal Reflux Disease; a Randomized Clinical Trial ABSTRACT: BACKGROUND Currently, it has been demonstrated that gastroesophageal reflux disease (GERD) is one of the most important disorders of the digestive system and the commixture of regular diet has a significant influence on its incidence, symptoms, and prognosis. The purpose of this study was to evaluate the effect of zinc supplementation, in combination with PPIs(Proton pump inhibitors), on the improvement of GERD symptoms. METHODS In a randomized double blind clinical trial, patients with reflux symptoms, who had obtained Reflux Disease Questionnaire (RDQ) score more than 8, were included and all the demographic features were recorded. Then, using upper gastrointestinal (GI) endoscopy, all the patients were divided into two groups as having non-erosive reflux disorder (NERD),or erosive reflux disorder (ERD). At the next step, based on random block statistical method, we divided the two groups into two subgroups; the drug subgroup [treated with PPIs (40 mg pantoprazole/daily), changing life style, and 220 mgzinc capsules daily] and the placebo subgroup [treated with PPIs, changing life style, and placebo]. After 3 months, we analyzed all data and the RDQ questionnaire was filled out for each patient. This project has been registered in Iranian Registry of Clinical Trials (IRCT) and all data were analyzed using SPSS software version 2. RESULTS A total of 140 patients (81 women and 59 men) with mean age of 42.78±11.5 years were included with 70 patients in each group. The most frequent presentations were heart burn (45.7%), and acid regurgitation (39.3%). The RDQ scores decreased after intervention in both drug (p<0.001) and placebo groups (p<0.001), which were statistically significant. But the difference of RDQ scores between the drug group and placebo group was not statistically significant (p=0.086). CONCLUSION Zinc supplementation cannot improve the severity of GERD. BODY.INTRODUCTION: One of the chronic and prevalent disorders of the digestive system is gastroesophageal reflux disease (GERD).Reflux of the gastric contents into the esophagus causes troublesome symptoms, including heartburn, regurgitation, and esophageal damages.1 In population-based studies, a remarkable increase in the incidence of GERD has been reported with the prevalence rates of 10-25% over the past decades globally.2,3 GERD can be classified based on the presence of symptoms without esophageal mucosal breaks on endoscopic examination [non-erosive reflux disease (NERD)] and the progression of NERD to erosive reflux disorder (ERD).1,4 Based on many studies, one of the most important risk factors, which are associated with the incidence and symptoms of GERD is human regular nutrition. Several studies have shown that the commixture of regular diet has a significant effect on the incidence, symptoms, and prognosis of GERD.5-7 Zinc is one of these critical components of regular diet, which is presented in all human tissues and body fluids (such as cornea, retina, skin, and parietal cells) and has an important role in cellular function.8 The effect of this mineral on GERD symptoms still remains unclear. Some studies have presented that zinc effectively inhibits gastric acid secretion,which provides a new prolonged therapy for those who suffer from overproduction of acid associated disorders such as heartburn and gastric acid reflux.9,10 On the other hand, there are other surveys that have shown that there is no relationship between acid reflux complications and intake of zinc supplements.11 Unfortunately, zinc deficiency is a frequent medical disorder worldwide, and therefore, it is necessary to know if its supplements could really improve the GERD symptoms. In this survey, we decided to evaluate the effect of zinc supplementationon GERD symptoms in patients who referred to gastrointestinal (GI) clinics. If there is a true relationship between zinc intake and improvement of GERD symptoms, this can be a good choice for adding it to routine GERD medical treatment. BODY.MATERIALS AND METHODS: In this randomized double blind clinical trial, all patients with acid reflux symptoms, referred to gastroenterology clinics in Rasht (a city in the northern Iran) from September 2014 to January 2015, were enrolled. Sampling method was easy and available. The patients were given the Reflux Disease Questionnaire (RDQ),12 for evaluating the symptoms before treatment. The RDQ is a self-administered questionnaire by which patients describe the frequency and severity of their GI symptoms by answering 15 questions scaled as Likert-type with scores ranging from 0 to 5. The individuals who obtained a RDQ score less than 8, patients aged below 18 years, pregnant women, patientswith positive H. pylori laboratory test, those with positive history of peptic ulcers, or patients with a history of previous abdominal surgery for any reason, were excluded from the study. BODY.MATERIALS AND METHODS. STEP-1: : Using upper GI endoscopy, two equal number groups were formed; 70 subjects with NERDas NERD group and 70 subjects with ERD as ERD group. A checklist based on the demographic features (such as age, sex, medical history, positive history of H2 blockers or PPIs consumption, duration of disease, more than 10 kg weight gain after maturity, weight loss during the past 3 months, insomnia, rapid eating, spicy foods, dinner time at 2 hours before sleep, smoking, and alcohol consumption) was filled out for each subject. BODY.MATERIALS AND METHODS. STEP-2: : At the next step, based on random block statistical method, we divided each group into two subgroups; the drug subgroup (n=35)[treated with PPIs (40 mg pantoprazole/daily), changing life style, and 220 mg zinc capsules daily], and the placebo subgroup (n=35)[treated with PPIs, changing life style, and placebo] (figure 1). Fig.1 The algorithm of the approach BODY.MATERIALS AND METHODS.MATERIALS: The zinc capsules contained 50 mg pure zinc, which was manufactured in Al-Havy Company. Both patients and the researchers did not know whether who received the drug or who received the placebo and all the patients were coded to prevent bias. After 3 months, we assessed all data and for the second time, RDQ questionnaire was filled out for each patient to evaluate the treatment result. BODY.MATERIALS AND METHODS. ETHICS AND STATISTICS: : This study was reviewed and approved by the Ethics Committee of Guilan University of Medical Sciences, and written informed consent was obtained from all the participants. Also, this project has been registered in Iranian Registry of Clinical Trials (IRCT registration number: IRCT201305281155N16).All the data were statistically analyzed using SPSS software for windows version 21.0 (SPSS Inc., Chicago, IL, USA). Percentage and frequency were used to report the results of the qualitative data and standard deviation and the mean were used to report the quantitative data. Chi-square, Fisher exact and independent t tests were used to compare the variables. In non-parametric results, Wilcoxon ranks test and Mann-Whitney test were used. p<0.05 was considered as statistically significant. BODY.RESULTS: Of the 140 patients with GERD who were enrolled in this survey, 81 patients were women (55.7%) and 59 patients were men (44.3%). The mean age of the subjects was 42.78±11.5 years. The most frequent underlying diseases were: Cardiac disease (n=13), diabetes (n=14), hypertension (n=13), and asthma (n=9).15 patients were smokers and 8 patients were alcohol consumer. 48 and 81 patients had a history of H2 blockers and PPIs consumption, respectively. The mean duration between the time of first symptoms presentation and diagnosis was 25.24±7.8 months. Heart burn (n=64), acid regurgitation (n=55), and retrosternal pain (n=49) were the most frequent GERD presentations. chart1 shows the general habits of the patients. Chart.1 The frequency distribution of the subject's general habits Based on random block method, the patients were divided into drug and placebo groups. Of the 70 patients included in the drug group, with mean age of 43.98±11.5 years,22 patients were men (31.4%) and 48 patients were women (68.6%). Of the 70 individuals included in the placebo group, with mean age of 41.48±12.4 years, 37 patients were men (52.9%) and 33 patients were women (47.1%). The statistical distribution of age and sex between the two groups had normal distribution. Statistical analysis showed no direct relationship between the results in the groups and age, sex, and general habits. Among underlying diseases, 4.3% of patients in the drug group and 14.3% of the patients in placebo group suffered from high blood pressure, and 20% of the patients in the drug group and 48.6% of the patients in the placebo group mentioned a history of H2 blockers consumption. But other underlying diseases such as diabetes, asthma, cardiovascular disease, and use of PPIs did not show significant difference. Table 1 shows the difference of the frequency of GERD presentations, before and after intervention, among all the subjects. As table shows, the frequency of presentations decreased in both drug and placebo groups after treatment. Based on the results obtained byFisher's exact test, the statistical difference between the drug and placebo groups in terms of presentations' frequencies was statistically significant in 6 items: Throat cleansing (p=0.05), chronic cough (p=0.04), postnasal drip (p=0.03), foreign body sensation (p=0.003), heart burn (p=0.003), and otitis media (p=0.003). Table1 Comparison of the frequency of GERD presentations among all subjects before and after intervention GERD presentations Drug group (n=70) Placebo group (n=70) Before intervention [n (%)] After intervention [n (%)] P value Before intervention [n (%)] After intervention [n (%)] P value Chest Pain 48 (68.6) 20 (28.6) 0.001 49 (70) 29 (41.4) 0.001 Regurgitation 49 (69.9) 21 (30) 0.001 48 (68.6) 21 (30) 0.002 Throat pain 43 (61.4) 12 (17.1) 0.002 30 (42.9) 13 (18.6) 0.004 Hoarseness 46 (65.7) 11 (15.7) 0.003 30 (42.9) 12 (17.1) 0.003 Throat cleansing 54 (77.1) 15 (21.4) 0.002 54 (77.1) 24 (34.3) 0.002 Sore throat 30 (42.9) 5 (7.1) 0.001 20 (28.6) 6 (8.6) 0.001 Chronic cough 50 (71.4) 12 (17.1) 0.001 44 (62.9) 26 (37.1) 0.001 Dysphagia 19 (27.8) 3 (4.3) 0.002 18 (25.8) 7 (9.9) 0.007 Postnasal drip 51 (72.8) 20 (28.6) 0.001 50 (71.5) 35 (50) 0.003 Foreign body sensation 43 (61.4) 12 (17.1) 0.002 52 (74.2) 27 (38.5) 0.001 Burning tongue 17 (24.2) 1 (1.4) 0.003 11 (15.8) 3 (4.3) 0.03 Throat tightness 20 (28.6) ---- 0.002 14 (20) 3 (4.3) 0.003 Otitis media 11 (15.7) ---- 0.001 91 (12.9) 2 (2.8) 0.02 Insomnia 29 (41.4) 9 (12.9) 0.001 23 (32.9) 5 (7.1) 0.001 Heart burn 64 (91.4) 23 (31.4) 0.002 57 (81.4) 32 (45.7) 0.002 Based on K-S test, RDQ scores did not follow the normal statistical distribution pattern.So to compare the results, before and after treatment, in each group and between the both groups, we used Wilcoxon ranked test and Mann-Whitney test, respectively (Chart 2). As the chart shows, the RDQ scores decreased after intervention in both drug group (p<0.001) and placebo group (p<0.001), which were statistically significant. On the other hand, based on Mann-Whitney test, the difference of RDQ scores between the drug group and placebo group was not statistically significant (p=0.086). To analyze the treatment effect, we assessed the effect size, which based on Cohen's classification,13 showed a large difference after treatment (0.8) as a proper influence on each group. But the comparison of standard response between the two study groups was not significant (p=0.72). These results show that due to pantoprazole, RDQ score decreased in both groups but zinc supplementation could not make a huge different results in drug group (table 2). Chart.2 The RDQ scores before and after intervention in both the study groups Table 2 Responsiveness indicators by zinc supplementation and placebo treatment RDQ Score Effect Size Standardized response mean Drug group Placebo group Drug group Placebo group 1.24 1.14 0.75 0.78 BODY.DISCUSSION: GERD is elucidated as symptoms or mucosal damages processed by the abnormal reflux of gastric contents into the esophagus.14 Affecting 33% of the world's populace, GERD incurs noteworthy immediate and backhanded expenses, huge loss of profitability of the affected individuals, and irreversible impacts on the quality of life.15 Due to the importance of GERD, design of more studies to identify its different aspects could be an effective task in the improvement of the general health. Our findings showed that heart burn, acid regurgitation, and retrosternal pain are the most frequent symptoms of GERD, which is similar to other surveys.16,17 This shows that when a patient presents with such symptoms, we have to consider GERD as the most important differential diagnosis and should refer him to a gastroenterology clinic. One of the most important risk factors thathave appeared to be connected with the side effects of gastroesophageal reflux is the mixture of daily nutrition.15-16 Our results showed that the highest frequency of general habits in patients with GERD belonged to rapid eating and eating spicy foods. Today, eating fast foods (Western foods) such as spicy and salty foods is increasing among Asians. This kind of nutrition carries a heavy load of nitrate, which irreversiblyaffectsthe GI system. As our study showed, such effects are more probable to make GI erosions with higher frequency in the ERD subjects. On the other hand, zinc deficiency is a common health problem in Asians. Zinc is an essential mineral in daily nourishment and plays a critical role in human tissue synthesis and repair.18 It has been shown that the essential nutrient zinc is highly concentrated in parietal cells (parietal cells are gastric cells that secrete gastric acid and intrinsic factor). So it may have beneficial effects on the treatment of gastric ulcers.13,19 Considers in the 1980s examined zinc's capability to mend and recover the defensive mucosal gel layer of the stomach.9 In addition zinc is rich in the muscularis of the gastric mucosa and appears to help this membrane to thicken. When the mucus membrane thickens it can better assist in buffering against excess gastric acid. It is known that zinc deficient patients are more susceptible to the development of gastric ulcers. Unfortunately proton pump inhibitors and H2-blockers inhibit the absorption of zinc, which may contribute to an unfortunate pathological cycle.15,18-19 Kirchhoff and colleagues,9 performed the first study to show the ability of zinc in the inhibition of acid secretion. They reported that zinc oral supplements (with or without PPIs) inhibited acid secretion and increased gastric PH in both human and rodent gastric glands. They showed that zinc delivered a fast and delayed hindrance of gastric corrosive discharge and offered a potential helpful contrasting option to PPIs. Similar to their findings, Tran and co-workers,20 presented that chronic gastritis could be constricted by fleeting treatment of zinc, which proposes that zinc alone might be powerful for the concealment of gastric mucosal irritation. These results are not matched with our findings. We found a significant improvement of GERD presentations among all the subjects after 3 months treatment, which shows the strong effect of the same treatment in both groups (40 mg pantoprazole/daily and changing life style). However, there was no huge distinction between the results of the drug and the placebo groups, which shows the non-effective role of zinc supplementation on GERD's symptoms and severity. Similar to our results, Murphy and colleagues,10 reported that intake of anti-oxidants and minerals (such as vitamin C and E, total carotenoids , zinc, copper, or selenium) had no effects on the symptoms of GERD or Barrett's esophagus. Our findings showed that zinc supplementation cannot improve the severity of GERD. Other surveys should be performed to assess the impact of zinc supplementation on patients with GERD andzinc deficiency. Although considering zinc supplementation for GERD patients with underlying zinc deficiency would lead to better probable results, this element was not evaluated in our study before intervention. It is also recommended to evaluate the relationship between other critical minerals and the severity of GERD to find other effective supplementations to replace or adjust the traditional treatment with PPIs and H2 blockers. BODY.ACKNOWLEDGEMENTS: This manuscript is a part of an internist thesis.We would like to thank all members of Gastrointestinal and Liver Diseases Research Center (GLDRC) who assisted us in this study. We thank Dr. Zahra Haghparast Ghadim Limudahi for critical reading of the manuscript and for the insightful suggestions. Funding/Support: This study was supported by the Gastrointestinal and Liver diseases Research Center of Guilan University of Medical Sciences. BODY.CONFLICT OF INTEREST: The authors declare no conflict of interest related to this work.

TITLE: Coconut-derived D-xylose affects postprandial glucose and insulin responses in healthy individuals ABSTRACT: Metabolic alterations including postprandial hyperglycemia have been implicated in the development of obesity-related diseases. Xylose is a sucrase inhibitor suggested to suppress the postprandial glucose surge. The objectives of this study were to assess the inhibitory effects of two different concentrations of xylose on postprandial glucose and insulin responses and to evaluate its efficacy in the presence of other macronutrients. Randomized double-blind cross-over studies were conducted to examine the effect of D-xylose on postprandial glucose and insulin response following the oral glucose tolerance test (OGTT). In study 1, the overnight-fasted study subjects (n = 49) consumed a test sucrose solution (50 g sucrose in 130 ml water) containing 0, 5, or 7.5 g D-xylose powder. In study 2, the overnight-fasted study subjects (n = 50) consumed a test meal (50 g sucrose in a 60 g muffin and 200 ml sucrose-containing solution). The control meal provided 64.5 g of carbohydrates, 4.5 g of fat, and 10 g of protein. The xylose meal was identical to the control meal except 5 g of xylose was added to the muffin mix. In study 1, the 5 g xylose-containing solutions exhibited significantly lower area under the glucose curve (AUCg) and area under the insulin curve (AUCi) values for 0-15 min (P < 0.0001, P < 0.0001), 0-30 min (P < 0.0001, P < 0.0001), 0-45 min (P < 0.0001, P < 0.0001), 0-60 min (P < 0.0001, P < 0.0001), 0-90 min (P < 0.0001, P < 0.0001) and 0-120 min (P = 0.0071, P = 0.0016). In study 2, the test meal exhibited significantly lower AUCg and AUCi values for 0-15 min (P < 0.0001, P < 0.0001), 0-30 min (P < 0.0001, P < 0.0001), 0-45 min (P < 0.0001, P = 0.0005), 0-60 min (P = 0.0002, P = 0.0025), and 0-90 min (P = 0.0396, P = 0.0246). In conclusion, xylose showed an acute suppressive effect on the postprandial glucose and insulin surges. BODY.INTRODUCTION: Obesity has been a major health concern worldwide, and the World Health Organization estimated that approximately 1.5 billion adults were overweight and that over 200 million men and nearly 300 million women were obese in 2008. The National Health and Nutrition Examination Survey of Korea has also reported that 34.0% of the population is either overweight or obese, raising a serious health hazard. Major metabolic disturbances due to excess body fat include insulin resistance, which is characterized by the impeded capacity of peripheral tissues to utilize glucose effectively [1], causing hyperglycemia and hyperinsulinemia. Increased circulating concentration of insulin also result in changes in lipid metabolism, cell growth, and cell-to-cell communication [2] which eventually lead to obesity-related disease development. The glycemic index (GI) was introduced to classify starchy foods according to their effect on postprandial glycemia [3]. Later, the glycemic load (GL) concept was introduced reflecting the quality (GI) and quantity of a specific food consumed that affect postprandial glycemic response [4]. Both low GI foods and low GL diets have been successfully applied to alleviate hyperglycemic and hyperinsulinemic conditions in prediabetic and diabetic individuals. Besides GI and GL, a growing body of research has focused on functional substances inhibiting carbohydrate digestion enzymes [5-6]. Previous preliminary studies have indicated that several monosaccharides act as glucosidase inhibitors [7-8]. D-xylose at a 10 mmol/L concentration inhibits intestinal sucrase activity by 52.1% [8]. Rats fed a combination of sucrose (2 g/kg) and xylose (0.1 g/kg) solution have significantly reduced postprandial blood glucose concentrations at 30 and 60 min compared to those of rats fed a sucrose solution alone [9]. One small-scale human study (n = 5) showed the efficacy of xylose to suppress the increase in postprandial blood glucose in which healthy males showed lowered postprandial blood levels glucose and insulin at 30 min after consuming the solution containing xylose (7.5 g) mixed with, 75 g of sucrose compared to levels after consuming a 100% sucrose solution. However, no significant difference was observed in blood glucose or insulin concentrations for the remaining time points. Gruzman et al. [10] reported that D-xylose increases the rate of glucose transport in a non-insulin-dependent manner in rats and human myotubes in vitro. These results suggest that xylose may be a good candidate functional food to control the postprandial blood glucose and insulin surges and possibly alleviate metabolic disturbances. Previous studies have reported that diet composition particularly quantitatively important macronutrients influence postprandial glycemia by affecting the rate of carbohydrate digestion and absorption. Dietary fat, carbohydrate, and protein in a single meal interact with each other to influence the rate of absorption [11]. Therefore, digestive enzyme inhibitors such as xylose may act differently when they are used as ingredients of specific food products. Thus, the objectives of this study were to assess the inhibitory effects of two different concentrations of xylose on postprandial serum glucose and insulin concentrations in a large group of healthy human subjects and to evaluate its efficacy in the form of a food product. BODY.SUBJECTS AND METHODS.SUBJECTS: Forty-nine subjects (25 males and 24 females) participated in study 1. Their average age was 35.4 ± 6.4 years (range, 25-49 years), and their body mass index was 23.8 ± 3.0 kg/m2. In study 2, 50 subjects (24 males and 26 females) participated. Their average age was 33.7 ± 6.0 years (range, 25-49 years), and their body mass index was 23.9 ± 3.2 kg/m2. The study subjects were recruited from a university website advertisement based on the following criteria: (1) fasting serum glucose < 7 mmol/L; (2) absence of any disease; (3) under no medications or dietary supplements; and (4) non-pregnant women. Thirty subjects (13 males and 17 females) were enrolled in both studies. This study was approved by the Institutional Review Board of Sookmyung Women's University (SM-IRB-11-0217-001), and eligible subjects gave informed written consent. Sample size was calculated based on a previous clinical trial [12-14], and a 1.0 mmol/L reduction in the peak glucose concentration during the oral glucose tolerance test (OGTT) was considered to be the primary efficacy point with the average standard deviation (SD) of 1.2 mmol/L at a two-tailed alpha = 0.05 and 1-β = 80%. BODY.SUBJECTS AND METHODS.STUDY DESIGN.STUDY 1: This study used a randomized double-blind three treatment cross-over design. The overnight fasted study subjects visited the trial center at the university once per week for 3 weeks and consumed a test sucrose solution (50 g sucrose in 130 ml water) containing 0, 5, or 7.5 g D-xylose powder each week in random order. Xylose concentrations were determined based on a previous report in which xylose mixed with sucrose at a 10% level showed a delay in glucose absorption [9]. Study subjects were asked to maintain their usual dietary and physical activity habits throughout the study. No significant differences in nutrient intake were observed (by 24-recall method) between visits (data not shown). BODY.SUBJECTS AND METHODS.STUDY DESIGN.STUDY 2: Study 2 was conducted as a randomized double-blind two treatment cross-over trial. The overnight-fasted study subjects visited the trial center once per week for 2 weeks and consumed a test meal in a given time (10 min) in random order. The test meal consisted of a muffin (60 g) and a sucrose-containing solution (200 ml). The control meal provided 64.5 g of carbohydrates (sucrose 50 g), 4.5 g of fat, and 10 g of protein. The xylose meal was identical to the control meal except 5 g of D-xylose was additionally added to the muffin mix. Study subjects were asked to maintain their usual dietary habits and physical activity level throughout the study. No significant differences in nutrient intake were observed (by 24-recall method) between visits (data not shown). BODY.SUBJECTS AND METHODS.BLOOD ANALYSES: A cannula was placed in the antecubital vein and blood samples were taken at the following times: 0, 15, 30, 45, 60, 90, and 120 minutes after taking the test solution or meal. The samples were left at room temperature for 30 min, centrifuged for 15 min at 2,500 rpm to separate the serum, and then stored at -70°C. Serum glucose concentrations were measured using the Pureauto S GLU reagent (Daiichi Pure Chemicals, Tokyo, Japan). Insulin was measured by electrochemiluminescence immunoassay (Roche Diagnostics GmbH, Mannheim, Germany). Serum glucose and insulin were measured at every time point (0, 15, 30, 45, 60, 90, and 120 minutes) after taking the test solution or meal, and the blood glycated hemoglobin (HbA1c), serum total cholesterol, triglyceride, and high density lipoprotein (HDL)-cholesterol were measured at only one time point (0 minutes). Blood HbA1c was determined by high performance liquid chromatography. An autoanalyzer (Hitachi 747 auto-analyzer, Tokyo, Japan) was used to quantify blood concentrations of serum total cholesterol, triglyceride, and HDL-cholesterol. Serum low-density lipoprotein (LDL) cholesterol concentration was calculated according to the Friedewald formula [15]. BODY.SUBJECTS AND METHODS.CALCULATIONS AND STATISTICAL ANALYSIS: All results are presented as mean ± SD. The areas under the glucose (AUCg) or insulin (AUCi) curves were calculated using the trapezoidal rule [16]. Statistical analyses were performed to compare the AUC of glucose and insulin and to compare concentrations of serum glucose and insulin between treatments at each time point. Differences were assessed using repeated-measure analysis of variance followed by Tukey's multiple comparisons test (study 1) or Student's paired t-test (study 2). The two-tailed significance threshold was set at P < 0.05. Statistical analyses were performed using SAS program version 9.1 (SAS Institute, Cary, NC, USA). BODY.RESULTS: Body compositions and clinical parameters of the study participants at baseline of each study are shown in Table 1. In study 1, the proportion of individuals with dyslipidemia was 12.2% for cholesterol (reference range, ≥ 230 mg/dl), 8.2% for triglycerides (reference range, ≥ 200 mg/dl), and 4.1% for LDL-cholesterol (reference range, ≥ 150 mg/dl), and the proportion of the impaired fasting glucose individuals was 8.2% for glucose (reference range, fasting glucose ≥ 100 mg/dl and < 126 mg/dl). In study 2, the proportion of individuals with dyslipidemia was 4.0% for cholesterol (reference range, ≥ 230 mg/dl), 8.0% for triglycerides (reference range, ≥ 200 mg/dl), and 4.0% for LDL-cholesterol (reference range, ≥ 150 mg/dl). The proportion of individuals with impaired fasting glucose was 2.0% for glucose (reference range, fasting glucose, ≥ 100 mg/dl and < 126 mg/dl). BODY.RESULTS.STUDY 1: The postprandial glucose and insulin responses after ingestion of 50 g sucrose with 5 g (low) xylose, 7.5 g xylose (high), or control (sucrose only) are presented in Figs. 1 and 2. The concentrations of serum glucose at 15 min (P < 0.0001), 30 min (P < 0.0001), and 45 min (P = 0.0005) were significantly lower after consuming both concentrations of xylose-containing solutions compared to those after consuming the control solution. However, glucose concentration was significantly higher at 120 min for those who consumed the xylose-containing solution (P < 0.0001). Serum concentrations of insulin at 15 min (P < 0.0001), 30 min (P < 0.0001), and 45 min (P = 0.0001) were also significantly lower after consuming both concentrations of xylose-containing solution than those after consuming the control solution. However, the xylose-containing solution significantly increased serum insulin at 120 min (P < 0.0001) compared to that in the control solution. No differences were observed for postprandial serum glucose or insulin concentration at either 60 or 90 min. The incremental area under the curve of postprandial serum glucose and insulin are shown in Tables 2 and 3. Xylose-containing solutions exhibited significantly lower AUCg values at 0-15 min (P < 0.0001), 0-30 min (P < 0.0001), 0-45 min (P < 0.0001), 0-60 min (P < 0.0001), and 0-90 min (P < 0.0001). However, the 0-120 min AUCg for in the 5 g xylose-supplemented solution was significantly lower than that of the control solution (P = 0.0071). The percent decreases in AUCg with 5 g xylose supplementation compared to those of the control were 47.3% (0-15 min), 39.9% (0-30 min), 35.5% (0-45 min), 32.3% (0-60 min), 26.2% (0-90 min), and 21.4% (0-120 min), respectively. Both 5 and 7.5 g xylose supplementation significantly reduced postprandial AUCi at 0-15 min (P < 0.0001), 0-30 min (P < 0.0001), 0-45 min (P < 0.0001), 0-60 min (P < 0.0001), 0-90 min (P < 0.0001), and 0-120 min (P = 0.0016). The percent decreases in AUCi with 5g xylose supplementation compared to those of the control were 45.2% (0-15 min), 39.2% (0-30 min), 35.2% (0-45 min), 30.7% (0-60 min), 24.5% (0-90 min), and 21.3% (0-120 min), respectively. BODY.RESULTS.STUDY 2: The mean postprandial serum glucose and insulin responses after ingestion of either the control meal or test meal are presented in Figs. 3 and 4. The concentrations of serum glucose at 15 min (P < 0.0001) and 30 min (P < 0.0001) after intake of the test meal were significantly lower than those after intake of the control meal. Additionally, the concentrations of insulin at 15 min (P < 0.0001) and 30 min (P = 0.0136) after intake of the test meal were significantly lower than those after intake of the control meal. No differences were observed for postprandial serum glucose or insulin concentrations at 45, 60, 90, or 120 min. The incremental area under the curve of postprandial serum glucose and insulin are shown in Tables 4 and 5. The test meal significantly lowered AUCg values at 0-15 min (P < 0.0001), 0-30 min (P < 0.0001), 0-45 min (P < 0.0001), 0-60 min (P = 0.0002), and 0-90 min (P = 0.0396). The percent decreases in AUCg with the test meal compared to those of the control meal were 37.0% (0-15 min), 32.4% (0-30 min), 26.5% (0-45 min), 21.4% (0-60 min), and 12.8% (0-90 min), respectively. The test meal significantly reduced postprandial AUCi at 0-15 min (P < 0.0001), 0-30 min (P < 0.0001), 0-45 min (P = 0.0005), 0-60 min (P = 0.0025), and 0-90 min (P = 0.0246). The percent decreases in AUCi with a test meal compared to those of the control meal were 47.8% (0-15 min), 39.5% (0-30 min), 32.7% (0-45 min), 28.9% (0-60 min), and 21.5% (0-90 min), respectively. BODY.DISCUSSION: Two acute randomized double-blind cross-over studies were conducted to examine the effect of D-xylose on postprandial glucose and insulin responses following an OGTT. Xylose is classified as a monosaccharide of the aldopentose type containing five carbon atoms and an aldehyde functional group. It is a precursor to hemicellulose, one of the main constituents of biomass. Corn cob, coconut, seed hulls, and straw are rich sources of D-xylose. D-xylose has been suggested as a potent sucrase inhibitor possibly by suppressing the postprandial glucose surge; however, no concrete scientific evidence has been provided for its efficacy in humans. Recent statistics indicate that a growing number of the population are either overweight or obese due to excess dietary energy intake, which promotes create insulin resistance. Evidence suggests that the initial pathophysiological changes during the progress of type 2 diabetes, cardiovascular heart diseases, and cancer are closely related to hyperinsulinemia, which lead to metabolic dysregulation. Post-challenge glucose levels of 108 mg/dL in mononuclear cells and monocytes results in a > two-fold elevation in reactive oxygen species [17]. High circulating levels of glucose can also generate glycation products from other important biological molecules that can cause undesirable functional and morphological changes [18]. In patients with type 2 diabetes, postmeal hyperglycemia induces diabetic complications [19]. Therefore, the homeostatic control of blood insulin and glucose possibly by regulating glucose absorption from the gut has been implicated as an important intervention target to suppress abnormal metabolic changes and prevent disease development. The GIs of various carbohydrate foods have been determined to develop a dietary means to control postprandial blood glucose. The GI is the increase in the area under the blood glucose curve produced by a standard amount of carbohydrate in a food, usually 50 g, relative to the area of increase produced by the same amount of carbohydrate from a standard source, usually white bread or glucose [16]. A low GI diet and low GI food intake delays or reduced rapid increases in blood glucose and improves insulin sensitivity. Elevated postprandial glucose excursions may increase the risk for cardiovascular disease even in nondiabetic persons [20]. Additionally, high GI diets have been associated with the development of insulin resistance [21], cardiovascular disease [22-23], and type 2 diabetes [24]. Thus, it is important to delay the rapid increase in blood glucose to improve insulin response and prevent other chronic metabolic diseases. Not only the diet itself but functional substances including different types of dietary fiber and monosaccharides have been developed to control postprandial glucose and insulin excursions. A recent human intervention trial using L-arabinose as a sucrase inhibitor indicated that a 75 g sucrose challenge supplemented with 4% w/w L-arabinose produces an 11% lower glucose peak and a 33% lower and delayed insulin peak [25]. A 23% reduction in the increase in the AUCi was also observed. In the present study, 5 g or 7.5 g xylose supplementation during a 50 g oral sucrose challenge effectively reduced serum glucose and insulin increases, and glucose absorption was delayed. D-Xylose possesses similar sucrase inhibitory activity as L-arabinose in vitro [26]. The 21.4% reduction in AUCg at 0-120 min and the 21.3% reduction in AUCi at 0-120 min after subjects ingested D-xylose as observed in the present study are close to a previous report on the efficacy of L-arabinose [26]. An earlier study indicated that 80% inhibition of sucrase activity is required to reduce the glycemic response by 50% following treatment with castanosperminem, an established strong sucrase inhibitor, suggesting the sucrase inhibition activity may partly explain the reduction in the glucose response. However, no adaptive response to sucrose digestion was observed after a long-term (14 days) treatment, indicating that sucrase blockers may be safely used as functional substances for glucose homeostatic control. The present study showed no dose-response inhibitory activity, suggesting that there may be an optimal dose to inhibit enzyme activity. The manner of sucrase inhibition by D-xylose has not been previously reported, whereas L-arcabose selectively inhibits sucrose activity in an noncompetitive manner with dose-responsive efficacy [8]. Therefore, further kinetics studies are required to understand the mechanism of enzyme inhibition. Because xylose is a sucrase inhibitor, it may be useful as a functional ingredient in sugar or other sucrose-rich food products to inhibit the blood glucose rise. In our second study we supplemented muffins with xylose to examine if xylose sustains its activity in a food matrix with large proportions of protein and fat. Previous studies have suggested that dietary protein increases insulin secretion and lowers blood glucose [11]. Additionally, the initial glycemic response is reduced after consuming a meal with higher fat content [27]. These results suggest that xylose supplemented in a meal with high fat or protein may not exert an identical effect as when supplemented in a sucrose solution. The results showed that a xylose-supplemented meal effectively reduced postprandial serum glucose and insulin responses; however, the extent of suppression was smaller than that of a xylose-supplemented solution. Xylose supplementation maintained its inhibitory activity up to 60 min for both AUCg and AUCi, and the increases in AUCg and AUCi at 0-120 min were reduced by 6.7% and 15.2 %, respectively. Previous studies have suggested that dietary protein increases insulin secretion and lowers blood glucose [11]. A shortened effectiveness may be explained by the presence of a higher amount of carbohydrate in test meals in study 2 compared to that in study 1 (65 g vs. 50 g) and/or the presence of starch in the test muffin, which may have less opportunity to counteract the sucrase inhibitor in a given time. As xylose is a sucrase inhibitor, it possesses no inhibitory effect on other carbohydrate digestive enzymes [9]. Several natural dietary substances have been tested for their inhibitory effects on the absorption of starch and sucrose in rats and pigs [28]. L-arabinose is effective for reducing postprandial blood glucose only after a sucrose challenge, but not with a rice starch challenge, whereas bean extract and hibiscus extract were more effective for reducing postprandial blood glucose after a rice starch challenge indicating that each substance may have different specificity to different carbohydrate digestive enzymes. We used a high-fat containing muffin as a test meal as opposed to a sucrose solution to observe possible changes in blood glucose responses due to high-fat content. Because we did not observe a significant difference except a loss of difference in AUCg and AUCi at one time interval (0-120 min), it was assumed that there was not a great deal of interaction between macronutrients and xylose. Further studies using different foods and food products are necessary to confirm the efficacy of xylose for postprandial glucose control. In conclusion, the present study indicated that coconut-derived D-xylose favorably affected postprandial glucose and insulin response when used as a supplement with sucrose. As repeated glucose and insulin excursions are closely related to metabolic stress, the proper application of xylose in different sucrose-based products may lessen the undesired effects of sucrose.

TITLE: Lidocaine spray alone is similar to spray plus viscous solution for pharyngeal observation during transoral endoscopy: a clinical randomized trial ABSTRACT: Background and study aims It is important to examine the pharynx during upper gastrointestinal endoscopy. Pharyngeal anesthesia using topical lidocaine is generally used as pretreatment. In Japan, lidocaine viscous solution is the anesthetic of choice, but lidocaine spray is applied when the former is considered insufficient. However, the relationship between the extent of pharyngeal anesthesia and accuracy of observation is unclear. We compared the performance of lidocaine spray alone versus lidocaine spray combined with lidocaine viscous solution for pharyngeal observation during transoral endoscopy. Patients and methods In this prospective, double-blinded, randomized clinical trial conducted between January and March 2015, 327 patients were randomly assigned to lidocaine spray alone (spray group, n = 157) or a combination of spray and viscous solution (combination group, n = 170). We compared the number of pharyngeal observable sites (non-inferiority test), pain by visual analogue scale, observation time, and the number of gag reflexes between the two groups. Results The mean number of images of suitable quality taken at the observable pharyngeal sites in the spray group was 8.33 (95 % confidence interval [CI]: 7.94 – 8.72) per patient, and 8.77 (95 % CI: 8.49 – 9.05) per patient in the combination group. The difference in the number of observable pharyngeal sites was – 0.44 (95 % CI: – 0.84 to – 0.03, P = 0.01). There were no differences in pain, observation time, or number of gag reflexes between the 2 groups. Subgroup analysis of the presence of sedation revealed no differences between the two groups for the number of pharyngeal observation sites and the number of gag reflexes. However, the number of gag reflexes was higher in the spray group compared to the combination group in a subgroup analysis that looked at the absence of sedation. Conclusions Lidocaine spray for pharyngeal anesthesia was not inferior to lidocaine spray and viscous solution in terms of pharyngeal observation. It was considered that lidocaine viscous solution was unnecessary for pharyngeal observation. UMIN000016073 BODY.INTRODUCTION: Upper gastrointestinal endoscopy (UGE) is a common diagnostic procedure. However, patients usually experience severe discomfort during transoral UGE because of the strong gag reflex and pain that occur when the endoscope is passed through the pharynx 1 2 3. To reduce transoral UGE-induced gag reflex and pain, topical pharyngeal anesthesia is administered, either alone or in combination with intravenous sedation 4 5. Lidocaine is the topical anesthetic of choice, administered as either a viscous solution or a spray. There is some literature comparing lidocaine viscous solution and spray in UGE. The Japan Gastroenterological Endoscopy Society (JGES) recommends use of lidocaine viscous solution prior to endoscopy; use of lidocaine spray is recommended only if the viscous solution is considered insufficient 6. However, topical lidocaine spray may be a better option than lidocaine viscous solution because of a higher procedural completion rate, greater ease of intubation, and greater satisfaction for both the patient and endoscopist 7. Recent advances in endoscopic instruments, including magnifying endoscopy and narrow band imaging (NBI), have enabled more in-depth examinations. Transoral UGE with NBI has been found to improve the early diagnosis of superficial squamous cell pharyngeal carcinomas 8 9 10. Early detection is associated with a better prognosis and use of minimally invasive treatment techniques such as endoscopic resection. However, endoscopic observation of the pharynx is difficult, and some pharyngeal tumors may remain undiagnosed. To reduce the rate of undiagnosed cancers, it is important to observe the pharynx carefully under less-active gag reflex, and to know the blind spots in the pharynx. To identify the best anesthetic for pharyngeal observation, we conducted a prospective, double-blinded, randomized clinical trial that compared lidocaine spray alone (spray group) with a combination of lidocaine spray and viscous solution (combination group). The viscous solution is hard to swallow because of its viscous texture and bitter taste 4 11. In addition, it prolongs the duration of pharyngeal anesthesia and increases the risk of lidocaine intoxication. In this study, if the spray group was not inferior to the combination group in terms of pharyngeal observation, lidocaine viscous solution would be considered unnecessary. We devised a non-inferiority trial to test this hypothesis. BODY.PATIENTS AND METHODS.PATIENTS: Patients age ≥ 20 years undergoing transoral UGE from January to March 2015 provided written informed consent prior to endoscopy. Only patients who agreed to participate in the study were enrolled. Patients who had previously undergone surgical or endoscopic mucosal resection for pharyngeal cancer were excluded, because their pharynxes had been removed or were too damaged to evaluate. Other exclusion criteria were a history of allergy of lidocaine, difficulty participating in the test because of psychosis or psychotic symptoms, and determined to be unable to carry out the study. BODY.PATIENTS AND METHODS.STUDY DESIGN AND ANESTHESIA PROTOCOLS: This prospective, double-blinded, randomized study was conducted with patients from a single center (Kanazawa University Hospital, Ishikawa, Japan). We inquired about study participants' expectation of sedation at the time of informed consent, before group allocation. If expectation of sedation is confirmed after group allocation, the patients in the spray group are more likely to expect more sedation than those in the combination group. The enrolled patients were randomized by computer-generated numerical codes to receive either spray or spray plus viscous solution (simple randomization, allocation ratio 1:1). A stratification factor (by sedation status or presence/absence of sedation) was included in the analysis because the results are likely to depend on sedation. The randomization list was stored in the clinical laboratory department, and could not be accessed by the study researchers. The combination group received lidocaine viscous oral solution 2 % (Xylocaine Viscous 2 %; AstraZeneca, Osaka, Japan). Lidocaine viscous solution was allowed to accumulate in the back of the throat for about 3 to 5 minutes. The spray group received a placebo (the patient could not distinguish the two by taste) that was allowed to accumulate in the back of the throat for about 3 to 5 minutes, similar to lidocaine viscous solution. After this step, participants of both groups inhaled five puffs (40 mg) of lidocaine (Xylocaine Pump Spray 8 %; AstraZeneca, Osaka, Japan). To remain blinded, the endoscopist entered the room only after pharyngeal anesthesia. According to a previously described method 12, the endoscopist took the first image immediately upon insertion of the endoscope, followed by 6 endoscopic images of the oropharynx, 4 images of the hypopharynx, and 1 image before insertion into the esophagus. The nasopharynx was not included in these examinations. Overall, 12 narrow band images were obtained per patient. We determined the time before the endoscopist was able to obtain any images. The endoscopist inserted the endoscope into the esophagus when it was impossible to obtain images as a result of a continuous gag reflex. Two endoscopists (T. H. and Y. A.) measured the time between the first and last images, and assessed whether the ten images of the oropharynx and hypopharynx were appropriate (Fig. 1). The endoscopists were blind to the study arm and independent from endoscopists who examined patients. The definition of an appropriate image required 3 criteria. First, the image was taken at the appropriate location. Second, the image was on focus. Third, mucus had been removed, and it was possible to evaluate the color of the pharyngeal mucosa in the images. Screening and pharyngeal examination were performed under unmagnified observation. Fig. 1 The 10 images of the oropharynx and hypopharynx. The definition of a high-quality image required 3 criteria: first, the image was taken at the appropriate location; second, the image was on focus; third, mucus had been removed, and it was possible to evaluate pharyngeal mucosa color. After the endoscopic procedure was carried out, the following data were recorded for each patient: age, gender, height, weight, body mass index (BMI), Eastern Cooperative Oncology Group (ECOG) performance status (PS), drinking and smoking habits, and the number of endoscopic procedures. Subjective symptoms of pain were evaluated by questionnaire after endoscopy. Pain was evaluated on an 11-point visual analogue scale (VAS), where 0 indicated no pain, and 10 points indicated the most severe pain. The researcher evaluated pharyngeal observation time and whether images of predetermined sites were clear. Two independent blinded expert endoscopists evaluated the quality of the images obtained at the predetermined sites. In case of disagreement, consensus was reached upon discussion with a panel of 3 other expert endoscopists. BODY.PATIENTS AND METHODS.ENDOSCOPIC EXAMINATIONS: All endoscopic procedures were carried out using a magnifying endoscope (GIF-H260Z; Olympus Medical Systems, Tokyo, Japan) with a hood attachment (MAJ-1990; Olympus Medical Systems). The videoendoscopy system used in this study comprised a video processor (EVIS LUCERA ELITE CV-290; Olympus Medical Systems) and a light source (EVIS LUCERA ELITE CLV-290SL; Olympus Medical Systems). Prior to the procedure, each patient was given 100 mL water containing 20,000 units pronase (Kaken Pharmaceutical, Tokyo, Japan), 1 g sodium bicarbonate, and 10 mL dimethylpolysiloxane (20 mg/mL; Horii Pharmaceutical Industries, Osaka, Japan). Afterward, pharyngeal anesthesia was performed as described in "Study design and anesthesia protocols." Patients were placed in the left lateral decubitus position, with endoscopic examinations carried out while they were awake or under conscious sedation with midazolam (Dormicum Injection; Astellas Pharma, Tokyo, Japan). The patient's wish to the use of a sedative was obtained before the procedure. The sedative was adjusted within the range of 2 mg to 5 mg based on the patient's body weight. The pharynx was assessed at the beginning of each examination, and standard endoscopy was carried out at the end of each pharyngeal examination. If pharyngeal lesions were detected, the examination was completed first and the lesions were evaluated at the end of endoscopy. BODY.PATIENTS AND METHODS.OUTCOME MEASURES: The main outcome was a difference in the number of pharynx observable sites between the spray group and combination group (non-inferiority trial). The secondary outcomes were (1) a difference in endoscopy-associated pain; (2) a difference in the pharyngeal observation time; (3) subgroup analysis of presence or absence of sedation; (4) adverse effects of lidocaine including decreased SpO2 (< 90 % or decrease of more than 4 % for < 94 %) decreased blood pressure (systolic blood pressure < 90 mmHg), and bradycardia (< 60/bpm or decrease of more than 10 %) 12; and (5) the percentages of suitable quality images obtained at the ten prescribed points. BODY.PATIENTS AND METHODS.STATISTICAL ANALYSIS: Sample size was calculated as follows. An experienced endoscopist is expected to be able to observe 90 % (9.0 sites) of the pharyngeal sites 13. A non-inferiority threshold [Δ] of 10 % (1.0 site) was selected to indicate that it was acceptable that the number of pharyngeal sites observed in the spray group was 10 % lower than that in the combination group. It was estimated that at least 310 cases were required to detect statistically significant differences, admitting a type I error rate of 0.025 (one-sided test) and a statistical power of 90 %. Therefore, we have determined that it was necessary to include a total of 320 cases in consideration of dropouts. Presence or absence of sedation was used as stratification factor. Continuous variables are expressed as mean (standard deviation [SD]), and comparisons between groups were performed using the Student's t-test or Mann Whitney U test (not approximately normally distributed). Categorical variables are expressed as percentage, and comparisons between groups were performed using Fisher exact tests. The level of statistical significance was defined as a P value < 0.05. Only the researchers performed the collection and aggregation of data. All statistical analyses were performed with SPSS II statistical software (SPSS Japan Inc., Tokyo, Japan). BODY.PATIENTS AND METHODS.ETHICS STATEMENT: The protocol and consent form for this study were approved by the institutional review board of Kanazawa University Hospital. The study conformed to the Declaration of Helsinki, complied with thical Guideline for Clinical Research of the ministry, and has been registered in the UMIN Clinical Trials Registry System as trial ID UMIN-CTR000016073. BODY.RESULTS: Fig. 2 shows a flow chart of patient selection and allocation. There were 468 eligible UGE patients who fulfilled the inclusion criteria. Of those, 141 patients refused to participate in the study. Finally, 327 patients were enrolled and randomly allocated to the spray group (157 patients) and the combination group (170 patients) by computer-generated codes. Fig. 2 Flowchart of patient enrollment. Patient demographics and clinical characteristics are shown in Table 1. The patients in the 2 groups were similar with regard to age, gender, height, weight, BMI, ECOG PS, drinking habits, smoking habits, the number of endoscopic procedures, and patients requiring sedation and antispasmodic drugs. Table 1 Patient demographics and clinical characteristics. Spray group(n = 157) Combination group(n = 170) Age, y 65.4 ± 15.5 65.3 ± 12.6 Gender (male:gemale) 84:73 97:73 Height, cm 160.5  ±  9.4 160.8 ± 9.2 Weight, kg 57.5 ± 12.2 59.2 ± 12.4 Body mass index 22.2 ± 3.6 22.7 ± 3.4 ECOG PS(0:1: ≥ 2) 105:43:9 120:39:11 Drinking habit(Never:Sometimes:One or more times per week:Every day) 87:26:17:27 83:34:20:22 Smoking habit: (no:yes) 86:71 101:69 Number of endoscopies(1st time: 2nd to 4th times: 5th times or more) 10:78:69 20:91:59 Sedation ( + :-) 108:49( + : 68.8 %) 115:55( + : 67.6 %) Antispasmodic ( + :-) 77:80( + : 49.0 %) 94:76( + : 55.3 %) The rate of agreement between the two endoscopists was 92.4 %, and the Kappa statistic was 0.767. The mean number of images with suitable quality of the pharyngeal observable sites per patient in the spray group was 8.33 (95 % confidence interval [CI]: 7.94 – 8.72), and that in the combination group was 8.77 (95 % CI: 8.49 – 9.05). The difference in the number of pharyngeal observable sites was – 0.44 (95 % CI: – 0.84 to – 0.03, P = 0.01) (Table 2). Table 2 Non-inferiority test of the difference of pharyngeal observable sites (based on a type I error rate of 0.025 [one-sided test], a statistical power of 90 %, and a non-inferiority threshold [Δ] of 0.1). Spray group (95 % CI) Combination group (95 % CI) Difference (95 % CI) P value 8.33 (7.94~8.72) 8.77 (8.49~9.05) –0.44 (–0.84~–0.03) 0.01 Table 3 shows the difference in pain and situation during endoscopy. There were almost no differences in pain evaluated by VAS (2.27 ± 2.88 vs. 2.33 ± 2.63, P = 0.85), pharyngeal observation time (72.0 ± 34.7 vs. 67.0 ± 27.8 seconds, P = 0.15), and the number of gag reflexes (2.12 ± 2.58 vs. 1.68 ± 2.27, P = 0.10) in the two groups. There were almost no differences in adverse events, decrease of SpO2 (2.55 % vs. 5.29 %, P = 0.20), decrease of blood pressure (0 % vs. 1.76 %, P = 0.27), and bradycardia (0 % vs. 0 %, P = 1.00) between the 2 groups. Table 3 Differences in pain and situation during endoscopy. Spray group(n = 157) Combination group(n = 170) P value Pain by visual analogue scale 2.27 ± 2.88 2.33 ± 2.63 0.85 Pharyngeal observation time, seconds 72.0 ± 34.7 67.0 ± 27.8 0.15 Number of gag reflexes 2.12 ± 2.58 1.68 ± 2.27 0.10 Adverse events (Decrease in SpO2) 4 (2.55 %) 9 (5.29 %) 0.20 (Decrease in blood pressure) 0 (0 %) 3 (1.76 %) 0.27 (Bradycardia) 0 (0 %) 0 (0 %) 1.00 Subgroup analysis for presence of sedation (223 cases) revealed almost no differences between the 2 groups in the number of pharyngeal observation sites (8.48 ± 2.16 vs. 8.79 ± 1.85, P = 0.25), pain evaluated by VAS (1.08 ± 2.06 vs. 1.33 ± 2.16, P = 0.79), pharyngeal observation time (73.3 ± 36.6 vs. 68.3 ± 30.9 seconds, P = 0.14), and number of gag reflexes (2.02 ± 2.44 vs. 1.86 ± 2.45, P = 0.63) (Table 4). Subgroup analysis of absence of sedation (104 cases) revealed almost no differences between the 2 groups in the number of pharyngeal observation sites (8.00 ± 3.12 vs. 8.73 ± 1.94, P = 0.63), pain evaluated by VAS (4.75 ± 2.79 vs. 4.30 ± 2.37, P = 0.20), and pharyngeal observation time (69.1 ± 30.1 vs. 64.1 ± 19.9 seconds, P = 0.16). The number of gag reflexes was significantly higher in the spray group than in the combination group (2.35 ± 2.87 vs. 1.27 ± 1.79), P = 0.03) (Table 4). Table 4 Subgroup analysis of presence or absence of sedation. Sedation ( + ) Spray group(n = 108) Combination group(n = 115) P value Age, y 64.5 ± 16.0 63.8 ± 13.6 Gender (male:female) 55:53 61:54 Ramsay score 4.83 ± 1.26 4.89 ± 1.25 Pharyngeal observable site 8.48 ± 2.16 8.79 ± 1.85 0.25 Pain by visual analogue scale 1.08 ± 2.06 1.33 ± 2.16 0.79 Pharyngeal observation time, seconds 73.3 ± 36.6 68.3 ± 30.9 0.14 Number of gag reflex 2.02 ± 2.44 1.86 ± 2.45 0.63 Sedation (–) Spray group(n = 49) Combination group(n = 55) P value Age, y 67.5 ± 14.1 68.3 ± 9.49 Gender (male:female) 29:20 36:19 Pharyngeal observable site 8.00 ± 3.12 8.73 ± 1.94 0.16 Pain by visual analogue scale 4.75 ± 2.79 4.30 ± 2.37 0.20 Pharyngeal observation time, seconds 69.1 ± 30.1 64.1 ± 19.9 0.16 Number of gag reflex 2.35 ± 2.87 1.27 ± 1.79 0.03 Table 5 shows the percentages of suitable quality images obtained at the ten prescribed points. The percentage of suitable quality images at the right pyriform sinus was significantly lower in the spray group compared to the combination group (82.8 % vs. 91.2 %, P = 0.02). There were no significant differences in the percentage of suitable quality images at any of the other points the between the 2 groups. Table 5 Percentages of suitable quality images obtained at the ten tested points. Spray group(n = 157) Combination group(n = 170) P value Uvula 78.3 84.1 0.18 Arch of the palate Right 84.7 87.6 0.44 Left 77.7 77.1 0.89 Wall of the oropharynx Right 87.9 91.8 0.25 Left 85.4 91.8 0.07 Posterior 92.4 96.5 0.10 Epiglottis 74.5 81.8 0.11 Vocal fold 86.6 90.0 0.34 Pyriform sinus Right 82.8 91.2 0.02 Left 82.8 85.3 0.54 BODY.DISCUSSION: This is the first study evaluating pharyngeal observation in patients receiving lidocaine spray alone versus lidocaine viscous solution and spray combination as topical pharyngeal anesthesia. According to a previous report, topical lidocaine spray may be described as a better option than lidocaine viscous solution because of higher procedural completion rate, greater ease of intubation, and greater satisfaction for both the patient and the endoscopist 7. If the spray group were not inferior to the combination group in terms of pharyngeal observation, lidocaine viscous solution would be considered unnecessary. We found that lidocaine spray alone performs similar to lidocaine spray plus viscous solution for pharyngeal observation during transoral endoscopy. For the observation of the pharynx, addition of lidocaine viscous solution was found not to be necessary. Side effects of lidocaine include an anaphylactoid reaction and poisoning after an increase in concentration of circulating lidocaine. The frequency of anaphylactoid reactions is low among amide-type local anesthetics such as lidocaine. However, methylparaben, which is added as a preservative, has similar structure and cross-antigenicity to para-aminobenzoic acid. Para-aminobenzoic acid can induce T-lymphocyte antibody production and sensitization, and eventually cause an allergic reaction. Thus, anaphylactoid reactions are probably non-immunoglobulin E-mediated anaphylactoid reactions in which the presence of methylparaben in the local anesthetic solution plays a major role 14. Methylparaben is included in lidocaine viscous solution, but not in lidocaine spray. Local anesthetics poisoning by lidocaine spray is thought to occur because the concentration of lidocaine is higher in the spray than in the viscous solution. However, 5 puffs of spray (40 mg lidocaine per puff) are usually sufficient for pharyngeal anesthesia. It is extremely rare to use more than 200 mg lidocaine per dose; a higher dosage could cause local anesthetics poisoning during pharyngeal anesthesia. In other words, the possibility of poisoning by lidocaine spray alone is considered to be extremely rare in pharyngeal anesthesia. However, the combined use of lidocaine spray and viscous solution increase the potential of poisoning. To prevent poisoning, it is important to perform minimal pharyngeal anesthesia with a valid method. In this study, the number of adverse events was higher in the spray group compared to the combination group, although this different was not significant because of the small number of events. Although the effect of sedation is suspected to decrease SpO2 or blood pressure, it is undeniable that adverse effects of lidocaine are stronger in the combination group. To prove this fact, it is necessary to perform the study in a larger number of cases. There were no significant differences in pain by VAS, pharyngeal observation time, or the number of gag reflexes between the 2 groups. These differences were also considered to be very clinically slight. The differences of 0.06 points for the VAS (max 10 points), 5.0 seconds of pharyngeal observation time, and 0.44 times of gag reflexes were not clinically meaningful. In the subgroup analysis of each pharyngeal site, the spray group was inferior to the combination group in terms of observation of the right pyriform sinus, statistically. But the difference of 8.4 % of observation rate of this structure was not clinically meaningful. This difference could be attributable to multiple comparisons, and there might be no statistically significant difference in left pyriform sinus observation. Although the possibility of the effect of anesthesia in the hypopharynx being stronger in the combination group cannot be denied, it is necessary to assess a larger number of cases because the results are not consistent between the left and right sides. Since the pyriform sinus is the predilection site of pharyngeal cancer 15, it is desirable to perform further studies on the relationship between the extent of hypopharyngeal anesthesia and pharyngeal observation. In this study, the number of gag reflexes was significantly higher in the spray group than in the combination group in subgroup analysis (no sedation). It is possible that the no-sedation group was more susceptible to pharyngeal anesthesia. Conscious sedation during endoscopic examination can provide more adequate anxiolysis and acceptance than wakefulness 16. Because of strong pain experienced by the patients, there are some opinions that pharyngeal observation is not needed. However, the overall rate of detection of pharyngeal cancer by screening endoscopy is as low as 0.26 % 16. In patients who are not at increased risk of pharyngeal cancer, the rate of detection of pharyngeal cancer is 0.11 % 17. Although this rate is lower than the 0.26 % of gastric cancer detection rate by regular screening 18, it is not a negligible value. Therefore, we are of the opinion that screening pharyngeal observation is necessary. Heuss reported that topical pharyngeal anesthesia does not seem to influence the ease of the procedure or patient or endoscopist satisfaction in adequately sedated patients 19. Nevertheless, it remains unclear whether pharyngeal anesthesia improves pharyngeal observation in adequately sedated patients. A future prospective, double-blinded, randomized clinical trial is necessary to clarify this issue. This study has some limitations. First, it was carried out at a single institution. Second, patients might be aware of the placebo of the viscous solution by detecting subtle differences in taste. Third, our study did not evaluate the postcricoid area. The nasal Valsalva and trumpet maneuvers with anterior neck skin traction cannot be carried out using transoral endoscopy 20. The sniffing position in the left decubitus position can improve examination of the postcricoid area 21. Future studies are needed to explore these methods. BODY.CONCLUSION: In conclusion, with and without sedation, using only lidocaine spray for pharyngeal anesthesia was not inferior to using lidocaine spray and viscous solution in terms of pharyngeal observation. While the overall findings support the hypothesis that lidocaine viscous solution is unnecessary for pharyngeal observation, the subgroup analysis of each pharyngeal site indicated that spray might be inferior to combination treatment with regard to pyriform sinus observation.

TITLE: Effects of lidocaine, ketamine, and remifentanil on withdrawal response of rocuronium ABSTRACT.BACKGROUND: Rocuronium has been well known to produce withdrawal response in 50-80% patients when administered intravenously. Several drugs are administered prior injection of rocuronium to prevent the withdrawal response. We compared the preventive effect of lidocaine, ketamine, and remifentanil on the withdrawal response of rocuronium. ABSTRACT.METHODS: A total of 120 patients undergoing various elective surgeries were enrolled. Patients were allocated into 4 groups according to the pretreatment drugs (Group N, normal saline; Groups L, lidocaine 40 mg; Group K, ketamine 0.5 mg/kg; Group R, remifentanil 1 μg/kg). Patients received drugs prepared by dilution to 3 ml volume before injection of rocuronium. Withdrawal responses after injection of rocuronium were graded on a 4-point scale. Hemodynamic changes were observed before and after administration of pretreatment drugs and after endotracheal intubation. ABSTRACT.RESULTS: Incidence of withdrawal response was significantly lower in group L (20%), group K (30%), and group R (0%), than group N (87%). Severe withdrawal response was observed in 5 of the 30 patients (17%) in group L, and in 9 of the 30 patients (30%) in group K. There was no severe withdrawal response in group R. Mean blood pressure and heart rate were significantly decreased in group R compared to other groups. ABSTRACT.CONCLUSIONS: It seems that remifentanil (1 μg/kg intravenously) was the strongest and most effective in prevention of withdrawal response after rocuronium injection among the 3 drugs. BODY.INTRODUCTION: Rocuronium is the most popular neuromuscular blocking agent because of rapid onset, relatively short duration of action, and less adverse effects on hepatobiliary system or autonomic system [1]. It produces sudden flexion movement on injection as a response to a pain [2]. The withdrawal response is a body reaction due to the pain in unconsciousness [3]. Therefore, drugs such as fentanyl, lidocaine, ketamine, remifentanil, tramadol, parecoxib, magnesium sulfate, and ondansetron have been introduced as a pretreatment to prevent the withdrawal response of rocuronium [4]. It has been reported that pretreatment of lidocaine 40 mg successfully reduces injection pain of rocuronium [5,6]. Ketamine (0.5 mg/kg intravenously) also effectively reduces injection pain without mental symptoms such as hallucination before injection of rocuronium [7,8]. Remifentanil (1.0 μg/kg) pretreatment, frequently used during induction of the anesthesia to improve intubation condition and reduce hemodynamic change, also prevents withdrawal response of rocuronium successfully [1]. According to the meta-analysis, opioids, lidocaine, and ketamine have been most frequently used as pretreatment to reduce injection pain of rocuronium [4]. Even though meta-analyses have been performed to compare the preventive effect of withdrawal responses to each drugs, it is an indirect comparison of numerous methods and dosages of pretreatment [4]. In addition, no clinical reports have directly compared the preventive effect of intravenous lidocaine, ketamine, and remifentanil on withdrawal response of rocuronium at an effective dose. Direct comparison can be helpful in the clinical setting, and it could be meaningful to identify which drug is the most effective when administered under the same condition. Therefore, we carried out the study to determine which drug had the most superior effect to reduce withdrawal response of rocuronium. BODY.MATERIALS AND METHODS: The study was conducted after approval by the Institutional Review Board. A total of 120 patients who were scheduled for elective surgery, aged 20 to 60 years, and American Society of Anesthesiologists class I or II were enrolled. Exclusion criteria were as follows: poor venous access, diabetes, allergies to anesthetic medications, neurologic deficit, psychiatric disorder, vasculitis, thrombosis, prior administration of analgesics within 24 hours, or pregnancy. Informed consent was obtained from all patients after full explanation of aim and method of the study and they were randomized into 3 groups according to pretreatment drugs through a computerized randomization. Patients in group N constituted the control group and received normal saline before injection of rocuronium. Those in group L, group K, and group R received lidocaine 40 mg, ketamine 0.5 mg/kg, and remifentanil 1 μg/kg, respectively [1,5,7]. The dosages of each group were selected from several published reports in which the drugs were administered intravenously and showed most effectiveness in preventing pain response after injection of rocuronium. All patients received midazolam 0.05 mg/kg intramuscularly 30 minutes before the induction of anesthesia. An 18 gauge venous cannula was kept at the main cephalic vein of the forearm which is proximal to the hand for the infusion of lactated Ringer's solution. After arrival at the operating room, standard monitoring devices such as electrocardiogram, pulse oximetry, and non-invasive blood pressure were attached to the patients. Preoxygenation with a facemask was applied for 5 min before the induction of anesthesia. Thiopental sodium (2.5%, 5 mg/kg) was injected for the loss of consciousness, and then ventilation with face mask was started. During ventilation, pretreatment drug was administered intravenously according to the groups. All drugs were diluted to a volume of 3 ml by an assistant nurse who had not participated during the induction of anesthesia for the double blind test. Each drug was administered during 15 seconds by another assistant nurse. Muscle rigidity which was defined as difficult ventilation with face mask due to increased tone of the trunk muscles and opioid induced cough was observed in the case of remifentanil injection. If severe muscle rigidity or cough occurred, the patient was dropped out and rocuronium (1.0 mg/kg) has administered immediately for the rapid sequence intubation. One minute after injection of the pretreatment drug, rocuronium (0.6 mg/kg) was administered for 10 seconds. The 4-point scale was used to assess the severity of withdrawal response during rocuronium injection (Table 1) [9]. Severe withdrawal responses were considered as the response > 2 on the 4-point scale. After injection of rocuronium, patients were ventilated with sevoflurane 2.5 vol% and 100% oxygen. Endotracheal intubation was done 2 minutes after rocuronium injection. Anesthesia was maintained with sevoflurane (2-4 vol% of end tidal concentration) with O2/N2O mixture (FiO2 = 0.5). Mean blood pressure and heart rate were measured according to the time sequence. Each measurement was done 1 minute before (control) and after the administration of pretreatment drug and 1 minute after endotracheal intubation. Focal reactions such as erythema, vasculitis, and thrombosis were also observed during the procedure. Sample size was calculated based on previous studies. The incidences of disappearance of withdrawal response or pain were 75, 60, and 100% after pretreatment with lidocaine [5], ketamine [7], and remifentanil [1], respectively. Sample size was calculated using G*Power software (ver. 3.1.5) which was based on the results of previous studies [1,5,7]. According to the analysis of variance, within-group standard deviation between 4 groups was 0.354 and effect size from variance was 0.437. Using α = 0.05 with a power 90%, the minimum sample size was predetermined as 24 patients per group. After considering drop-out rate as 20%, we included 30 patients in each group. Statistical analysis was done with SPSS 12.0 (SPSS, Chicago, IL, USA). Incidence of withdrawal response was expressed with number (%) and other data were expressed as mean ± standard deviation (SD). Demographic data were analyzed by ANOVA. Gender and weight which were non-parametrically distributed were analyzed by Kruskal Wallis test. The incidence of withdrawal response and 4-point scale were analyzed by the Chi-square test. P < 0.00833 was considered statistically significant accounting for the Bonferroni correction. Hemodynamic changes were analyzed by repeat measures ANOVA, and when between-group differences were observed, Mann-Whitney U test was used to analyze between pairs of group. Post hoc tests were done with Turkey's HSD. P < 0.05 was considered statistically significant. BODY.RESULTS: There were no dropouts and the data from all 120 patients could be analyzed. There were no significant differences among the groups in demographic data (Table 1). The incidence of withdrawal response was 87% (26/30), 20% (6/30), 30% (9/30), and 0% in group N, group L, group K, and group R, respectively (Table 2). Severe withdrawal response which is >2 on the 4-point scale occurred in 25 patients (84%) in group N (P < 0.00833). However, it was observed in 5 patients (17%) in group L, in 9 patients (30%) in group K, and in no patient in group R, respectively. The number of patients needed to be treated (NNT) to prevent pain in one who would have had pain was 1.49, 1.75, and 1.15 in group L, group K, and group R, respectively. Lidocaine (40 mg), ketamine (0.5 mg/kg), or remifentanil (1 μg/kg) decreased the withdrawal response significantly and remifentanil (1 μg/kg) was the most effective in the prevention of withdrawal response among the drugs (P < 0.00833) (Table 2). There were no significant differences in mean blood pressure and heart rate before injection of pretreatment drugs among the groups (Figs. 1 and 2). However, there were significant decreases in group R compared to control and other groups after injection of pretreatment drugs (Figs. 1 and 2). After intubation, mean blood pressure was increased in group N, group L, and group K compared to control (Fig. 1). However, in group R, mean blood pressure was significantly lower compared to other groups (P < 0.05) (Fig. 1). Heart rate increased significantly compared to control in all groups but, it was significantly lower in group R compared to other groups (P < 0.05) (Fig. 2). No erythema, venous sequels, or adverse event were observed in any patient. BODY.DISCUSSION: In the current study, we compared the preventive effect of lidocaine, ketamine, and rocuronium on withdrawal response of rocuronium injection. We showed that the 3 drugs decreased the withdrawal response caused by rocuronium injection. However, remifentanil was the most effective drug in prevention of withdrawal response among the drugs. Pain during intravenous injection of rocuronium and distressing side effect are very common [10,11]. The pain is sometimes severe such as burning sensation [6,10,11], and causes withdrawal movement of the arm which can create secondary injury or pulmonary aspiration due to gastric regurgitation [12]. However, the mechanisms of injection pain of rocuronium are not clear. Lidocaine is a common used amide local anesthetic agent which has short duration of action. Previous studies have reported that lidocaine is effective in reducing the pain on injection of rocuronium because of its local analgesic effect [6,11]. Cheong and Wong [11] reported that only 7% of patients had complained of pain after rocuronium injection when treated with lidocaine 30 mg before administration of rocuronium. Ahmad et al. [6] reported that pretreatment with lidocaine 40 mg reduced the incidence of withdrawal reaction to 30%. In our study, the incidence of withdrawal response was reduced to 20% after pretreatment with lidocaine 40 mg intravenously. It seems that the preventive effect of lidocaine against injection pain is dependent on the timing between the administration of lidocaine and rocuronium. According to the previous reports, peripheral analgesic effect of lidocaine is rapid and disappears quickly, not lasting beyond 2 minutes [6,11]. The time of administration of rocuronium in this study was 60 seconds after intravenous injection of lidocaine, which was earlier than the study of Ahmad et al. (more than 120 seconds) [6] and later than the Cheong and Wong's study (10 seconds). Ketamine is a phencyclidine derivative hypnotic agent with strong analgesic properties [7,13]. Ketamine activates N-methyl-D aspartate receptors not only in the vascular endothelium but also in the central nervous system [13]. It may also increase the pain threshold in the central nervous system. Previous reports showed that ketamine administration before rocuronium injection reduced injection pain of rocuronium effectively [7,8,14]. The incidence of withdrawal response after rocuronium administration was 27% when ketamine 0.2 mg/kg has pretreated [14]. In another case, it was reported that rocuronium injection pain occurred in 40% of patients even if ketamine 0.5 mg/kg was used as pretreatment [7]. In our study, pretreatment of ketamine 0.5 mg/kg showed that the incidence of withdrawal response was 30%, comparable to lidocaine. However, severe withdrawal response with generalized movement occurred more frequently in the ketamine group (30%) than the lidocaine group (17%). Thus, lidocaine may be superior to ketamine in preventing rocuronium injection pain. In addition, it is well known that ketamine produces sympathetic stimulation or psychomimetic emergence reactions. However, we did not observe significant increase in blood pressure or heart rate compared to control, and psychomimetic reactions in our patients. We think that this was due to the low dose of ketamine (0.5 mg/kg) [15]. Remifentanil is an opioid agent with analgesic effect. It is very suitable for pretreatment during injection of rocuronium because it has very rapid onset time and rapid clearance [16]. In general, opioid decreases pain through both central and peripheral opioid receptors [16]. However, remifentanil acts mainly on the central opioid receptors for reducing pain during injection of rocuronium. When the venous occlusion technique with a tourniquet on the forearm was used for the remifentanil pretreatment, 9.1% adult patients and 60% pediatric patients showed pain responses [17,18]. Rocuronium showed significant prevention (0% of incidence) of withdrawal response or injection pain of rocuronium when administered intravenously [1], which was in agreement with our results. The incidence of withdrawal response was also 0% in the remifentanil group in our study. Although they have different time interval between remifentanil and rocuronium injection (90 vs. 60 seconds), it seems that 60 seconds may be enough to reach the central effect of remifentanil to prevent rocuronium injection pain in adults. Its peak effect after IV administration, occurs in 70 seconds [1]. Furthermore, remifentanil is frequently used for blunting hemodynamic changes during endotracheal intubation [19,20]. In this study, mean blood pressure and heart rate were significantly lower in the group which had remifentanil 1 μg/kg compared to other groups. The preventive effect of remifentanil on hemodynamic changes during endotracheal intubation is dose dependent. Optimal effect-site concentration of remifentanil for preventing development of hypertension during endotracheal intubation is 3.3 ng/ml [20]. However, calculated effect-site concentration of remifentanil in the current study was 2.21-2.29 ng/ml according to the Minto kinetic model when it was assumed that intubation was done about 2 minutes after remifentanil 1 μg/kg administration [21]. However, remifentanil which was used to reduce pain of rocuronium injection was also effective in blunting hemodynamic changes during endotracheal intubation in our study. The reasons of this difference are not clear, but the following conditions may have affected the results. Firstly, we used the bolus injection instead of target-controlled infusion since we focused on the pain of the rocuronium injection and accordingly modulated the administration method to be equivalent with other drugs. Secondly, time interval between remifentanil injection and endotracheal intubation was shorter in our study than previous study (about 120 vs. 250 second) [20]. It is well known that lidocaine was also effective for blunting the hemodynamic response to endotracheal intubation. However, our study did not show the effectiveness of lidocaine similar to the report of Suzuki et al. [19]. Relative low dose than known effective dose (1.0 mg/kg vs. 1.5 mg/kg) and shorter time after lidocaine injection than recommended timing (2 vs. 3 minutes) could be the reasons why lidocaine was not effective in preventing hemodynamic changes after endotracheal intubation [19]. There are several limitations to the current study. Firstly, latent time of preventive effect after injection of drug may be different according to the drug. Lidocaine acts more strongly on peripheral receptors and the effects are rapid and transient [6,11], whereas, analgesic effect of ketamine or remifentanil works mainly on the central nervous system and lasts longer [13,16]. Thus, the time of drug effects could be different. However, we set the timing of rocuronium injection to 60 seconds after administration of preventive drugs. Secondly, analgesic effects of the 3 drugs used in this study were not equipotent. As mentioned earlier, there is no clear comparison of the analgesic potency of lidocaine, ketamine, and remifentanil during injection of rocuronium. This makes it difficult to set the appropriate dose of each drugs and may lead to discrepancy in the study result. However, this study focused on the comparison of drugs which are used frequently for prevention of rocuronium injection pain in the clinical setting. Thirdly, venous occlusion technique is also used frequently for lidocaine; however, we used intravenous injection only. According to meta-analysis, pretreatment with lidocaine is effective whether venous occlusion is used or not [4]. Furthermore, pretreatment of remifentanil with venous occlusion is not effective as injection only [4]. The aim of this study was to determine the most effective pretreatment drug for rocuronium injection pain in the same condition, thus, we selected intravenous administration only. Finally, individual variations could have affected the withdrawal responses. Those responses following injection of drugs that causes pain can be different according to clinical factors such as gender, age, and cannulation site [22]. Moreover, while the cephalic vein was selected in the study, various anatomical variations exist in individuals [23]. In conclusion, we found that remifentanil (1 μg/kg intravenously) was the most effective drug in the prevention of the withdrawal response on rocuronium injection among the 3 drugs (lidocaine, ketamine, and remifentanil). Moreover, remifentanil reduced the hemodynamic changes after endotracheal intubation, effectively.

TITLE: Effects of efforts to intensify management on blood pressure control among patients with type 2 diabetes mellitus and hypertension: A pilot study ABSTRACT: There continues to be a need for improved medical management of diabetes patients with hypertension in primary care. While several care models have shown effectiveness in achieving various outcomes among these patients, it remains unclear what care model is most effective in improving blood pressure control in primary care. In this prospective study, 54 patients with type 2 diabetes mellitus and blood pressure of >140/90 identified through the registry, were randomized into three groups. Group A attended a nurse educator-conducted class on diabetes and hypertension, group B attended the same class and was asked to monitor their home blood pressure using provided device, and group C served as control (usual care). Of the 24 subjects who completed the study, only 20% achieved the target blood pressure of <130/80 and there was no statistical difference in mean systolic and diastolic blood pressures among the three groups (p > 0.05). Efforts to intensify management of hypertension among type 2 diabetes patients did not result in better blood pressure control compared to usual care. Studies looking into factors which limit patients' participation in group classes and determining patients' preferences in disease management would be helpful in ensuring success of any chronic disease management program. BODY.BACKGROUND: Cardiovascular disease is the leading cause of premature mortality among patients with diabetes, with heart disease accounting for more than half of these deaths. Hypertension is common in patients with type 2 diabetes, with a prevalence of 40%–60% over the age range of 45 to 75 and contributes to their risk of cardiovascular disease.1 The association of elevated blood pressure with risk is amplified in patients with diabetes who have roughly a doubling of absolute risk compared with patients without diabetes at each systolic blood pressure level. Several trials have documented the importance of blood pressure control in reducing the risk of cardiovascular and renal disease among patients with diabetes.2–5 Results of these trials supported an aggressive approach to the treatment of hypertension among patients with diabetes leading to a recommended blood pressure goal of <130/80.5–7 This requires the use of at least two agents in most patients with the consensus of having a renin–angiotensin system blocker as first-line treatment.6–8 Effective control of blood pressure among patients with diabetes continues to pose a clinical practice challenge. Hypertensive diabetes patients are still frequently not treated to their goal blood pressure; studies demonstrated that this group of patients has worse blood pressure control than patients with hypertension but without diabetes mellitus.9,10 Clinical uncertainty about the true blood pressure value was a major reason identified as to why providers do not intensify antihypertensive therapy among diabetes patients.11 Clearly, there is room for outpatient practice improvement. Several care models have been shown to be effective at improving outcomes among diabetes patients. Group education classes have been successful in enhancing diabetes care.12,13 A case study reported achievement of blood pressure control in majority of patients with diabetes secondary to nursing staff involvement in care management and use of medications concomitant with guidelines.14 Another study found that a nurse-led hypertension clinic was more effective for patients with type 2 diabetes and uncontrolled hypertension compared to conventional care.15 It is unclear which of these models is most effective in achieving hypertension control among diabetes patients in a primary care setting. In the recent years, the SHEAF trial and other studies have thrown another complexity into hypertension control by showing that office blood pressure readings were inaccurate in 22% of treated hypertensive patients. Subsequent studies showed that use of home blood pressure measurement by a physician/nurse team has the potential to significantly improve long-term hypertension control rates and that self-monitoring of blood pressure promoted achievement of target blood pressure in primary health care.16,17 One study even showed ambulatory blood pressure to be a better marker than clinic blood pressure in predicting cardiovascular events in patients with or without type 2 diabetes.18 Thus, self-monitored or home blood pressure measurement is becoming a potentially very powerful and cost-effective tool in the management of hypertension. Disease registries are powerful tools that allow identification of high-risk patients within a defined population.19 A previously published study utilized the diabetes registry to evaluate implementation strategies aimed at improving glycosylated hemoglobin and low-density lipoprotein testing rates among poorly controlled diabetes patients.20 Using the diabetes registry to identify diabetes patients with hypertension who meet our target population, we conducted a study to compare three practice care models, two using intensified management and one using usual care, with the aim of achieving target blood pressure as recommended by current practice guidelines. We hypothesized that (1) participation of diabetes patients with uncontrolled blood pressure (blood pressure >140/90) in an intensified care management model using a specific intervention would result in improved blood pressure control among this group of patients compared to conventional care, that (2) the percentage of diabetes patients with uncontrolled hypertension achieving target blood pressure readings (blood pressure <130/80) will be significantly higher among those randomized to an intensified model compared to conventional care, and that (3) different care delivery models would lead to varying degrees of blood pressure improvement among diabetes patients with uncontrolled hypertension. BODY.METHODS.DESIGN: This was a prospective randomized control trial conducted in a multispecialty clinic in the midwestern United States. Eligible subjects were primary care paneled patients with diabetes mellitus type 2 and blood pressure above determined cut-off who were identified using the diabetes registry. Nursing home patients were excluded. Study duration was six months. The study protocol was approved by the Institutional Review Board. BODY.METHODS.REGISTRY TOOL: The registry is an institutionally developed centralized database of diabetes patients who were identified based on administrative billing data that used International Classification of Diseases 9th revision codes (ICD-9 CM) for the diagnosis of diabetes. Provider patient lists are pulled from the clinic's generalized patient appointment system, which identifies a patient's primary care provider. Before being included in the registry, all patient records were reviewed by a registered nurse to verify that they are diabetics and are assigned to the correct physician. The registry is interfaced with other clinical information systems which allow entry of patient data such as blood pressure and laboratory test results. Blood pressure data are captured and updated weekly from the electronic medical record (EMR). It became available to providers beginning in early 2000. BODY.METHODS.BLOOD PRESSURE CUT-OFF: To determine the blood pressure threshold for identification of our target population from the diabetes registry, we did a retrospective analysis of three separate blood pressure readings in three groups of 20 randomly selected patients using targets of 130/80, 140/90, and 150/90. Using 130/80 as cut-off, 100% of 20 patients had blood pressure of greater than 130/80 with one reading but only 45% was consistently above target with two out of three readings. At a cut-off of 140/90, 50% were consistently above target with two readings. With 150/90 as cut-off, 100% was above it at first reading and 65% was still above target in two of three readings. However, this group constitutes less than a third of patients in the registry with above target blood pressure. We therefore selected to use a cut-off of 140/90 as at least 50% of the 60 randomly selected diabetes patients in the registry were consistently above this target in two out of three readings. BODY.METHODS.INTERVENTION: Four hundred ten patients qualified and were sent a letter of invitation to participate. There were 197 responders of whom 143 refused to participate and only 54 consented (Figure 1). After stratification based on gender, age (≤60 years, >60 years) and hemoglobin A1C level (≤7%, >7%), each of the participant was randomized to one of three groups with two arms using practice care models and the third arm serving as control group. Group A patients were invited to participate in a class focusing on hypertension in diabetes. Group B patients were also invited to participate in the class; in addition, they were given automated blood pressure devices and asked to track their home blood pressure readings and record them in a booklet to be submitted at the end of the study. Three classes were set up to allow for flexibility in scheduling and each participant in the two groups was asked to attend one class. The classes were conducted by a diabetes nurse educator using structured format. The automated blood pressure device (Life Source UA-767 Plus; Life Source, San Jose, CA, USA) had been clinically validated according to British Hypertension Society (BHS) standards and had met the limits set by the American National Standards Institute. Group C patients did not receive any invitation. All study participants continued to see their primary care physicians for usual care. No interim follow-up was done during the study period. Since the study focused on comparing practice models, participants' use of pharmacologic therapy for blood pressure control was not captured. Seventeen patients were randomized to group A, 19 to group B, and 18 to group C. Group A and B subjects had their blood pressure checked by a registered nurse (MH) during class attendance using standardized blood pressure measurement protocol used in the clinic (copy available upon request). Clinic blood pressure readings correlate closely with registry data. Those in group B received additional instruction on home blood pressure monitoring from the registered nurse based on the clinic's patient education pamphlet entitled "Measuring Your Blood Pressure at Home" (MC3031-03). A copy of the pamphlet was also provided. All study participants were asked to return for a nurse blood pressure recheck after six months, again following the standardized blood pressure measurement protocol. Group B patients were also asked to bring back their booklet with home blood pressure readings. Primary outcome measure was the percentage of study subjects who achieved blood pressure goal of <130/80 within six months as measured in clinic during the return visit. Secondary outcome measures were mean arterial pressures of participants in each study arm, and number of nurse (RN) and physician (MD) visits for each patient in each group. BODY.METHODS.STATISTICAL METHOD: Mean ± standard deviation (SD) for continuous variable or frequency (percentage %) for categorical variable was compared among the three groups using one-way analysis of variance (ANOVA) or Pearson's chi-squared test, respectively. Post hoc pair-wise comparisons were applied using the Scheffe's significant criteria.21 Table 1 was prepared using all available data with intention-to-treat analysis while Table 3 showed data only on those who had completed the study. Multivariate logistic model approach was also used to find possible factors that predicted achievement of target blood pressure goals. The analysis was performed using intent-to-treat and per protocol approaches. Participants who did not complete the entire six-month study period or failed to return at six-month follow-up were considered dropouts and were excluded in the per protocol analysis, but included in the intent-to-treat analysis. When sample size is 18 in each group, we would have 80% power to detect at the 0.050 level an effect size of 0.1893 using one-way ANOVA. With the same sample size, a 0.050 level Pearson's chi-squared test will have 80% power to detect an odds ratio of 9.4 in proportions. All analyses were handled by SAS software (version 9.1.3; SAS institute, Cary, NC, USA). Any three-group comparison p-value less than 0.05 was considered as statistically significant. BODY.RESULTS: Of the 410 eligible patients who were invited to participate, 143 (34.9%) declined participation and 54 (13.2%) consented. There were 213 (52.0%) nonresponders. We did not have gender and age information for the nonresponders as well as age information for nonparticipants. Gender was not different between those who consented to participate and the decliners (p = 0.87). The state where the patients lived were not different among participants, decliners, and nonresponders (p = 0.95). The participants however had slightly lower systolic and diastolic blood pressures than others (p = 0.03 and p = 0.06, respectively). Twenty-eight (52%) of the 54 participants are female; 41 (76%) are aged over 60 years and 22 (41%) have hemoglobin A1C level above 7% (Table 2). From the registry, study participants had a mean systolic blood pressure of 149.79 and a mean diastolic blood pressure of 73.89. There was no difference in baseline mean systolic and diastolic blood pressure among the three groups. Seven patients randomized to group A attended the class; only five returned after six months for blood pressure recheck. Ten patients in group B attended the class and took home an automated blood pressure device; seven returned after six months. Of the 18 patients randomized to Group C, 12 returned, one died, and two were too ill to leave home (Figure 1). After six months, only 20% (n = 5) of the 24 subjects who completed the study achieved the target blood pressure of <130/80. There was no difference in the percentage of subjects achieving target blood pressure among the three groups (p > 0.05). Using both intention-to treat and per protocol analyses, there was no significant difference in mean systolic and diastolic blood pressures among the three groups of study participants after six months (Tables 1, 3). Interestingly, those in groups B and C had lower mean systolic and diastolic blood pressure than those in group A; however they are still not significant. Seven participants in group B returned their booklet with home blood pressure recordings at the end of the study period. There was variation in the number of home blood pressure readings among the seven participants with total readings ranging from 38 to 156. The mean systolic and diastolic home blood pressure readings in five of the seven participants was <130/80. We compared the number of RN and MD visits during the study period for each study participant and found no difference among the three groups. We did not find any significant factor, using the multivariate logistic model that predicted achievement of target blood pressure goal. We considered each participant's baseline systolic blood pressure, baseline diastolic blood pressure, BMI, RN, and MD visits. BODY.DISCUSSION: In this study, only 10% of randomized subjects (n = 54) with uncontrolled hypertension and diabetes achieved the target blood pressure of <130/60 after six months. Our study failed to show a statistical difference in mean blood pressure readings after six months among the subjects randomized into three study groups. Those randomized into a more intensive management; ie, groups A and B did not have significant improvement in blood pressure control compared to those under usual care. Different care delivery models (usual care plus education, usual care plus education and home blood pressure self monitoring, or usual care alone) did not result in varying degrees of blood pressure improvement among diabetes patients with uncontrolled hypertension. Given our small sample size, it was not surprising to see no statistically significant differences among the study groups. We were also underpowered as only half of the randomized participants completed the study. Seven out of the 19 participants who were randomized to group B returned after six months with recorded home blood pressure readings, five subjects had mean blood pressure readings below 130/80. Outcome blood pressure readings in this study were obtained during a return clinic visit using standard protocol. Only two study participants in group B met target pressure on return clinic blood pressure check. This observation is consistent with previously reported discrepancies between office and home blood pressure readings, home or ambulatory readings potentially being more accurate. Evaluating the achievement of recommended blood pressure among diabetics with uncontrolled hypertension through different delivery care models using home blood pressure recordings may therefore be a more effective outcome measure. Self-management is an essential component of chronic disease model. We failed to see a trend towards increased self-activation among participants in the practice model arms. Participation in education and tracking blood pressure readings at home did not increase patient's likelihood of seeing a health care provider more often than those under conventional care. A characteristic defined as "I can take charge" and reflected in an individual's proactive behavior of seeking more information from health care providers has been equated with high self-efficacy and adherence to medication.22 In this study, we incorporated chronic disease model components such as use of registry and allied professionals to achieve our stated aim.23 It was apparent that without the component of patient self-management, attainment of target blood pressure would be difficult. Indeed, evidence has supported the effectiveness of self-management training in diabetes care.24,25 We are not aware of any previously reported study that has compared blood pressure control among patients with diabetes mellitus and uncontrolled hypertension who were identified through the registry and randomized into different delivery care models. Despite its negative outcome, our study is the first of such kind. Our study was limited by its lack of power due to a small sample size and short follow-up of six months. We would recommend redesigning a larger study with a longer follow-up duration based on our experience in this pilot study. As we continue to be challenged on how to best manage our patients with chronic diseases in the changing health care environment and patient population profile, there remains a great need to create innovative practice models. Given our study results, the next step perhaps is to focus on identifying factors that create barriers to patients' participation into care initiatives and surveying patients regarding their preferences in care delivery and disease management. Valuable data can then be obtained that may help guide educators and practitioners into structuring a practice care model that enhances patients' engagement in their health and deliver efficient care with sustained outcomes.

TITLE: Effect of intranasal dexmedetomidine on emergence agitation after sevoflurane anesthesia in children undergoing tonsillectomy and/or adenoidectomy ABSTRACT.BACKGROUND:: Emergence agitation (EA) after sevoflurane anesthesia is common in children during recovery from general anesthesia and may result in postoperative complications. This study investigated safety and effectiveness of intranasal dexmedetomidine in reducing the incidence and severity of EA. ABSTRACT.METHODS:: This prospective, randomized double-blinded controlled trial included 86 patients scheduled for the tonsillectomy and/or adenoidectomy under general anesthesia with sevoflurane. They were randomly allocated into two groups. Group D received intranasal dexmedetomidine at 1 μg/kg, and Group C received intranasal saline 0.9% after the induction of general anesthesia. Four-point agitation scale and Face, Legs, Activity, Cry and Consolability (FLACC) scale for pain assessment were measured at six time points (after extubation, leaving the operating room, on arrival to postanesthesia care unit [PACU], 10, 20, and 30 min after arrival in PACU). Extubation, emergence, and discharge times were recorded in addition to any adverse effects. ABSTRACT.RESULTS:: There was a significant difference in the incidence of EA between Groups D and C (6.98% and 58%, respectively, with P = 0.001). The median four-point agitation scales and the median scores of FLACC pain scales of Group D were significantly lower than those of Group C at the all six time points with P < 0.05. Extubation, emergence, and discharge times were comparable in both groups, and none of the subjects reported any adverse effects. ABSTRACT.CONCLUSION:: This study demonstrates that a 1 μg/kg dose of intranasal dexmedetomidine administered after the induction of anesthesia reduces post-sevoflurane incidence and severity of EA in children undergone tonsillectomy and/or adenoidectomy with no adverse effects and smooth recovery profile. BODY.INTRODUCTION: Emergence agitation (EA) in pediatrics is defined as a postoperative negative behavior that may be accompanied by symptoms as combative movements, excitability, thrashing, disorientation, and inconsolable crying.[1] The definite cause and pathophysiology of EA are not fully elucidated but risk factors include preschool age, preoperative anxiety, postoperative pain, nausea, vomiting, otolaryngology procedures, and inhalational anesthetics specially sevoflurane. Due to its low blood/gas partition coefficient (0.68) and weak airway irritation, sevoflurane is the most popular anesthetic used for children.[2] However, it is associated with higher incidence of (EA) (up to 80%) and this incidence is not related to the duration of exposure and the dose of sevoflurane.[3] Multiple drugs and techniques used to control this problem as benzodiazepines, propofol, fentanyl, and α-2 agonists to improve quality of recovery profile in pediatric age group.[45] Among these drugs dexmedetomidine, the dextroenantiomer of medetomidine; the methylated derivative of etomidine is a highly specific α-2 adrenoceptor agonist and has sedative and analgesic properties without significant respiratory depression at the clinically approved dosage.[67] There is now an increasing evidence to support the use of dexmedetomidine as a premedication, sedative, anesthetic adjunct, and for EA management in pediatric age group[89] for nonpainful[10] and painful procedures[11] despite the lack of the United States' Food and Drug Administration approval for use in this age group. Intranasally administered dexmedetomidine could be tolerated safely in the pediatric age group for sedation in high doses up to 4 μg/kg.[1213] The objective of the current study was to determine the influence of intranasally administered dexmedetomidine on EA incidence and severity in children undergoing tonsillectomy and/or adenoidectomy after sevoflurane anesthesia. BODY.METHODS: This prospective, randomized controlled double-blinded clinical trial was conducted between November 2015 and March 2016. The study obtained an approval from the local Institution Research and Ethics Committee and registered at The Pan African Clinical Trials Registry (www.pactr.org) by the identification number of registry (PACTR201604001572340). Informed written consent was obtained from the parents of all the children. The study included 86 patients ASA I and II physical status, and their ages ranged between 3 and 7-years. These patients were scheduled for an elective tonsillectomy and/or adenoidectomy under general anesthesia with sevoflurane. Patients with obstructive sleep apnea, mental retardation or developmental delay, chest, cardiac or neurological diseases, known allergy or hypersensitivity to dexmedetomidine, and patients receiving medications known to interact with dexmedetomidine such as furosemide, lorazepam, and diphenhydramine were excluded from the study. All patients were admitted to hospital on the morning of surgery. They waited in pediatric preanesthesia holding area with their parents. They received no premedication and moved to the operating room (OR) accompanied by one of their parents. The parent was permitted to be present during the induction of anesthesia. All patients fasted 6 h for solids and 2 h for clear fluids. Patients were randomly allocated using a computerized random number generator into two groups. Group D (Study group) received intranasal dexmedetomidine (Precedex; Hospira Inc., Lake Forest, IL, USA) at 1 μg/kg after induction of general anesthesia. Intranasal dexmedetomidine was prepared from the 100 μg/ml parenteral preparation in a 1-ml syringe (with 0.9% saline added to make a final volume of 1 ml.), 0.5 ml installed in each nostril. Group C (control group) received intranasal saline 0.9% after induction of general anesthesia, 0.5 ml in each nostril. Dose calculation, drug preparation, and administration were done by attending anesthesiologists who were not involved or had not participated in this trial. The observers and data collectors were blinded to the study drug given also. General anesthesia induction was done by gradual increase of sevoflurane concentration to a maximum of 6 Vol. % in 100% oxygen (6 L/min) via facemask. An intravenous (IV) catheter was inserted after the loss of eyelash reflex, then dexamethasone at a dose of 0.3 mg/kg given, and the airway was secured with oral endotracheal tube after an adequate depth of anesthesia reached with fentanyl 1 μg/kg and cis-atracurium 0.1 mg/kg. Electrocardiogram, oxygen saturation (SpO2), mean arterial pressure, heart rate (HR), end-tidal CO2 concentration (EtCO2), and end-tidal sevoflurane concentration were monitored continuously. Sevoflurane concentration was maintained at 2–4 Vol. % in 50% oxygen air mixture (2 L/min) after intubation, then adjusted according to the patient's response to provide a stable HR, blood pressure (BP), and SpO2 with pressure-controlled ventilation, inspiratory pressure, and respiratory rate adjusted to maintain EtCO2 between 35 and 45 mmHg. All patients received acetaminophen (200 mg) suppository after induction of general anesthesia. During surgery, the surgeon infiltrated the operative site by 1% lidocaine with epinephrine (1:100,000) for pain and bleeding control (1 ml in each tonsillar bed). At the end of surgery, sevoflurane was discontinued, reversal of neuromuscular block facilitated by neostigmine bromide 20 μg/kg with atropine sulfate 20 μg/kg, and endotracheal tube was removed in lateral decubitus when the patients met the criteria of extubation (return of gag reflex, facial grimace, and purposeful motor movements). The time between the insertion and removal of the mouth gag recorded as the duration of surgery, and the time from sevoflurane mask induction till the extubation time was recorded as the duration of anesthesia. The time to extubation defined as the time from the end of surgery to tracheal extubation and the emergence time defined as the time of first response to command or eye opening on command after extubation were also recorded. On admission to postanesthesia care unit (PACU), the patients were monitored for HR, noninvasive BP, SpO2, and respiratory rate continuously for 30 min by anesthesia nurses who were blinded to groups' allocation. The primary outcome of this trial was the incidence of EA (highest score) which was assessed at six time points (after extubation, leaving the OR, on arrival to PACU, 10, 20, and 30 min after arrival in PACU) by four-point agitation scale[14] [Table 1]. Table 1 Four-point scale for the assessment of emergence agitation[ 14 ] Agitation scores of 3 and 4 were defined as an agitation episode and treated by nalbuphine (0.1 mg/kg) as a rescue therapy to control agitation episodes. The total dose of nalbuphine was calculated and compared for significance between both groups. Pain assessment was done using Face, Legs, Activity, Cry, and Consolability (FLACC) scale[15] [Table 2] at the same six time points (after extubation, on leaving the OR, on arrival to PACU, 10, 20, and 30 min after arrival in PACU). Nalbuphine as a rescue analgesic at a dose of 0.1 mg/kg was given if FLACC scores ≥5. The four-point agitation score and FLACC scale score assessment were done by anesthesiologists blinded to groups' allocation. In addition, time to discharge from PACU, defined as time started from patient's arrival to PACU till modified Aldrete score[16] ≥9, and the incidence of adverse events (nausea, vomiting, somnolence, apnea, desaturation, hypotension, and bradycardia) were recorded. Ondansetron (0.15 mg/kg) was given to control nausea and vomiting if any and total dose was calculated and compared for significance between both groups. Bradycardia (≤60 bpm) treated by atropine 20 μg/kg, and hypotension (≤20% of baseline reading) treated by ephedrine 5 mg increments. Patients were transferred to the ward after being fully conscious with stable vital signs for 30 min, and the absence of bleeding, pain, nausea or vomiting. Table 2 Face, Legs, Activity, Cry, and Consolability scale score for pain assessment[ 15 ] BODY.METHODS.STATISTICAL METHODS AND ANALYSIS: Based on the results of Aono et al.,[14] a sample size of 36 children per study group was estimated to have an 80% power (α= 0.05, two-tailed) and to detect a difference of 30% in the incidence of EA (primary outcome). Forty-three patients were included in each group to account for possible dropouts. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 16 (SPSS Inc., Chicago, IL, USA). Comparison of quantitative variables between the study groups was done using unpaired Student's t-test when the data were normally distributed and Mann–Whitney rank sum (when indicated). For comparing categorical data, Chi-square test was performed, and Fisher's exact test was used when appropriate. Continuous variables are presented as mean ± standard deviation, ordinal data presented as median (interquartile range [range]), and categorical data are presented as numbers and frequencies. P ≤ 0.05 was considered statistically significant. BODY.RESULTS: From the 102 patients assessed for eligibility, 11 patients did not meet the inclusion criteria and five patients did not participate in the study since their parents refused. So, the remaining 86 patients were enrolled in the study [Figure 1]. Figure 1Consort flowchart showing the number of patients at each phase of the study Demographic characteristics, duration of surgery, duration of anesthesia, and type of surgery were comparable in both groups [Table 3]. There was no statistically significant difference between the two groups regarding extubation, emergence, and discharge times with a P > 0.05 [Table 4]. Table 3 Demographic characteristics and intraoperative parameters Table 4 Recovery profile in operating room and postanesthesia care unit Postoperative nalbuphine dose consumption as a rescue medication for agitation episodes and pain was significantly higher in Group C compared with Group D with a P = 0.001 [Table 4]. There was statistically insignificant difference between the two groups with regard to nausea, vomiting, and total dose of ondansetron used during emergence or in the PACU with a P > 0.05 [Table 4]. There were no complications as somnolence, apnea, desaturation, hypotension, and bradycardia reported during emergence or in PACU in both groups before discharge to the ward. The incidence of EA was significantly lower in children allocated to Group D (three participants, 6.98%) compared to those allocated to Group C (25 participants, 58.14%) with a P = 0.001 [Table 5]. Table 5 Incidence of agitation The median agitation scales score of Group D were significantly lower than those of Group C at all time points with a P < 0.001 [Table 6] and also the median FLACC scales score of Group D were significantly lower than those of Group C at all time points with a P < 0.01 [Table 7]. Table 6 Severity of agitation at six points of time Table 7 The scoring system for pain scale at six points of time BODY.DISCUSSION: Confirming the previous trials,[1171819] this trial emphasized that dexmedetomidine markedly decreases the incidence of EA and curtail its severity in children after sevoflurane anesthesia without complications and with smooth postoperative recovery course. The intranasal route of administration was characterized by being noninvasive with relatively delayed onset (30–45 min), extended duration of action although short elimination half-life (1.8–3 h),[2021] and with no or little adverse effects in comparison to IV route, its safety and efficacy documented in various studies in comparison with other drugs and placebo when used as a premedication, anesthetic adjunct, or for the management of EA.[2022] In their trial to detect the appropriate dose of dexmedetomidine for the prevention of EA in children undergone tonsillectomies or adenoidectomies after desflurane anesthesia, Kim et al. reported that IV dose of 0.25 μg/kg and 0.38 μg/kg could prevent EA in 50% and 95%, respectively.[23] They have used five-point scale for EA scoring different that we have used in our trial, and their suggested smaller doses for EA prevention could be explained by the fact that desflurane may induce EA with shorter duration than sevoflurane.[23] In our study, we used a dose of 1 μg/kg for the prevention of EA post-sevoflurane for children undergone tonsillectomy and/or adenoidectomy. Akin et al. used the same dose and route of administration in comparison to intranasal midazolam, their primary end-point was satisfactory mask induction which was significantly better in midazolam group, and they were unable to find statistically significant difference in incidence and severity of EA between groups when administration of the drugs was 45–60 min before induction. In contrary, our trial was powered primarily to detect the preventive effect of dexmedetomidine on incidence and severity of EA. Nevertheless, Akin et al. reported that the number of children requiring postoperative analgesia was significantly lower in children allocated to the dexmedetomidine group, and these data go in line with our findings.[24] In the same context, Guler et al. reported that an IV bolus of 0.5 μg/kg of dexmedetomidine given 5 min before the end of surgery could significantly decrease incidence and severity of EA in children undergone adenotonsillectomy with sevoflurane.[18] In line with our findings, Olutoye et al. reported that there was no statistically significant difference in the analgesic effects of dexmedetomidine 0.5 μg/kg and morphine 50 μg/kg in children underwent tonsillectomies and adenoidectomies, and they added that postoperative sedation and incidence of nausea and vomiting were less with dexmedetomidine.[25] In our trial, we used the FLACC scale to assess pain score, and it worked efficiently in nonverbal pediatric patients, and it was easily estimated in OR and PACU. The previously mentioned trials used other pain scales such as objective pain score,[1824] children and infants postoperative pain scale (CHIPPS),[17] Wong–Baker FACES pain rating scale,[23] and the Children's Hospital of Eastern Ontario Pain score.[25] The pain after this type of surgical procedures could be severe and this may increase the incidence and aggravate the severity of EA, in addition, assessment of pain could be mistaken as an agitation episode and vice versa. Furthermore, most of the children with high-FLACC scale score ≥5 had also high agitation score of 3 or 4 and require nalbuphine rescue dose. The clinical and statistical significant difference of nalbuphine use in control group emphasized both the sedative and the analgesic properties of dexmedetomidine which are also used for sedation and analgesia. The comparable recovery times between both groups could be attributed to the sedative effects of dexmedetomidine in Group D and on the other hand excess use of nalbuphine in Group C [Table 4]. There was relative delay of anesthesia time over surgery time in both groups; this delay could be attributed mainly to the period from removing mouse gag till extubation (extubation time). Although the extubation time was longer in Group D than in Group C by about 2 min (mean difference), it was statistically insignificant difference [Table 3]. Of note, this relative delay was comparable between both groups [Table 4]. There was no statistically significant difference in the incidence of nausea and vomiting between both groups and also there were no reported cases of perioperative hypotension, bradycardia, apnea, desaturation, and excessive somnolence reflected high safety profile of the drug even with higher doses (2 μg/kg) as those used by Yuen et al. for better sedation and parent separation.[12] In this trial, we choose to administer the drug intranasally after the induction of anesthesia to compensate for delayed onset (30–45 min) to procedure lasting for 25–45 min with peak effect at 90–105 min after administration.[26] Although the anesthesia time is not matched perfectly in this trial (33.6 ± 6.5 and 35.1 ± 5.9 in Groups D and C, respectively) [Table 3] with the reported onset time of intranasal dexmedetomidine (30–45 min),[26] it is fairly acceptable in clinical practice. The role of dexmedetomidine in the reduction of incidence and severity of EA in children after sevoflurane anesthesia is documented with different dose regimens, timing, techniques, and route of administration in two meta-analyses done by Gyanesh et al. and Sun et al.[89] As a limitation to this study, we use one milliliter syringes dripping for intranasal drug installation due to lack of intranasal drug delivery systems as an atomizer and nasal spray which improve drug absorption, hasten onset time, and optimize bioavailability instead we use 1-mL syringe dripping. In addition, we did not measure serum concentration of dexmedetomidine repeatedly after intranasal installation, and future studies are needed to elucidate detailed pharmacokinetics and pharmacodynamics of the drug in pediatric age group via the intranasal route. BODY.CONCLUSION: This study demonstrates that a dose of 1 μg/kg intranasal dexmedetomidine administered after the induction of anesthesia reduces post-sevoflurane incidence and severity of EA in children undergone tonsillectomy and/or adenoidectomy with no adverse effects and smooth postoperative course. BODY.CONCLUSION.FINANCIAL SUPPORT AND SPONSORSHIP: Authors would like to acknowledge the financial support provided by Benha University Hospital and Fayoum University Hospital. BODY.CONCLUSION.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: Vitamin D Supplementation and High-Density Lipoprotein Cholesterol: A Study in Healthy School Children ABSTRACT.BACKGROUND: The high-density lipoprotein cholesterol (HDL-C) level has been shown to have a significant role in the prevention of cardiovascular diseases and atherosclerosis. Low vitamin D levels have been shown to be correlated with dyslipidemia, but limited data exist on indigenous children. ABSTRACT.OBJECTIVES: We aimed to investigate the effect of vitamin D supplementation on HDL-C levels in school-aged Iranian children. ABSTRACT.METHODS: In this prospective controlled clinical trial, 47 healthy children (23 boys) aged 10 - 14 years, students of Birjand (Iran) elementary schools, were selected and randomly divided into two groups. The study group received a vitamin D supplement (1000 mg capsule) daily for one month, and placebo tablets were prescribed to the controls. Before and after the treatment course, the serum HDL-C and 25-hydroxy vitamin D levels of both groups were measured. The data were analyzed by SPSS, ver. 16, and Chi-square tests, Fisher's exact test, paired-sample t-tests, and Pearson's correlation were used, wherever appropriate. The significance level was set at P < 0.05. ABSTRACT.RESULTS: Forty children completed the study; their mean age was 11.5 ± 1.175 years. The mean serum levels of both HDL-C and vitamin D showed a significant rise following the treatment in the study group (P = 0.007 and P < 0.001, respectively), whereas both variables decreased slightly in the control group (P = 0.27). There was no statistically significant difference in the mean serum levels of HDL-C and vitamin D between the two groups after the intervention (P = 0.11 and P = 0.20, respectively). ABSTRACT.CONCLUSIONS: Vitamin D supplements seem to have a positive impact on serum HDL-C levels and may be effective in reducing the risk of cardiovascular diseases in the long term. BODY.1. BACKGROUND: Vitamin D belongs to a group of fat-soluble steroids, which are mainly responsible for calibrating serum calcium, iron, magnesium, phosphate, and zinc. Although vitamin D may be ingested through diet and supplement use, newly published articles have classified vitamin D as a hormone (1, 2). It is becoming increasingly clear that vitamin D has a much broader range of actions in the human body, in addition to its well-known effects on calcium homeostasis and bone metabolism. Interestingly, vitamin D also has antiatherogenic functions, inhibiting the formation of foam cells and cholesterol uptake by macrophages and enabling HDL transport (3). Race, female gender, season, pubertal status, and visceral adiposity are independent predictors of plasma 25 (OH)D status (4). Vitamin D deficiency is associated with vascular stiffness, which is a known predictor of cardiovascular diseases, and a marker of subclinical atherosclerosis (3, 5). Cardiovascular diseases are the most common noncommunicable diseases responsible for morbidity and mortality worldwide (6). Blood lipid disorders, including low high-density lipoprotein cholesterol (HDL-C) levels, are considered one of the major risk factors for coronary artery diseases, such as atherosclerosis (7, 8). HDL-C prevents the blockage of blood vessels by preventing low-density lipoprotein (LDL) sedimentation. Therefore, increased HDL-C levels reduce the risk of heart attack and stroke (9). Most of the variation in disease progression of atherosclerosis, which starts in childhood, occurs with aging and eventually leads to morbidity and mortality in old age (10). Vitamin D insufficiency is a common public health problem but is often unrecognized and untreated. It is associated with rickets, dental caries, and growth retardation in children (11). Today, vitamin D deficiency is rising in many parts of the world due to urbanization and dietary changes, resulting in a 90% prevalence of hypovitaminosis in some countries (1, 3, 12). In a study by Kelishadi et al. of 1095 Iranian students, 40% were vitamin D deficient, and 39% had vitamin D insufficiency (12). Two other studies of urban populations in Iran reported moderate-to-severe deficiency of 65% and 50%, respectively (13, 14). Despite receiving abundant sunlight, Saudi Arabia has a high prevalence of vitamin D insufficiency, mainly due to reduced outdoor activity and lack of regular consumption of vitamin D-fortified foods (3, 5). Screening and treating vitamin D deficiency would most probably be reflected in reduced cardiovascular morbidity and mortality. Fortunately, vitamin D deficiency is treatable, and supplementation is inexpensive (11), with common food sources including fortified milk and dairy products (3). In several studies, higher levels of vitamin D were associated with lower rates of cardiovascular disease, perhaps through improved lipid profiles. However, the results are inconsistent, and the nature of the association between vitamin D and lipid levels remains unknown (15). This relationship is also unclear among prepubertal children (16). Williams et al. stated that higher circulating 25(OH)D was associated with cardioprotective levels of HDL-C, Apo-A1, and adiponectin in children (17). Kelishadi et al. also reported that higher serum 25(OH)D levels were related to more favorable lipid profiles in the pediatric age group (18). BODY.2. OBJECTIVES: Due to the high risk of vitamin D deficiency in Iran and the need to assess its impact on levels of HDL-C, a cardioprotective factor, we investigated the effect of vitamin D supplementation on HDL-C levels in school-aged children. BODY.3. METHODS: This randomized controlled clinical trial was conducted in 2014 with primary schoolchildren in Birjand, Iran. The study protocol was approved by the medical ethics committee of Birjand University of Medical Sciences and submitted to the clinical trials registry system (1N2014050717608). Fifty-four children aged 10 - 14 years who consented to participate in the study were randomly selected from primary schools (28 girls and 27 boys). All the children underwent a physical examination. Those who were symptomatic, had an underlying disease, and used any type of drug or supplement were excluded from the study. The serum levels of vitamin D and HDL-C of the enrolled participants were measured, and any child with vitamin D deficiency was excluded from the study. Finally, 47 children were included in the study (23 boys and 24 girls). A questionnaire, the validity of which was confirmed by five professionals, was used to collect demographic and laboratory data and record the medical histories of the children. The data included the patient's weight and history of drug use, congenital cardiovascular disease, cancer, hypothyroidism, hyperthyroidism, and kidney and liver diseases. The children were then randomly divided into two groups: a study group (n = 24) that received vitamin D tablets at a dose of 1,000 IU daily for one month and a control group (n = 23) that received placebo tablets for the same duration (Figure 1). The placebo tablets were the same size and color as the vitamin D tablets and provided in the same type of package. At the end of the treatment course, 5cc of blood was taken from each participant and re-examined in the same lab using the same testing kits. The serum HDL-C concentration was measured using commercially available enzymatic reagents (Roche Kits, Germany) with a closed fully automated analyzer system (Roche Cobas Integra). The 25-hydroxy vitamin D level was measured using an electro-chemiluminsence system (the system close company Roche E411 Series kits made in Germany in 2013). The collected data were analyzed using SPSS, ver. 16. A chi-square test and Fisher's exact test were used for qualitative data comparisons between the two groups. A paired sample t-test and Pearson's correlation coefficient were used, wherever appropriate. The significance level was set at P < 0.05. Figure 1.Flowchart of the Study Patients BODY.4. RESULTS: In total, 40 children completed the study: 20 boys and 20 girls. Their mean age was 11.5 ± 1.175 years. Thirteen (65%) and 7 (35%) children in the study and control groups were female, indicating no meaningful difference between the two groups in terms of sex (P = 0.06). Moreover, no significant difference existed between the two groups regarding age (P = 0.79). The mean serum HDL-C level before the intervention was 47.30 ± 11.15 mg/dL, with an average level of 43.55 ± 11.25mg/dL among the boys and girls, respectively, indicating no significant difference based on sex (P = 0.30). The mean serum vitamin D level of the boys was 12.34 ± 4.85 ng/mL, whereas it was 4.92 ± 2.15 ng/mL for the girls, revealing a meaningful difference based on sex, with a significantly higher level among the boys (P < 0.001). As demonstrated in Table 1, there was no significant difference in the mean level of HDL and vitamin D between the study and control groups before the intervention (P = 0.95 and P = 0.20, respectively). Similar results were obtained for mean HDL and vitamin D levels after the intervention (P = 0.11 and P = 0.20, respectively). The mean serum levels of both HDL-C and vitamin D showed a significant rise following the treatment in the study group (P = 0.007 and P < 0.001, respectively), whereas they decreased slightly in the control group (P = 0.27) (Table 1). When the post-interventional level of serum HDL and vitamin D was compared based on sex, a statistically significant difference was observed only for vitamin D, indicating a significantly higher level among boys (P < 0.001). Table 1. Comparison of Changes in Serum Levels of HDL-C and Vitamin D in Both Groups Before and After the Intervention Group Before the Intervention (Mean ± SD) After the Intervention (Mean ± SD) P Value Mean Change Before and After the Intervention Vitamin D Study 7.55 ±4.96 11.50 ± 5.84 < 0.001 3.95 ± 2.15 Control 9.71 ± 5.48 9.26 ± 4.97 0.27 -0.45 ± 1.74 P Value (two-group comparison) 0.20 0.20 - < 0.001 HDL Study 45.55 ± 12.28 49.65 ± 11.53 0.007 4.10 ± 6.10 Control 45.30 ± 10.38 43.65 ± 11.47 0.27 -1.65 ± 6.44 P Value (two-group comparison) 0.95 0.11 - 0.006 After the intervention and before adjustment, the mean serum level of vitamin D in the study and control groups was 11.50 ± 5.84 ng/mL and 9.26 ± 4.97 ng/mL, respectively. The same values after adjusting for confounding variables were 12.54 ng/mL and 8.21 ng/mL, respectively. The mean serum level of HDL before adjustment was 49.65 ± 11.53 mg/dL and 43.65 ± 11.47 mg/dL in the study and control group, respectively. The same values were 49.54 mg/dL and 43.76 mg/dL after adjustment. Based on an ANCOVA, the serum HDL levels were correlated before and after the intervention (P < 0.001). After adjustment for confounding variables, the mean serum HDL level of the treatment group was significantly higher than that of the control group (P = 0.04). The same correlation was found for vitamin D levels (P < 0.001). After adjustment, the level of the study group was significantly higher than that of the control group (P < 0.001). BODY.5. DISCUSSION: Activated vitamin D can influence cellular growth, proliferation, and apoptosis; oxidative stress; cell membrane transport; cell adhesion; and immune system functions (11, 19, 20). It can also regulate a large number of genes and healthy aging. Various factors, such as ethnicity, geographical location, culture, and dietary habits, can influence vitamin D levels (3). Besides therapeutic measures to correct high rates of vitamin D deficiency in youth, the benefit of vitamin D optimization adiposity measures and lipid profiles needs to be established (4). Herein, we aimed to investigate the effect of vitamin D supplementation on HDL-C levels in school-aged Iranian children. To date, several studies have shown that higher 25(OH)D3 concentrations in childhood are associated with higher levels of HDL in adolescence (21). In a national population-based study of 1095 students in Iran, the median 25(OH)D level was 12.70 ng/mL in boys and 13.20 ng/mL in girls, corresponding to vitamin D deficiency of 40% and vitamin D insufficiency of 39% (12). The study found no significant differences in the median 25(OH)D level between boys and girls (12). In the present study, we observed a significant sex-related difference in vitamin D, with a significantly higher level among the boys (P < 0.001). The adjusted regression analysis in the latter mentioned study revealed a significantly positive correlation with HDL-C. Kelishadi et al. recently performed a systematic review and meta-analysis of the relationship of serum 25-hydroxy-vitamin D with lipid profiles in the pediatric age group (18). They concluded that a higher serum 25(OH)D level was related to more favorable lipid profiles in this population (P < 0.001). In a prospective 2-year study by Hirschler et al. of 60 children (29 males) who received 100,000 units of vitamin D and 36 children (16 males) who received 50,000 units, vitamin D and lipid levels significantly improved in the first group compared to the latter one, suggesting that optimal vitamin D levels were associated with healthier lipid profiles (22). Vitezova et al. suggested that total cholesterol may be associated with decreased 25(OH)D concentrations but that the inverse did not hold true (15). They concluded and that the observed inverse association between HDL-C and 25(OH)D may be bidirectional. In a retrospective review of children aged 2 - 18 years, Johnson et al. revealed a positive association between 25(OH)D and HDL-C (P ≤ 0.001), reporting lower levels of HDL-C in children with vitamin D deficiency (23). Nwosu et al. concluded that vitamin D levels varied inversely with non-HDL, TC/HDL, and LDL (16). In their study, a 25(OH)D level of 30 ng/mL was associated with optimal cardioprotection in children, resulting in significantly lower non-HDL-C, TC/HDL, TG, and LDL compared to a level < 20 ng/mL (P = 0.006). In the present study, serum HDL-C and vitamin D levels showed no statistically significant difference before and after the intervention in the control group (P = 0.27), whereas they showed a significant increase in the study group (P = 0.007 and P < 0.001, respectively). On the other hand, Birken et al. reported nonsignificant associations between 25(OH)D, LDL, and HDL (24). In this study, 25(OH)D concentrations showed an inverse association with circulating lipids in early childhood, suggesting that vitamin D exposure in early life may be an early modifiable risk factor for cardiovascular disease. In a study of 1504 Korean adolescents aged 12 - 18 years, Nam et al. found no significant relationship between the serum 25(OH)D concentration and hyperglycemia, reduced HDL-C, or hypertriacylglycerolemia, with or without adjustment for confounding variables (25). In contrast, in the Caspian III study, the highly prevalent disorders of low 25(OH)D and low HDL-C in children and adolescents of the Middle East and North Africa region had a significant association (12). In the present study, after adjustment for confounding variables, the mean serum HDL level of the treatment group was significantly higher than that of the control group (P = 0.04). Accordingly, the mean serum vitamin D level in the study group was significantly greater than in the control group (P < 0.001). In line with this result, Maki et al. reported a significant increase in the serum HDL level of recipients of vitamin D, as well as a significant reduction in TC/HDL (26). Another study found that obese children and adolescents with vitamin D deficiency had lower levels of HDL-C (27). An observational study showed that high levels of 25(OH)D were associated with an increase in desirable fat (2). A study of healthy adolescents reported a significant inverse relationship between 25(OH)D and serum LDL and HDL-C levels (28). The above results and those of the present study point to positive effects of vitamin D supplementation on serum HDL. Experiments on liver cells confirmed these effects, showing that vitamin D metabolites potentially had an inhibitory effect on the production of apoA-I (29, 30). Taken together, vitamin D consumption is supported in patients with low serum vitamin D levels, with very positive results in terms of general health and cardiovascular health achievable with minimal intervention and minimal costs. The main limitations of this study were the small sample size, lack of cooperation in proper use of drugs, and not visiting the clinic for second sampling as expected. BODY.5. DISCUSSION.5.1. CONCLUSION: Based on the current state of knowledge, it may be too early to consider or to rule out vitamin D as a tool to estimate or mitigate residual cardiovascular risk. Despite the small sample size, the present study provides consistent support for a relationship between vitamin D and HDL-C, indicating that vitamin D supplementation results in increased blood levels of HDL and that it can be regarded as a protective factor to reduce the risk of cardiovascular disease.

TITLE: Medical Student Bias and Care Recommendations for an Obese versus Non-Obese Virtual Patient ABSTRACT.OBJECTIVE: This study examined the independent effect of a patient's weight on medical students' attitudes, beliefs, and interpersonal behavior toward the patient, in addition to the clinical recommendations they make for her care. ABSTRACT.DESIGN: Seventy-six clinical-level medical students were randomly assigned to interact with a digital, virtual female patient who was visibly either obese or non-obese. ABSTRACT.METHODS: Interactions with the patient took place in an immersive virtual clinical environment (i.e., virtual reality) which allowed standardization of all patient behaviors and characteristics except for weight. Visual contact behavior was automatically recorded during the interaction. Afterward, participants filled out a battery of self-report questionnaires. ABSTRACT.RESULTS: Analyses revealed more negative stereotyping, less anticipated patient adherence, worse perceived health, more responsibility attributed for potentially weight-related presenting complaints, and less visual contact directed toward the obese version of a virtual patient than the non-obese version of the patient. In contrast, there was no clear evidence of bias in clinical recommendations made for the patient's care. ABSTRACT.CONCLUSION: Biases in attitudes, beliefs, and interpersonal behavior have important implications because they can influence the tone of clinical encounters and rapport in the patient-provider relationship, which can have important downstream consequences. Gaining a clear understanding of the nature and source of weight bias in the clinical encounter is an important first step toward development of strategies to address it. BODY.MEDICAL STUDENT BIAS AND CARE RECOMMENDATIONS FOR AN OBESE VERSUS NON-OBESE VIRTUAL PATIENT: The worldwide prevalence of obesity is high and has steadily grown1. Accordingly, the proportion of patients with obesity seen in primary care systems is also quite high. Despite the frequency of encounters between clinicians and patients who are obese, many providers hold negative attitudes toward these individuals2-6. In turn, a number of studies demonstrate that obese patients report negative clinical experiences, poor treatment, and stigmatizing behavior by health care providers5, 7-13. These experiences are posited to contribute to avoidance of cancer screening and other preventive health services among persons who are obese14-16. Health care providers' attitudes toward people who are obese are consistent with those of the general public. Indeed, research indicates that many physicians perceive individuals who are obese to be highly responsible for causing their condition2, 17, 18 and endorse stereotypes that they are lazy and lacking in self control2, 19. A corollary to these beliefs is providers' tendency to perceive obese patients as being unmotivated and non-compliant20. These attitudes and beliefs can shape the clinical interaction making it more negative and uncomfortable for patients who are obese21, 22 and decreasing patient-provider rapport. Negative attitudes can also be subtly conveyed in interpersonal behaviors23, 24. The degree to which a provider makes eye contact with a patient, for example, can reflect the level of his or her regard for that patient. These nonverbal behaviors color the tone of the medical encounter and influence patient satisfaction25. Though provider attitudes and beliefs about patients with obesity can clearly be impactful in the clinical encounter, previous studies have largely been limited to surveys of general attitudes. Each interaction between a provider and a patient occurs in a complex clinical context with a multitude of clinical and interpersonal variations. Thus, there are numerous characteristics of the patient and aspects of the interaction aside from patient weight that could initiate or exacerbate negative attitudes. Many of these variables, however, can be confounded with a patient's weight, making it difficult to pinpoint the source of negative attitudes reported in surveys. For example, patients who are obese tend to have different co-morbidities or different health status than patients who are not obese26. Patients who are obese may also exhibit different interaction styles,27 developed through previous experiences with weight bias and negative treatment. Similarly, providers' expectations about patients who are obese may elicit negative interaction styles from these patients (i.e., self-fulfilling prophecies)28. General surveys do not account for these factors. Thus, there is little evidence on the extent to which bias against and negativity toward obese patients is activated by the patient's weight, as opposed to factors that arise during the medical encounter. In addition to affecting rapport with patients, providers' attitudes toward obese patients might also influence their decisions about patient care. Indeed, individuals' biased attitudes are often linked to related behaviors29. Only two known studies have attempted to examine the link between provider attitudes and patient care behavior. Thus far, findings are mixed. Wigton and McGaghie found no differences in providers' psychiatric-related recommendations in response to viewing video tapes of actors playing obese or non-obese patients30. Hebl and Xu reported finding differences in providers' test ordering for obese versus non-obese patients based on hypothetical chart review31. If provider biases about obese patients do lead to differences in clinical decision making, this could clearly have important implications for patient health. Gaining a clear understanding of the nature of weight bias in the clinical encounter is an important first step toward development of strategies to address it. It is extremely difficult to examine the effect of a single clinical variable, like patient weight, in a real medical setting. Actual clinician-patient encounters are un-standardized and cannot be manipulated for experimental purposes. Even standardized patients or actors are unable to completely standardize their verbal and nonverbal behavior. There is therefore a need for a highly controlled examination of the effects of patient weight within the context of a medical encounter. To accomplish this, we employed immersive virtual environment (IVE) technology (i.e., virtual reality) which allows experimental manipulation of clinical variables, while maintaining total patient standardization in a realistic clinical context32, 33. In IVEs, users are immersed in a digitally created environment. This is realized using a combination of graphics software and a carefully designed user interface to create three-dimensional environments that users can navigate in a natural way (e.g., by walking). This technology also allows tracking of users' visual gaze, which can serve as a subtle indicator of bias34. To examine the effect of patient weight on provider attitudes, beliefs, and behavior, we immersed clinical-level medical students (in their third or fourth years of training) in an IVE interaction with a virtual female patient who was visibly either obese or non-obese. The patient presented with two potentially weight-related complaints, knee pain and shortness of breath, and one non-weight-related complaint, eczema. We chose to focus on a medical student population because they are a likely target of intervention or education efforts aimed at improving clinical interaction with obese patients. We had three main hypotheses: Students will exhibit more negative attitudes and have more negative beliefs about the obese patient than the non-obese patient (negative stereotyping, labeling her as less likely to be adherent, labeling her as less healthy, and assigning her more responsibility for causing her potentially weight-related presenting complaints).Students will exhibit less visual contact with the obese patient than the non-obese patient.Students who interact with the obese patient will exhibit more reliance on lifestyle-related clinical recommendations for her potentially weight-related concerns, and thus make fewer recommendations for diagnostic follow-up for her shortness of breath, and for symptom management for her knee pain. BODY.METHODS.DESIGN: This study was an experiment in which students were randomly assigned to one of two conditions: interaction with an obese or a non-obese version of the same digital patient (see Figure 1). Data for this study were obtained from a larger experimental project examining the impact of genetics information provision to medical students. BODY.METHODS.PARTICIPANTS: Participants were recruited from the Washington, DC and Baltimore, MD metropolitan areas. They included 76 third and fourth year medical students. Thirty-seven were randomly assigned to the obese patient, 39 to the non-obese patient condition. Because we used IVE technology, exclusion criteria included having a seizure or vestibular disorder, being highly prone to motion sickness, and having poor, uncorrected hearing or vision. Participants were compensated $100 for their participation. BODY.METHODS.PROCEDURE: This study was approved by the governing Institutional Review Board. Each student completed one experimental session lasting approximately an hour. The study was described to students as an investigation of patient-provider interaction in virtual environments; we did not communicate any study aims related to obesity until debriefing. After students consented to participate in the study, they completed a task in which they were asked to read and describe a short article on a medical topic. The content of the article was unrelated to any other portion of the study. Following this task, students engaged in an IVE-based interaction with a virtual female patient who was either obese or not depending upon assigned condition. Students wore a head-mounted display to interface with the virtual environment. Their head and body movements were tracked using an optical and inertial tracking system to render the appropriate scene in real time. Scenes were rendered stereoscopically, producing a three-dimensional virtual world. Students' movements in the virtual environment were recorded for later analysis. The virtual encounter with the patient was a primary care-type clinical encounter. We constrained communication and interaction so that each student would receive the same type and amount of information from the patient. All aspects of the virtual patient and her history were identical between the obese and non-obese versions with the exception of her reported body weight, her reported BMI, and her visible body size. Students 'entered' the virtual clinic room by wearing the head-mounted display and were directed to look at a virtual computer monitor within the environment. This monitor provided information and instructions to guide students though the interaction. The patient's chart information was then displayed on the computer monitor (see Figure 2). All details were identical for the obese and non-obese patients (blood pressure, pulse, temperature, medications, etc.) with the exception of weight and BMI. The obese patient's weight was reported as 247 pounds with a BMI of 39.9, and the non-obese patient's weight was reported as 134 pounds (BMI=21.6). When students were finished reviewing the patient information, they saw the patient for the first time. Next, a turn-taking clinical interaction between the student and the patient took place. The student introduced him or herself to the patient. The patient verbalized information about her current health symptoms and concerns. Specifically, the patient reported that she currently had a rash on her hand that she believed to be eczema, that she was experiencing knee pain previously diagnosed as osteoarthritis, and she had been experiencing some intermittent shortness of breath. After the patient finished speaking, the student had an opportunity to visually examine the patient (e.g., taking a closer look at the eczema rash on her hand). The student responded verbally to the patient with whatever content he or she felt was appropriate, and the interaction was ended. Following the virtual encounter, students completed a battery of computerized questionnaires. Afterward they were weighed and measured for height. Finally, students were fully debriefed and dismissed. BODY.METHODS.MEASURES: Students completed several measures indicating their attitudes toward and beliefs about the patient following the interaction. All scale responses were collected using seven-point Likert-type scales. Attitude and belief measures are described in the order of presentation. BODY.METHODS.MEASURES.BELIEFS ABOUT PATIENT'S HEALTH: We measured students' beliefs about how healthy the patient was with four items previously used by Hebl and Xu31. Originally, each was a single item that was part of a larger battery of assessments. We created a scale from the four items related to perceptions of the patients' health status. Items assessed beliefs about how healthy the patient is, how well the patient takes care of herself, the patient's self-discipline, and the seriousness of the patient's health condition. Scale endpoints included "not at all" and "extremely." The scale we created from these items showed good reliability (Cronbach's alpha = .76). BODY.METHODS.MEASURES.PERCEPTIONS OF PATIENT'S ADHERENCE: We measured students' perceptions of the patient's likelihood to adhere to their advice with a single item taken from the same battery of items described above31. The item was worded, "this patient would follow my advice." Scale endpoints included "not at all" and "extremely." BODY.METHODS.MEASURES.NEGATIVE STEREOTYPING: We measured students' attitudes about the patient using a negative stereotype scale based on the Obese Persons Trait Survey35 (Cronbach's alpha = .91). The scale consists of ten negative traits (e.g., laziness). Students were asked to indicate the extent to which each trait described the patient. Scale endpoints were "strongly disagree" and "strongly agree." BODY.METHODS.MEASURES.PERCEPTIONS OF PATIENT'S RESPONSIBILITY: The extent to which students believed the patient was responsible for causing each of her three presenting complaints (eczema, shortness of breath, and knee pain) was assessed with a single item for each, e.g., "how responsible is your patient for causing her eczema?" Scale endpoints were "not at all responsible" and "entirely responsible". BODY.METHODS.MEASURES.VISUAL CONTACT: We unobtrusively measured the extent of visual contact students made with the virtual patient during the interaction. The IVE system recorded the direction of each student's gaze twice every second. These data were run through a computer program to determine the extent to which the patient's face was central in the participant's view over the course of the exam period. BODY.METHODS.MEASURES.CLINICAL RECOMMENDATIONS: Prior to answering any of the attitude or belief items, students were asked to generate an open-ended list of the follow-up recommendations they would make after this initial visit for each of the patient's complaints (eczema, shortness of breath, and knee pain). Students were prompted with three broad categories: diagnostic tests, medication-related treatment, and non-medication-related treatment. Responses were tabulated. To capture the most common recommendations, we included recommendations in analyses only if at least 20% of students in either condition made a given recommendation. BODY.METHODS.MEASURES.DEMOGRAPHICS: Demographic variables such as gender, age, race and ethnicity, year in medical school, and family history of obesity were collected last among the self-report measures. Students' weight and height were measured directly using a scale and tape measure at the conclusion of the visit. BODY.METHODS.STATISTICAL ANALYSIS: Analyses were performed using SPSS version 17.0 (SPSS Inc., Chicago IL). Statistical significance was assessed at p<.05. Descriptive analyses of all variables were performed. For clinical recommendations, chi square analyses were used to assess differences between the two conditions. For all other attitude and behavior variables, comparisons between the two conditions were performed using 1-way ANOVAs. We included participant gender as a covariate in attitude and belief analyses as these can differ between male and female providers19, 36. We also initially performed analyses with student BMI (kg/m2) as a covariate, however, BMI was not a significant covariate so it was removed from the analyses reported here. BODY.RESULTS: Participant characteristics are presented in Table 1. Students assigned to each condition did not significantly differ from one another on any demographic characteristic. BODY.RESULTS.ATTITUDES AND BELIEFS: Unadjusted means and standard deviations are reported in Table 2. Students endorsed significantly higher levels of negative stereotypes when they interacted with the obese version of the patient than the non-obese version of the patient, F(1,73)=49.64, p<.0001. Students also rated the obese patient as less likely to adhere to their advice, F(1,73)=7.42, p<.01. Finally, students rated the obese patient as being less healthy than the non-obese patient, F(1,73)=54.16, p<.0001. In addition, the obese patient was rated as more responsible for causing the presenting complaints that could be construed as weight-related, but not the unrelated condition. That is, the obese patient was held significantly more responsible for causing her shortness of breath, F(1,73)=26.37, p<.0001, and her knee pain, F(1,73)=36.11, p<.0001, but there was no difference in student ratings of the obese and non-obese patients' responsibility for causing the eczema. BODY.RESULTS.VISUAL CONTACT: Students had the patient's face in view a lower percentage of the time (i.e., made less visual contact) when the patient was obese (M=29%, SD=13) than when she was not obese (M=37%, SD=18), F(1,74)=6.31, p<.05. BODY.RESULTS.CLINICAL RECOMMENDATIONS: We assessed students' clinical follow-up recommendations for the two presenting complaints that could be construed to be weight-related; knee pain and shortness of breath. Comparisons of recommendation rates between the two conditions are presented in Table 3. We assessed the extent of reliance on lifestyle-related follow-up recommendations (weight loss, diet, exercise) versus symptom management recommendations for knee pain, and versus diagnostics for shortness of breath. Comparisons of recommendation categories for shortness of breath by condition revealed that students were more likely to recommend lifestyle changes when the patient was obese than when she was not obese, χ2 (1, N =76) =14.6, p<.0001. They were less likely to recommend symptom management (i.e., bronchodilator prescription) for shortness of breath when the patient was obese, χ2 (1, N =76) =4.8, p<.05. There was, however, no significant difference in recommendation rates for diagnostic tests. Comparison by condition of recommendation categories for knee pain resulted in no significant differences; students in the obese and non-obese patient conditions recommended lifestyle, symptom management, and diagnostic follow-up at similar rates. BODY.DISCUSSION: In this study we found increased negative stereotyping, less anticipated patient adherence, worse perceived health, more responsibility attributed for potentially weight-related presenting complaints, and less visual contact directed toward the obese version of a virtual patient than the non-obese version of the patient. This pattern occurred in response to the size of the patient alone, as all other interaction variables and all potential confounders were held constant. Unlike previous studies that used videotaped actors or written vignettes30, 31, use of an IVE clinical 'simulation' allowed us to disentangle the effect of patient weight from other factors while maintaining psychological realism and immersion in the experimental scenario. Previous research has shown that experiences in virtual environments are psychologically compelling37. Virtual patients generally elicit reactions similar to standardized patients in training scenarios38, 39. Behavior in these simulations can also translate to behavior in real clinical interactions39, 40. The attitude and belief outcomes reported here are important in their own right and can have substantial impact on patients' experiences of the medical encounter. Furthermore, Epstein and Street41, 42 posit a model through which negative interpersonal interactions in the clinic can contribute to obese individuals' avoidance of preventive care, as demonstrated in the literature13-15. In the model, factors like clinician-patient rapport and patient satisfaction are considered 'proximal outcomes' of the clinical interaction. These proximal outcomes can influence health directly and also influence 'intermediate outcomes' such as a patient's commitment to treatment, which impacts health. The current study demonstrated one concrete example of negative interpersonal behavior in that students exhibited decreased visual contact with the obese patient. Visual contact is a proxy for eye contact, a behavior that is linked to empathy expression by providers. Eye contact is part of a constellation of nonverbal behaviors that reflect providers' regard for patients and that are linked to patient satisfaction25. We anticipated that differences in beliefs and attitudes would affect students' clinical recommendations for the obese patient. Although there were some individual clinical recommendations that differed between the obese and non-obese versions of the patient in the current study, we did not find pervasive patterns indicative of bias. Though we found no clear evidence of bias, there were some differences in rates of individual recommendations that are of interest. The differences we found between conditions (e.g., for lifestyle-related treatment recommendations) were not inappropriate when the patient's body weight was used as clinical data in the decision-making process. It is worth noting, however, that some of the individual recommendations that differed between the obese and non-obese patient, weight loss and lipid profile in particular, though medically reasonable, may be less germane to short-term workup or symptom relief. Other recommendation differences (minimizing knee stress, heat and cold application, and bronchodilator prescription) might also indicate weight-related assumptions about factors causing the patient's symptoms. Such recommendations, therefore, may be indicative of an understanding that the obese patient is less healthy, less active, or should lose weight generally. Further research should explore beliefs and assumptions behind these subtle differences in recommendation patterns. We did not find evidence to support our hypothesis that students would rely on lifestyle recommendations to address the obese patient's potentially weight-related symptoms and would thus be less thorough with respect to symptom management and diagnostics. There are several possible reasons for this. In general, diagnosis, treatment, and management of conditions like knee pain and shortness of breath involve protocols43, 44 that these students are likely to have recently learned. Behaviors that largely depend upon protocol and habit are less likely to be influenced by one's attitudes and beliefs45. Thus, it may be the case that the particular symptoms examined here did not leave students leeway to express their attitudes. Previous research has similarly shown little influence of patient characteristics on management plans for shortness of breath whereas these characteristics influenced recommendations for a different symptom46. Alternatively, some students in our sample may not yet have been well-versed in these protocols and thus may have made more recommendations across the board so as not miss anything. More research is warranted to disentangle these issues. Regardless, in this study, the attitudes and beliefs students reported toward obese patients seem to hold more implications for the quality of the clinical encounter than for care recommendations. In examining these findings it is also of note that participant BMI was not a significant predictor in our analyses. Thus, students who had higher BMIs did not exhibit lower levels of bias. This finding extends the currently mixed literature6 on whether providers' weight impacts their attitudes and/or beliefs about patients who are obese. The current study has several limitations. This study focused on medical students. Although we chose this sample because students are a clear target for potential interventions, they are still in the midst of their clinical training. Thus, the current findings may be less generalizable to practicing physicians and other clinicians. Furthermore, although we were able to measure and report students' nonverbal visual contact during the clinical interaction, we did not include other measures of interpersonal behavior or interaction quality. This is in part because the communication between the students and the virtual patient was constrained to keep it constant between participants and between conditions. In the future, examining additional verbal and nonverbal behaviors could shed more light on how attitudes and beliefs impact interpersonal behavior during a medical encounter. In addition, several of our measures consisted of a single item. More in depth assessments may increase validity of belief and attitude measurement in future studies. Another limitation was the fact that we did not include assessments of whether and the extent to which participants perceived the patient as being obese. The virtual patient did appear to have a somewhat smaller body type than would be typical of someone with a BMI of 39.9. Based on the fact that the vast majority of students recommended weight loss for the patient, however, it clear that the patient was generally perceived as being overweight or obese. Finally, we did not allow for a true physical examination or interview during the clinical interaction. We provided students with several pieces of clinical information (e.g., blood pressure, smoking status) and included a visual examination period in which students could take a closer look at the patient. However, students were not able to perform any other type of examination that might have informed their diagnostic and treatment recommendations. For this initial study we opted to keep the interaction simple. In the future, however, making the flow of the interaction more similar to reality may increase external validity. Further exploration of the patient-provider relationship and how obesity stigma plays out in this complex interaction is warranted. Experimental work focusing on the patients' experiences in the interaction will aid in understanding processes at work when they report negative encounters with providers. In turn, explication of these processes will help to identify points of intervention where we might improve patient-provider interactions for patients who are obese. The current findings demonstrate that patients who are obese can trigger negative, biased attitudes, beliefs, and differential interpersonal behavior based on their size alone, in the absence of particular interaction styles, health characteristics, or other differences. Even though these attitudes and beliefs did not translate into biases in patient care recommendations, they have important implications in their own right. Negative attitudes and biases can influence the tone of clinical encounters and rapport in the patient-provider relationship, both of which can have important downstream consequences. It is therefore important to develop strategies for mitigating the effects of these reactions to patients who are obese.

TITLE: Possible Long-Term Efficacy of Sitagliptin, a Dipeptidyl Peptidase-4 Inhibitor, for Slowly Progressive Type 1 Diabetes (SPIDDM) in the Stage of Non-Insulin-Dependency: An Open-Label Randomized Controlled Pilot Trial (SPAN-S) ABSTRACT.INTRODUCTION: We tested the hypothesis that dipeptidyl peptidase-4 (DPP-4) inhibitors are effective in preserving the β-cell function for long-term periods in patients with slowly progressive type 1 diabetes (SPIDDM) or latent autoimmune diabetes in adults (LADA). ABSTRACT.METHODS: In the present open-label, randomized, controlled trial, 14 non-insulin-requiring diabetic patients with glutamic acid decarboxylase autoantibodies (GADAb) were randomly assigned to receive either sitagliptin (S group) or pioglitazone (P group). As a historical control, the Tokyo Study, in which non-insulin-dependent patients with SPIDDM were assigned to receive treatment by either insulin or sulfonylurea (SU), was used. ABSTRACT.RESULTS: On average, the ∑C-peptide values during the oral glucose tolerance test through the follow-up periods showed a nonsignificant increase in the S group (n = 6, n = 5 at 48 months) compared to the P group (n = 5, n = 2 at 48 months). In comparison to the data in the Tokyo Study, treatment by sitagliptin significantly influenced the longitudinal changes in the ∑C-peptide values with a more increased direction than insulin or SU, especially in patients with 48 months of follow-up (p = 0.014 and p = 0.007, respectively). Although the titers of GADAb were not significantly different between the S and P groups during the study, the change ratio of the GADAb titers from baseline was significantly inversely correlated with the change ratio of the ∑C-peptide values from baseline in the S group (p = 0.003); in particular, when the GADAb titers decreased from baseline, the ∑C-peptide values frequently increased. ABSTRACT.CONCLUSION: The present pilot trial suggests that treatment of SPIDDM/LADA by sitagliptin, a DPP-4 inhibitor, may be more effective in preserving the β-cell function than insulin treatment for at least 4 years, possibly through the immune modulatory effects of DPP-4 inhibitors. ABSTRACT.CLINICAL TRIAL REGISTRATION: Japanese Clinical Trials Registry UMIN000003693. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13300-017-0299-7) contains supplementary material, which is available to authorized users. BODY.INTRODUCTION: Type 1 diabetes (T1D) is caused by the autoimmune destruction of pancreatic β-cells, but it has been revealed that human T1D is substantially heterogeneous in its pathogenesis and natural history [1]. Slowly progressive insulin-dependent (type 1) diabetes mellitus (SPIDDM) [2, 3] or latent autoimmune diabetes in adults (LADA) [4–6] is characterized by the clinical phenotype of type 2 diabetes (T2D), such as adult onset, non-insulin independence at diagnosis, obesity and features of metabolic disorders, but has glutamic acid decarboxylase autoantibodies (GADAb) and/or islet cell antibodies (ICA); other antibodies such as insulinoma-associated antigen-2 autoantibodies (IA-2Ab) and insulin autoantibodies (IAA) are rather infrequent. Patients with SPIDDM/LADA frequently progress to an insulin-dependent state but with a slower rate of β-cell loss than in acute-onset T1D, probably because of the mild autoimmune attack. The prevalence of SPIDDM/LADA has been reported to range from 3% to 12% [7–10] of patients initially diagnosed with T2D in Western and other countries. The generally unsatisfactory results of recent prevention/intervention trials for T1D have highlighted its heterogeneity [11, 12], and it has therefore been proposed that SPIDDM/LADA is an attractive target for immune intervention approaches [13, 14], given its milder autoimmune process and the longer preservation of the β-cell function than in acute-onset T1D. We previously conducted the Tokyo Study in Japanese patients with SPIDDM [15], which showed that, compared with sulfonylurea (SU), treatment with insulin slowed progression to an insulin-dependent state. The effect of early insulin therapy on SPIDDM/LADA has been confirmed [16]. Subsequent to the Tokyo study, we focused on dipeptidyl peptidase-4 (DPP-4) inhibitors as a promising treatment option for SPIDDM, since several studies have reported the beneficial effects of DPP-4 inhibitors in the NOD mouse, a mouse model of T1D [17–19]. We conducted the "study for the prevention of autoimmune non-insulin-dependent diabetes mellitus with sitagliptin" (SPAN-S) to evaluate the effects of sitagliptin, the first DPP-4 inhibitor released in Japan, on the glycemic control, preservation of β-cell function and longitudinal changes in islet cell antibodies. Although several studies in patients with SPIDDM/LADA have reported favorable results with DPP-4 inhibitors since the launch of this study [20–22], we continued our study for a longer period to assess the long-term efficacy of sitagliptin for SPIDDM/LADA. BODY.METHODS.PATIENTS: This study was conducted in accordance with the International Conference on Harmonization Guidelines for Good Clinical Practice and the Declaration of Helsinki. The Saitama Medical University Ethics Committee approved this study, and the protocol was approved by the institutional review boards of the collaborating hospitals or the directors of the clinics. All of the procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964, as revised in 2013. Written informed consent was obtained from all patients for being included in the study. This was an open-label, prospective, randomized, controlled trial (Fig. 1). The protocol of this trial and supporting CONSORT checklist are available as Protocol S1 (original language), S2 (English translation) and Checklist S1. Participants were recruited from 10 June 2010, and follow-up data were collected until 23 February 2017. Fourteen SPIDDM patients were enrolled, and 11 of them were followed for at least 12 months (Fig. 1b). The inclusion criteria were as follows: (1) diabetes diagnosis and HbA1c level between 6.9% and 8.4%, (2) GADAb-positive, (3) fasting serum C-peptide level ≥1.0 ng/ml, (4) age 20–79 years of age, (5) no treatment with oral glucose-lowering drugs except metformin within 2 months before enrollment and (6) no history of insulin or GLP-1 receptor antagonist treatment. The exclusion criteria were: (1) women who were pregnant or planning on becoming pregnant, (2) renal dysfunction with serum creatinine ≥1.5 mg/dl for males and ≥1.3 mg/dl for females, or creatinine clearance ≤50 ml/min, and (3) patients who were deemed unsuitable for participation by their attending physician because of associated disease, complications or any other reasons. All subjects included in the study were Japanese.Fig. 1Trial design and participant flow. a Schematic illustration of the trial design. b Participant flow. Data are presented as n. α-GI alpha-glucosidase inhibitor, Lab laboratory test, M month, OGTT 75-g oral glucose tolerance test The eligible patients, who were recruited from the collaborating hospitals or clinics, were all randomly assigned using a centralized internet web system to receive either sitagliptin or pioglitazone based on minimization adjusting for age, sex, HbA1c level, GADAb titer and metformin use before entry. Because the inferiority of SU compared to insulin treatment had been revealed in the Tokyo study, pioglitazone, a thiazolidinedione (TZD) approved in Japan, was selected as the comparator. The efficacy of TZD on SPIDDM/LADA has been evaluated in several studies with conflicting results [23, 24]. In addition, from an ethical point of view, patients under treatment with metformin were allowed to be enrolled because a possible favorable effect of metformin on SPIDDM/LADA was suggested in our previous study [23]. BODY.METHODS.STUDY DESIGN: Patients were scheduled for visits at 2-month intervals for at least 12 months of follow-up. The dosage for the sitagliptin group was started at 50 mg once per day and that for the pioglitazone group was 15 mg once per day; if metformin was used at enrollment, the dosage was not changed. The target for glycemic control was set at an HbA1c level <7.0%. If glycemic control was not achieved during each visit, the regimens were intensified by either (1) increasing the dosage of sitagliptin or pioglitazone to 100 or 30 mg, respectively, and/or (2) adding metformin at 250–2250 mg or increasing the dosage up to 2250 mg and/or adding an alpha-glucosidase inhibitor (acarbose, voglibose or miglitol). Notably, SU agents were never used. The patients in both groups were switched to an insulin injection regimen when the HbA1c levels became ≥9.4% despite the above protocol of medication. BODY.METHODS.FOLLOW-UP ASSESSMENT AND ENDPOINT: The patients' body weight, blood pressure, plasma glucose (PG) and HbA1c levels were measured every 2 months. All of the patients received an annual 75-g oral glucose tolerance test (OGTT) without receiving their morning dose of medication, and their islet antibodies (GADAb, IA-2Ab and IAA) were determined annually. The primary endpoints were the HbA1c values, the proportion of patients whose HbA1c values increased to ≥9.4%, and the period from enrollment in which their HbA1c values increased to ≥9.4%. The secondary endpoints included the sum of the serum C-peptide levels at 0, 30, 60, 90 and 120 min during the OGTT (∑C-peptide), body mass index (BMI) and islet autoantibodies. BODY.METHODS.AUTOANTIBODIES: GADAb, IA-2Ab and IAA levels were determined in this study via radioimmunoassay (RSR Ltd., Cardiff, UK). With regard to the thyroid autoantibodies (TAb), thyroid peroxidase autoantibodies (TPOAb) and thyroglobulin autoantibodies (TGAb) were determined by an electrochemiluminescence immunoassay (Roche Diagnostics GmbH, Mannheim, Germany). Regarding the present GADAb assay, 10 U/ml corresponded to 180 U/ml (WHO units), the cutoff value that predicted further progression of β-cell dysfunction in the Tokyo study [25]. BODY.METHODS.SAFETY ASSESSMENTS: Any adverse events associated with the medications used in this study were evaluated and treated properly by physicians. Hypoglycemia was defined as a blood glucose level <70 mg/dl (3.9 mmol/l), irrespective of hypoglycemic symptoms. BODY.METHODS.SAMPLE SIZE: Based on our previous intervention trial (Tokyo Study) [15], in which 60 SPIDDM patients (30 each in the insulin and SU groups) were followed and the primary endpoint was the time at which the patient reached an insulin-dependent state (i.e., ∑C-peptide <4 ng/ml), and on the more preserved β-cell function in the inclusion criteria of this trial, which would show deleterious progression of the function more clearly, we anticipated that a total of 40 patients (20 each in the S and P groups) would be a sufficient population in our initial plan for the SPAN-S. Although the primary endpoint in the present study was defined by HbA1c values, worsening of HbA1c values was expected to correspond well to worsening of ∑C-peptide levels. BODY.METHODS.STATISTICAL ANALYSIS: Continuous data at baseline (n = 11) were first checked for their normality by the D'Agostino and Pearson test, revealing that the age, GADAb titer and change ratio of the GADAb titer from baseline significantly deviated from a normal distribution. Thus, the Mann-Whitney U test and Wilcoxon signed rank test were applied to these data for comparisons between groups and comparison from the baseline within the same group, respectively. An unpaired t test and paired t-test were used for duration, BMI, HbA1c, ∑PG, ∑C-peptide and change ratio of ∑C-peptide from baseline, respectively. Categorical variables were compared using Fisher's exact test. To evaluate the difference in the longitudinal ∑C-peptide data or their change ratios between the treatment groups, the interaction between time and treatment assignment at entry was examined using a repeated measures analysis of variance (ANOVA). The correlation between the change ratio of the GADAb titers and that of the ∑C-peptide values was assessed using a nonparametric Spearman's correlation coefficient. Statistical significance was defined as p < 0.05. The GraphPad Prism software program ver. 6 (GraphPad Software, Inc., La Jolla, CA, USA) was used for these tests. BODY.RESULTS.PATIENTS AND FOLLOW-UP: As shown in Fig. 1b, 14 patients were allocated: 7 were assigned sitagliptin (S group) and 7 pioglitazone (P group). Among them, six patients in the S group and five patients in the P group were followed for more than 1 year and analyzed. The baseline characteristics of these 11 patients are shown in Table 1. There were no significant differences between the two groups in age, sex, duration, metformin use before entry, BMI, HbA1c levels, GADAb titers or positivity of other autoantibodies (IA2Ab, IAA and TAb).Table 1Baseline characteristics of study patients with SPIDDMSitagliptin group (n = 6)Pioglitazone group (n = 5) p valueAge (years)56 ± 1657 ± 80.93Sex (male:female)2:42:30.85Duration of diabetes (years)4.3 ± 2.93.8 ± 4.10.81Metformin use before entry, n (%)5 (83)3 (60)0.48BMI (kg/m2)23.2 ± 6.223.9 ± 3.90.73HbA1c (%)7.5 ± 0.57.3 ± 0.40.59GADAb (U/ml)2.65 (1.6–250)8.4 (1.8–14,000)0.93IA-2Ab positive, n (%)2 (33)1 (20)0.70IAA positive, n (%)0 (0)0 (0)naTAb (TPOAb and/or TGAb) positive, n (%)2 (33)3 (60)0.46Data are n (%), mean ± SD or median (range) BMI body mass index, GADAb glutamic acid decarboxylase autoantibodies, IA-2Ab insulinoma-associated antigen-2 autoantibodies, IAA insulin autoantibodies, TAb thyroid autoantibodies, TPOAb thyroid peroxidase autoantibodies, TGAb thyroglobulin autoantibodies, na not applicable Six and five patients in the S group were followed for 24 and 48 months, respectively, while five, four and two patients in the P group were followed for 12, 24 and 48 months, respectively (Table S1). The intensification of the regimens after entry is shown in Table S2. Briefly, the sitagliptin dosage was not increased in any patients, and the pioglitazone dosage was increased in three patients (cases P2, P4 and P5). Metformin was added to two patients (case S5 and P5) among those who had not been treated by metformin at entry (cases S5, P1 and P5), and an alpha-glucosidase inhibitor (voglibose) was added to one patient (case S4). The metformin dosage was changed in four patients (cases S2, S4, P3 and P4). BODY.RESULTS.LONGITUDINAL CHANGES IN THE BMI, BLOOD PRESSURE AND HBA1C: The BMI (or body weight) of the S group did not significantly differ from that at baseline; the BMI (or body weight) of the P group showed an increasing trend from the value at enrollment, but the change was not significant (Table S1). The blood pressure of both groups did not significantly differ from that at baseline (data not shown). The HbA1c levels were decreased in both groups, and significant reductions from baseline were observed at several points in the S group: at 12 months in the patients with both 24 and 48 months of follow-up (n = 6 and n = 5, respectively) and at 36 months in those with 48 months of follow-up (Table S1). The worst HbA1c levels in the S and P groups were 8.2% and 8.2%, respectively (Table S2). The values of BMI (or body weight), blood pressure and HbA1c did not significantly differ between the two groups during the study. BODY.RESULTS.LONGITUDINAL CHANGES IN THE C-PEPTIDE RESPONSE TO THE OGTT: On average, the ∑C-peptide values throughout the follow-up periods were greater in the S group than in the P group, although a repeated-measure ANOVA showed that the influence of the treatment assignment on the longitudinal changes in the ∑C-peptide values was not significant in any of the follow-up periods (Table S1). In the six patients in the S group who were observed for at least 24 months, the ∑C-peptide values increased at 12 and 24 months (six patients) and decreased at 36 and 48 months (5 patients) but were still higher than those at baseline; furthermore, the increases at 12 months from baseline were significant (p = 0.049) in the patients with 12 and 24 months of follow-up (Table S1). In the five patients in the P group, of whom only two were followed more than 36 months, the ∑C-peptide values were mostly unchanged from baseline (Table S1 and Table S2). BODY.RESULTS.COMPARISON OF THE Β-CELL FUNCTION WITH HISTORICAL CONTROL: We compared our present data with those in the Tokyo Study as a historical control. In the Tokyo Study [15], the GADAb-positive non-insulin-requiring diabetic patients (SPIDDM), the same target subjects as in the present SPAN-S trial, were randomly assigned to receive either insulin (I group) or SU (SU group) and were followed up for a maximum of 60 months. The longitudinal changes in the ∑C-peptide values of the S and P groups were compared with those of the I and SU groups in the Tokyo Study. Of note, no significant differences were observed between the S or P group in the present study and the I or SU group in the Tokyo Study in the baseline characteristics of age, sex, duration, BMI, HbA1c levels, GADAb titers and ∑C-peptide values. Although the I group demonstrated better preservation of ∑C-peptide values than the SU group, as reported previously, the preservation of ∑C-peptide values in the S group in the present study appeared to be even better. As shown in Fig. 2a, a repeated-measures ANOVA revealed a significant interaction between time and treatment assignment (sitagliptin or insulin) as well as between time and treatment assignment (sitagliptin or SU) in the patients with 48 months of follow-up (p = 0.030 and p = 0.00004, respectively), indicating that treatment by sitagliptin significantly influenced the longitudinal changes of the C-peptide responses to the OGTT with a more increased direction than insulin and SU. Differences between the S group and the I and SU groups were further assessed based on the change ratios of the ∑C-peptide values from baseline (Fig. 2b). The significant superiority of treatment by sitagliptin to that by insulin or SU in the preservation of the ∑C-peptide values appeared to be more evident in the patients with 48 months of follow-up (p = 0.014 and p = 0.007, respectively), although the significant superiority of sitagliptin to insulin or SU was also observed in the patients with 24 and 36 months of follow-up (data not shown). As for the P group, no significant superiority of pioglitazone compared to insulin or SU was observed in a repeated-measures ANOVA (data not shown).Fig. 2Longitudinal changes in the C-peptide response to the OGTT for 48 months in the patients with sitagliptin treatment and in the Tokyo study. Patients with 48 months of follow-up are shown. The data are expressed as the mean ± SEM. In both the ∑C-peptide values (a) and change ratios from baseline (b), a repeated-measures ANOVA revealed a significant interaction between time and treatment assignment (sitagliptin or insulin; p = 0.030 and p = 0.014, respectively) as well as between time and treatment assignment (sitagliptin or SU; p = 0.00004 and p = 0.007, respectively) in the longitudinal changes BODY.RESULTS.INDIVIDUAL LONGITUDINAL ∑C-PEPTIDE VALUES, GADAB TITERS AND IA2AB TITERS OF THE PATIENTS IN THE S GROUP WITH 48 MONTHS OF FOLLOW-UP: We were able to observe the clinical course for 48 months in 5 patients of the S group (cases S1, S2, S3, S4 and S5 in Table S2). Individual longitudinal change ratios of their ∑C-peptide values, GADAb and IA-2Ab titers from baseline are shown in Fig. S1. Among these patients, four were heterozygous for the susceptible and neutral HLA haplotypes (S/N), and the other one (case S1) was homozygous for the neutral haplotype (N/N) [26]. Regarding TAb (TPOAb or TGAb), two patients (cases S3 and S5) were positive for TPOAb. The ∑C-peptide values of all five patients were preserved during the 48 months of follow-up. Except for cases S2 and S5 whose ∑C-peptide values remained almost the same throughout the study (S2) or slightly decreased from 24 to 48 months (S5), the values of the other three patients showed a tendency to increase from 12 to 24 months and then decreased from 36 to 48 months (Fig. S1). In comparison to cases S2, S3 and S5, who all had GADAb >10 U/ml and two of whom (S2 and S3) were IA2Ab-positive, the ∑C-peptide values of cases S1 and S4, who both showed GADAb <10 U/ml and were IA2Ab-negative, were increased to a greater degree from baseline, even at 48 months from entry. The relative increase from baseline was highest in case S1, who carried the HLA N/N genotype as well as both GADAb <10 U/ml and IA2Ab-negative status. BODY.RESULTS.GADAB TITERS: The titers of GADAb were not significantly different between the S and P groups at any time point in the present study (Table S1). However, as shown in Fig. 3, the change ratios of the GADAb titers from baseline were significantly inversely correlated with the change ratios of the ∑C-peptide values from baseline in the S group by a nonparametric analysis (Spearman's rank correlation coefficient = −0.600, p = 0.003). In particular, when the change ratios of the GADAb titers from baseline were negative, those of the ∑C-peptide values were mostly positive, i.e., when the GADAb titers decreased from baseline, the ∑C-peptide values frequently increased. In contrast, when the change ratios of the GADAb titers from baseline were positive, those of the ∑C-peptide values tended to show values around zero (Fig. 3). In the P group, a nonsignificant inverse correlation was observed (Spearman's rank correlation coefficient = −0.478, p = 0.101; Fig. S2).Fig. 3The correlation between the change ratios of the GADAb titers and of the ∑C-peptide values from baseline in the S group. Spearman correlation calculations revealed a significant inverse correlation between the change ratios of the GADAb titers and those of the ∑C-peptide values from baseline in the patients with sitagliptin treatment (Spearman's rank correlation coefficient = -0.600, p = 0.003) BODY.RESULTS.HYPOGLYCEMIA AND ADVERSE EVENTS: No drug-related adverse events, including hypoglycemia, were observed. BODY.DISCUSSION: In the present report, although the initial outcomes based on comparisons between sitagliptin and comparator (pioglitazone) were not evaluated enough—mostly because of the recruited sample size with low power—we presented our study as a pilot trial focusing on sitagliptin-treated patients with long-term (4 years) follow-up and further compared the effects of sitagliptin treatment on preserving the β-cell function with those of insulin or SU treatment in the patients enrolled in the Tokyo Study. The present findings suggested that treatment by sitagliptin for SPIDDM/LADA is effective with respect to the preservation of the β-cell function as well as glycemic control. On average, the ∑C-peptide values of the sitagliptin group were consistently higher than baseline for 48 months, with an apparent peak at 24 months. Compared to the data of the Tokyo Study as a historical control, in which SPIDDM patients were randomly assigned to treatment by either insulin or SU, the preservation of the β-cell function following treatment by sitagliptin was significantly superior to that achieved with insulin and SU therapy, with the differences being evident from 24 months (Fig. 2b). We should be cautious of bias associated with using a historical control. Nevertheless, as described in the results section, no significant differences in the baseline characteristics were observed between the present study and the Tokyo Study. Furthermore, in the Tokyo Study, several patients showed a low β-cell function at baseline (defined as ∑C-peptide <10 ng/ml), possibly because the enrolled patients in the Tokyo Study were recruited among those treated by SU, while none of the patients in the sitagliptin group of the present study showed such a low function. However, even when patients with a low β-cell function at baseline were excluded, the longitudinal changes in the ∑C-peptide values in patients with 48 months of follow-up were significantly higher in the sitagliptin group than in the insulin and SU groups (p = 0.019 and p = 0.014 by repeated-measures ANOVA, respectively; data not shown). The favorable effect of sitagliptin on the β-cell function may be because of the reduced glucotoxicity due to the improvement of glycemic control, as shown by the decline in the HbA1c values from baseline (Table S1). However, DPP-4 inhibitors may also have roles in the immune system. In the NOD mouse model of T1D, sitagliptin improved the rate of the islet graft survival partially by reducing the CD4 T cell numbers [19]. In another study, twice-daily treatment with NVP-DPP, a short-acting DPP-4 inhibitor, was able to reverse new-onset diabetes in NOD mice by increasing the number of CD4+CD25+FoxP3+ regulatory T cells [18]. Recently, Alonso et al. reported that a DPP-4 inhibitor, MK626, prevented T1D in NOD mice in association with changes in the CD8+ T effector memory lymphocyte subset [27]. The superiority of sitagliptin to insulin in the preservation of the β-cell function might come from the immune regulatory effects of DPP-4 inhibitors on the autoimmune process. Intriguingly, in the present study, we also observed that the change ratio of GADAb titers from baseline was significantly inversely correlated with that of ∑C-peptide in the S group (Fig. 3). A higher GADAb titer in SPIDDM is a marker of both an activated T cell response to β-cell destruction and a high risk for progression to insulin dependence [28, 29]. Therefore, the inverse correlation observed in the present study, especially the obvious association between the increase in the ∑C-peptide values from baseline and the decrease in the GADAb titers (Fig. 3), may reflect changes in the active autoimmune process of the patients influenced by the treatment with sitagliptin, which resulted in the alteration of the β-cell function in an inverse direction. As demonstrated in the individual longitudinal change ratios of their ∑C-peptide values from baseline (Fig. S1), the long-term preservation of the β-cell function (∑C-peptide values) of patients in the S group appeared to be associated with both GADAb <10 U/ml and IA2Ab-negative status (cases S1 and S4) or the absence of the HLA class II susceptibility haplotype (case S1). However, further validation in a larger population of patients will be required to determine the immunologic and genetic factors influencing the efficacy of sitagliptin on the β-cell function. Recently, based on the potential beneficial effects of DPP-4 inhibitors and the promising results in animal studies, DPP-4 inhibitors have been clinically tested in patients with T1D or SPIDDM/LADA. Favorable effects of sitagliptin were suggested in several cases with T1D [30] or SPIDDM/LADA [31, 32]; very recently, 4-year clinical remission of T1D in two young patients treated with sitagliptin and vitamin D3 were reported [33]. However, the results of clinical trials for T1D patients have been conflicting and appear to be inconclusive [34–36]. In contrast, the trials for patients with SPIDDM/LADA, in whom the effects of sitagliptin for 1 year [20], linagliptin for a maximum of 2 years [21] and saxagliptin for 6 months [22] have been evaluated, all reported favorable results concerning the preservation of the β-cell function, suggesting that DPP-4 inhibitors may be effective in SPIDDM/LADA patients as a class effect and that SPIDDM/LADA might be a good target in prevention/intervention trials. The present findings lend support to the previous reports on SPIDDM/LADA and further suggest that the beneficial immunologic effects of DPP-4 inhibitors may persist for a longer period in non-insulin-dependent patients with SPIDDM/LADA than was observed in previous studies. The major limitation of the present study is its small sample size, which substantially reduced the power of the study. The low number of enrolled patients may be due in part to the inclusion criteria of certain HbA1c values and no history of insulin treatment, since Japanese patients with SPIDDM under suboptimal glycemic control tend to be treated by insulin based on the results of the Tokyo Study. It should be noted that due to the small sample size, the present study lacks sufficient power to discriminate between the effects of sitagliptin and pioglitazone on SPIDDM and also insufficiently characterized the patients with SPIDDM/LADA suitable for treatment with DPP-4 inhibitors. However, the observed efficacy of sitagliptin treatment on glycemic control and the preservation of the β-cell function appeared to be notable, particularly in the two patients who had GADAb >10 U/ml and were IA2Ab-positive and heterozygous for susceptible and neutral HLA DRB1-DQB1 haplotypes [26] (Fig. S1B and S1C). Another limitation is that metformin use was not prohibited in the present study from an ethical point of view. Metformin was used to treat five of the six patients in the S group at baseline; during the study period, the dosage was increased in two patients and was added in one patient (Table S2). We previously reported that metformin may suppress the disease course of SPIDDM/LADA more effectively than pioglitazone [23], and we subsequently experienced a patient with SPIDDM/LADA whose β-cell function was maintained for 5 years with treatment of metformin alone [37]. Metformin increases the GLP-1 level [38], which regulates lymphocyte proliferation and the maintenance of peripheral regulatory T cells [39]; given that metformin was recently reported to improve gut microbiome flora in type 2 diabetes patients [40], metformin treatment for non-insulin-dependent SPIDDM might have exerted immunomodulatory effects through gut flora modification. As such, metformin may be effective in preserving the β-cell function in addition to its glucose-lowering effects and thus the usage of metformin may have influenced the favorable results in the S group. However, possible favorable effects of metformin on SPIDDM/LADA need to be verified in extensive clinical studies. Finally, although the use of an alpha-glucosidase inhibitor was also allowed in this trial, its influence would be minimal since only one patient received the medication and little is known of its role in SPIDDM/LADA. BODY.CONLUSION: The present study suggested that treatment of SPIDDM/LADA by sitagliptin, possibly as a class effect of DPP-4 inhibitors, may be more effective in the preservation of the β-cell function than insulin treatment for at least 4 years, possibly through the immune modulatory effects of DPP-4 inhibitors. We therefore believe that this class of drugs may be an attractive choice in the treatment of SPIDDM/LADA, especially in the stage of non-insulin-dependency. Further large-scale and long-term studies are necessary to clarify the details of the beneficial effects of DPP-4 inhibitors in SPIDDM/LADA patients and possibly in acute-onset T1D patients with a residual β-cell function. BODY.ELECTRONIC SUPPLEMENTARY MATERIAL: Below is the link to the electronic supplementary material. Supplementary material 1 (PDF 427 kb) Supplementary material 2 (PDF 306 kb) Supplementary material 3 (PDF 145 kb) Supplementary material 4 (PDF 1356 kb)

TITLE: Effect of integrated yoga therapy on pain, morning stiffness and anxiety in osteoarthritis of the knee joint: A randomized control study ABSTRACT.AIM:: To study the effect of integrated yoga on pain, morning stiffness and anxiety in osteoarthritis of knees. ABSTRACT.MATERIALS AND METHODS:: Two hundred and fifty participants with OA knees (35–80 years) were randomly assigned to yoga or control group. Both groups had transcutaneous electrical stimulation and ultrasound treatment followed by intervention (40 min) for two weeks with follow up for three months. The integrated yoga consisted of yogic loosening and strengthening practices, asanas, relaxation, pranayama and meditation. The control group had physiotherapy exercises. Assessments were done on 15th (post 1) and 90th day (post 2). ABSTRACT.RESULTS:: Resting pain (numerical rating scale) reduced better (P<0.001, Mann–Whitney U test) in yoga group (post 1=33.6% and post 2=71.8%) than control group (post 1=13.4% and post 2=37.5%). Morning stiffness decreased more (P<0.001) in yoga (post 1=68.6% and post 2=98.1%) than control group (post 1=38.6% and post 2=71.6%). State anxiety (STAI-1) reduced (P<0.001) by 35.5% (post 1) and 58.4% (post 2) in the yoga group and 15.6% (post 1) and 38.8% (post 2) in the control group; trait anxiety (STAI 2) reduced (P<0.001) better (post 1=34.6% and post 2=57.10%) in yoga than control group (post 1=14.12% and post 2=34.73%). Systolic blood pressure reduced (P<0.001) better in yoga group (post 1=−7.93% and post 2=−15.7%) than the control group (post 1=−1.8% and post 2=−3.8%). Diastolic blood pressure reduced (P<0.001) better in yoga group (post 1=−7.6% and post 2=−16.4%) than the control group (post 1=−2.1% and post 2=−5.0%). Pulse rate reduced (P<0.001) better in yoga group (post 1=−8.41% and post 2=−12.4%) than the control group (post 1=−5.1% and post 2=−7.1%). ABSTRACT.CONCLUSION:: Integrated approach of yoga therapy is better than physiotherapy exercises as an adjunct to transcutaneous electrical stimulation and ultrasound treatment in reducing pain, morning stiffness, state and trait anxiety, blood pressure and pulse rate in patients with OA knees. BODY.INTRODUCTION: Patients with osteoarthritis (OA) of knee are characterized primarily by articular cartilage degeneration and a secondary peri-articular bone response.[1] Worldwide, the prevalence rate of OA is 9.6% for men and 18% for women >60 years.[2] In India OA is the second most common rheumatologic problem and has a prevalence rate of 22 to 39.[3] Clinically it presents as pain in and around the joint, joint stiffness usually after rest, crepitation and restricted joint movements associated with muscle weakness.[4] The strongest risk factors for OA are age[5] and genetics.[6] Other risk factors include female gender, obesity, cigarette smoking, intra-articular fractures, chondrocalcinosis, crystals in joint fluid/cartilage, prolonged immobilization, joint hypermobility, instability, peripheral neuropathy, prolonged occupational or sports stress.[7] Chrousos and Gold observed that the development of age-related diseases occurs at different rates in different individuals and psychological distress appears to be an important factor promoting earlier onset of age-related diseases.[8] Aging being a strong risk factor for OA, psycho emotional stresses would also have a contributory role in initiation and aggravation of degenerative changes of OA. It has been suggested that the presence of depressive symptoms predicts future musculoskeletal disorders but not vice versa.[9] Linton S.J reviewed the psychological risk factors in back and neck pain which indicated a clear link between psychological variables with neck and back pain. Stress, distress or anxiety as well as mood and emotions, cognitive dysfunction and pain behavior were found to be significant factors.[10] Relieving pain and stiffness and improving physical function are the important goals of present day therapy for OA.[11] Non-opioid analgesics such as acetaminophen and non-steroidal anti-inflammatory drugs (NSAIDs), including cyclo-oxygenase II inhibitors have been the mainstay of drug treatment.[12] They reduce both pain and inflammation quite effectively, but their long-term use is associated with increased risk for gastrointestinal bleeding,[13] hypertension[14] congestive heart failure,[15] renal insufficiency and other adverse effects.[16] Before deciding on specific non-pharmacologic and pharmacologic options, it is important to understand the degree of the patient's symptoms, concerns, disability, and what the arthritis means to him or her.[17] Emotional responses are a component of any pain and hence the first step in osteoarthritis pain management is to respect pain, treat it intensively and address the psyche. Inadequately treated pain can lead to other serious co-morbidities, including depression, sleep disturbances, anxiety, fatigue, impaired ambulation, decreased socialization and poor quality of life.[18] Yoga is an ancient Indian science and way of life which talks about the origin of diseases.[19] The texts describe the mechanism of how the suppressed emotions (called adhis) percolate into the physical body manifesting as diseases (adhija vyahdis). These texts go on to describe the conceptual basis for reversibility of mind body disease (prasava-pratiprasava model) and offer the necessary principles to design specific postures, breathing and meditation techniques for different diseases.[20] Hence, yoga is fast advancing as an effective therapeutic tool in physical, psychological and psychosomatic disorders. Several studies point to the psychological benefits of yoga during health and disease. In a study on healthy adults, Vempati et al. showed that the yoga-based guided relaxation can reduce the sympathetic activity as measured by autonomic parameters, oxygen consumption and breath volume.[21] Medical and pre-medical students showed lesser anxiety and stress during an examination period after eight weeks of meditation.[22] The relaxation component of yoga has shown significant reduction in heart rate and blood pressure in different conditions.[23] Transcendental meditation (TM) was compared to muscle relaxation in its effectiveness in controlling stress with significantly better reduction in blood pressure in the TM group.[24] Yogitha et al. showed reduction in blood pressure, pulse rate and state anxiety levels in patients with common neck pain after integrated yoga.[25] Yoga therapy has shown significant reduction in pain, functional disability with improved strength, balance and gait, when used as an adjunct in the management of rheumatoid arthritis,[26] hand OA[27] and OA knees.[2829] Although we know that the psychological components such as depression and anxiety are important aspects to be addressed in pain management and yoga has shown its contributory role, there are no yoga studies that have looked at the relationship between anxiety and pain in patients with OA knees. Hence the present study was planned. BODY.MATERIALS AND METHODS: Patients with OA knees from the outpatient department of Ebnezar Orthopedic Center, Bengaluru were recruited for the study. A sample size of 250 was obtained on G power software by fixing the alpha at 0.05 powered at 0.8 and an effect size of 0.38 considering the mean and SD of an earlier study.[30] Two hundred and fifty patients, 76 males and 174 females in the age group of 35 to 80 years (yoga –59.56 ± 9.54) and (control –59.42 ± 10.66) with OA knees(one or both joints) satisfying the ACR Guidelines[31] for diagnosis were included. The inclusion criteria were (i) persistent pain for three months prior to recruitment, (ii) moderate to severe pain on walking, (iii) Kellegren and Lawrence[32] radiological grading of II to IV in X-rays taken within six months prior to entry, and (iv) those fully ambulant, literate and willing to participate in the study. Those with (i) grade I changes in X-ray (ii) acute knee pain, (iii) secondary osteoarthritis due to rheumatoid arthritis, gout, septic arthritis, tuberculosis, tumor, trauma or hemophilia were excluded. The study was approved by the institutional review board (IRB) and ethical committee of SVAYSA university. Signed informed consent was obtained from all the participants. BODY.MATERIALS AND METHODS.DESIGN: This was a prospective randomized parallel active control study on patients with OA knees in the age range of 35 to 80 years. After the initial screening, patients who fulfilled the entry criteria were assigned to either yoga or control group. A computer generated random number table (www.randomizer.org) was used for randomization. Numbered envelopes were used to conceal the sequence until the intervention was assigned. Both groups were given the conventional physiotherapy using transcutaneous electrical stimulation and ultrasound followed by supervised practices at the center for 40 min daily (6 days/week) for two weeks. The study group was taught integrated yoga and the control group the non-yogic physiotherapy exercises by certified therapists. After this, they were asked to practice only the supervised practices of 40 min daily at home for the next three months. Compliance was supervised by telephone calls once in three days and a weekly review class at the center. All patients were asked to tick the practices daily after the home practice in the diary provided for the purpose; at every visit their clinical progress and therapy received on the day were documented. All assessments were carried out on 1st , 15th and 90th days. BODY.MATERIALS AND METHODS.BLINDING AND MASKING: As this was an interventional study, double blinding was not possible. The answer sheets of the questionnaires were coded and analyzed only after the study was completed. The statistician who did the randomization and data analysis and the researcher who carried out the assessments were blinded to the treatment status of the subjects. BODY.MATERIALS AND METHODS.INTERVENTION FOR YOGA GROUP: The daily routine practiced at the center in the yoga group included 40 min of integrated yoga therapy practice after 20 min of physiotherapy with transcutaneous electrical stimulation and ultrasound for 15 days. The integrated yoga therapy practice included shithilikarana vyayamas (loosening practices), sakti vikasaka (strengthening practices) followed by yogasanas and relaxation techniques with devotional songs. Later patient was advised to continue the integrated yoga therapy practice of 40 min at home for the next three months. The concepts used to develop a specific module of an integrated approach to yoga therapy (IAYT) for knee pain were taken from the traditional yoga scriptures (Patanjali Yoga Sutras, Yoga vasishtha and Upanishads) that highlight a holistic life style for positive health at physical, mental, emotional and intellectual levels.[33] Yoga is defined as mastery over the modifications of mind (Chitta Vritti Nirodhah-definition of yoga by Patanjali). It helps to remove the unnecessary surges of neuromuscular activation resulting from heightened stress responses that may contribute to aging[34] The daily routine included a 1 h practice as follows [Table 1]: Table 1 Yoga module for osteoarthritis of knees Yogic sukshma vyayamas(loosening and strengthening practices): These are safe rhythmic repetitive stretching movements synchronized with breathing. These practices mobilize the joints and strengthen the periarticular muscles.Relaxation techniques: Three types of guided relaxation techniques were interspersed between the physical practices of sukshmavyayamas and asanas.Asanas (physical postures): Asanas are featured by effortless maintenance in the final posture by internal awareness. We selected asanas in standing and supine position that would relax and strengthen the knee joints.Pranayama: The practice of voluntary regulated breathing while the mind is directed to the flow of breath is called Pranayama. These practices promote autonomic balance through mastery over the mind.[35]Meditation: Patanjali defines meditation (dhyana) as effortless flow of a single thought in the mind without distractions (pratyaya ekataanata dhyanam). This has been shown to offer physiological benefits through alertful rest to the mind body complex.[36]Lectures and Counseling: Yogic concepts of health and disease, yama, niyama, bhakti yoga, Jnana yoga and karma yoga were presented in the theory classes. These sessions were aimed at understanding the need for life style change, weight management and prevent early aging by yogic self management of psychosocial stresses. BODY.MATERIALS AND METHODS.INTERVENTION FOR CONTROL GROUP: The daily routine practiced at the center in the control group included 40 min of a therapeutic exercise practice after 20 min of physiotherapy with transcutaneous electrical stimulation and ultrasound for 15 days. The therapeutic physical exercises (40 min) included loosening and strengthening practices for the hands, elbows, arms and shoulders followed by a brief period of rest and specific knee practices followed by supine rest with light music. Later patient was advised to continue the therapeutic exercise practice of 40 min at home for the next three months [Table 2]. Table 2 Control module for osteoarthritis of knees BODY.MATERIALS AND METHODS.MEASUREMENTS: a.Numerical pain rating scale (NRS): Pain at rest was recorded by the patient on numerical pain rating scale prepared for the purpose by drawing a 10 cm line in the center of a white sheet with '0' as nil pain and '10' as worst possible pain.[37–39] Separate sheets were used at each assessment time.Early morning stiffness in minutes as reported by the patients during clinical interview was documented.State and trait anxiety inventory (STAI-1 and STAI-2): STAI developed by Spielberger et al.,[40] consisting of 2 forms each comprising of 20 items rated on a four point scale (0-3) was used for assessing the anxiety levels. Form STAI-1 assesses the state anxiety which is defined as 'a transitory emotional state that varies in intensity, fluctuates over time and characterized by feelings of tension and apprehension and by heightened activity of the autonomic nervous system′. It evaluates how respondents feel 'right now' at this moment. Form STAI-2 evaluates trait anxiety, which is defined as 'a relatively stable individual predisposition to respond to situations perceived as threatening′. It assesses how the respondents feel most of the time. The scores for each of the forms range from 20 to 80, with high scores indicating presence of high levels of anxiety. We used both Y1 and Y2 in our study.Quek et al .,[41] have reported a high degree of internal consistency for STAI with Cronbach's alpha of 0.38 to 0.89 for each of the 40 items and 0.86 for the total scores. test-retest correlation coefficients for the 40 items score were highly significant. Intra-class correlation coefficient was also high (ICC=0.39 to 0.89).Blood pressure (BP): BP was measured using a mercury sphygmomanometer (Diamond Company) on day one, 15th day and on 90th day.Pulse rate (PR) - Pulse rate was counted manually for 1 min on first, 15th day and on 90 th days. It was ensured that the BP and pulse were recorded after completing the intervention in both groups at all points of time. BODY.MATERIALS AND METHODS.STATISTICAL METHODS: The data were analyzed using SPSS Version 16. The base line values of the two groups were checked for normal distribution by using Shapiro-Wilk's test. The baseline values were not normally distributed. Hence Wilcoxon's signed ranks test and Mann –Whitney U test were used to compare means within and between the two groups respectively. Spearman's Rho test was used to observe the correlations between all variables at all three points in time (pre, 15th and 90th days). Figure 1 shows the trial profile. 7 patients dropped out in the yoga group and 8 in the control group. Table 3 denotes the demographic data. There was no significant difference between groups at baseline on all variables (P>0.05, Mann–Whitney test for pre values). Figure 1Trial profile Table 3 Demographic data BODY.RESULTS: Table 4 shows the results within yoga group and between the groups. Table 5 shows the results within control group and between the groups after 15th and 90th days. Table 4 Results within yoga group Table 5 Results within control group BODY.RESULTS.RESTING PAIN: Mann–Whitney U test showed a significant difference between and within groups in resting NRS (Wilcoxon's, P<0.001) after the intervention on 15th and 90th day with higher effect sizes in yoga than control group. BODY.RESULTS.EARLY MORNING STIFFNESS: There was a significant difference in early morning stiffness within groups (Wilcoxon's, P<0.001) and between groups (Mann–Whitney, P<0.001) after the intervention at both points in time with higher effect sizes in yoga than control group. BODY.RESULTS.STATE AND TRAIT ANXIETY SCORES: There was better reduction in both state and trait anxiety scores (Wilcoxon's, P<0.001) and between groups (Mann–Whitney, P<0.001) in the yoga group with significant differences within and between yoga and control groups at two weeks and three months. BODY.RESULTS.SYSTOLIC AND DIASTOLIC BLOOD PRESSURE: There was significant difference within (Wilcoxon's, P<0.001) and between groups (Mann–Whitney, P<0.001) in the systolic and diastolic blood pressure in the two groups with better reduction in yoga group. BODY.RESULTS.PULSE RATE: There was significant reduction in both groups with (Wilcoxon's, P<0.001) and between groups (Mann–Whitney, P<0.001) significantly better reduction in yoga than control group at 15th and 90 th days. BODY.RESULTS.CORRELATIONS: There was a significant positive correlation (Spearman's Rho test) between early morning stiffness and pain at 2 weeks (P<0.001, r=0.35). Pulse rate also showed positive correlation with pain (P=0.05, r=0.18) at 2 weeks. State anxiety (STAI-1) was positively correlated with pain (P<0.001, r=0.34) at 90th day; Trait anxiety also showed a positive correlation with pain (P=–0.013. r=0.23) at 90 days. BODY.DISCUSSION: This randomized two armed parallel control trial on 250 patients with osteoarthritis of knees of both genders (F=175) in the age range of 35 to 80 years showed significantly better improvement in yoga than control group on all variables (P<0.001, Mann–Whitney) including resting pain, early morning stiffness, state and trait anxiety scores, blood pressure and pulse rate. BODY.DISCUSSION.RESTING PAIN: In a pilot study on OA of knees, Kolasinski et al.,[28] used a specific sequence of asanas based on the teachings of Iyengar for eight weeks. They measured only the pain and physical functions by WOMAC with a significant reduction (P=0.04) in pain by 46.7%. In another pilot study on yoga for OA knees, Ranjita et al.,[42] used a set of integrated yoga therapy program in a non-residential camp set up for one week without any physiotherapy intervention. She showed a 40% reduction in resting pain after yoga. In our study, we added yoga after the standard physiotherapy which showed a reduction in resting pain scores by 33.6 and 71.8% after 15 and 90 days respectively. Looking at the degree of changes in all the three yoga studies which is similar (37-47%), we may speculate that yoga is beneficial when used with or without a session of physiotherapy. BODY.DISCUSSION.EARLY MORNING STIFFNESS: Haslock et al.,[26] showed the beneficial effects of specific integrated yoga practices in patients with rheumatoid arthritis who had secondary OA in several joints. They observed better increase in hand grip strength (63%, left, 66% right) in yoga group than non yoga controls (8% left and 5% right) indicating reduced stiffness. Our study showed a reduction in early morning stiffness scores by 69 and 98% after 15 and 90 days respectively. None of the other yoga studies have noted morning stiffness as an outcome variable. BODY.DISCUSSION.STATE TRAIT ANXIETY: There was better reduction in state and trait anxiety scores in the yoga group with significant differences within and between yoga and control groups at two weeks and three months. In our earlier study on patients with chronic neck pain,[25] we had observed significant 19.3% reduction in STAI-1 scores as compared to 8.2% in control group within 10 days of intervention. In the present study, we used both state and trait anxiety measures since this was a long-term follow up of three months. The reduction in anxiety scores after yoga in both STAI-1 (36% -post 1 and 58% -post 2) and STAI-2 scores (post 1-35% and post 2-57%) were much higher in these patients with chronic knee pain as compared to those with neck pain. BODY.DISCUSSION.BLOOD PRESSURE AND PULSE RATE: In a randomized controlled study in patients with chronic neck pain, we observed a reduction in both systolic and diastolic blood pressure by 16% after ten days' of add -on IAYT for neck pain.[25] In the present study also, we observed similar reduction of 16% in both systolic and diastolic BP after 90 days of intervention. The pulse rate reduced by 10% in the chronic neck pain study which is similar to the present study with 12.4% reduction after 90 days of IAYT for knee pain. A significant correlation observed in this study between pain and early morning stiffness after yoga points to the global improvement in the patient's condition. Again, a significant correlation between pulse rate and anxiety with pain shows the mind body interaction.[43] We know that there exists an etiological relationship between an aging disease such as OA and life style, obesity and stress.[5] Whether this positive correlation has an etiologically predictable relationship needs to be studied in future studies. There are some studies that have looked at anxiety and autonomic variables in normal healthy volunteers after yoga for promotion of positive heath. Raghuraj et al.,[44] observed significant reduction in heart rate variability and blood pressure immediately after 20 min practice of alternate nostril breathing (nadishuddhi pranayama). In a study by Vempati et al.,[21] in healthy adults, the yoga-based guided relaxation was shown to reduce the sympathetic activity as measured by reduced heart rate and skin conductance, oxygen consumption and breath volume. Reduction in heart rate has also been observed after meditation.[3645] The immediate effect of a 30 min practice of a meditation technique called cyclic meditation on STAI 1 was measured in normal healthy volunteers, which showed significantly better reduction in state anxiety after the cyclic meditation session as compared to a session of supine rest. Studies on different types of meditation have consistently shown increased mental alertness even while the patients are physiologically relaxed. These autonomic changes of reduced sympathetic arousal, reduced anxiety and alertful rest points to stress reduction after yoga both in health and disease which seems to be the inner healer.[36] BODY.DISCUSSION.MECHANISMS: The experience of pain in OA patients is not only due to activation of sensory nociceptive fibers in the arthritic joint but it is compounded by other factors such as affective, behavioral and cognitive factors.[17] Stress reducing effect of yoga seems to be a major mechanism of its efficacy in pain management in patients with OA knees.[46] The multi-factorial approach of yoga includes not only physical practices (asanas) but also has the components of breathing (pranayama), meditation (dharana and dhyana), introspective intellectual (jnana yoga) and emotional (bhakti yoga) practices. These practices help in bringing about mastery over the modifications of the mind (chittavritti nirodhah) which is the definition of yoga according to Patanjali.[21] This may contribute to a consistent change in behavior and life style that can reduce anxiety and the resultant effect on pain reduction. Baser et al.[47] have shown the association between cognitive behavioral therapy (CBT) with physical, psychological and social well being. The concepts in philosophy of yoga are similar to those of CBT which says that chronic pain is not simply a neurophysiologic state but is influenced by the way a person sees the world and attributes meaning to the events. Lip chick et al.,[48] showed that the increased sense of personal control over pain following a pain management program of CBT was accompanied by a reduction in negativity. Thus the present study gives evidence to the relationship between reduction in pain, anxiety and sympathetic tone after yoga in patients with OA knees. This may offer preliminary evidence to the reversibility theory of yoga in a degenerative disease. BODY.DISCUSSION.STRENGTHS OF THE STUDY: Good sample size, randomized control design, active supervised intervention for the control group for the same duration as the experimental group and follow up for three months with good compliance (6% dropout) are the strengths of this study. The result of this study that has shown marked differences between groups on all variables offers strong evidence for incorporating this module of IAYT for OA knees by the clinicians. BODY.DISCUSSION.LIMITATIONS OF THE STUDY: The study was on a selected group who presented to a specialty orthopedic center and hence not generalizable. BODY.DISCUSSION.SUGGESTIONS FOR FUTURE WORK: A longer follow up of ≥12 months is necessary to check the long-term efficacy and acceptability. Studies using MRI and biochemical variables may throw light on the mechanisms. BODY.CONCLUSIONS: Adjunctive program of integrated approach of yoga therapy for OA knees reduces 'rest pain′, early morning stiffness, anxiety, and blood pressure and pulse rate. Yoga offers a good value addition as a non-pharmacological intervention in management of OA knees.

TITLE: A randomized, double-blind, placebo-controlled, crossover trial of “on-demand” tramadol for treatment of premature ejaculation ABSTRACT.OBJECTIVES:: The objective of this study is to assess the dose-related effects of tramadol on a group of patients with premature ejaculation (PE). ABSTRACT.SUBJECTS AND METHODS:: During the period of months between June 2010 and July 2012, 180 PE patients presented to outpatient clinic of our hospital. Patients were randomized in a 1:1:1 fashion to receive different sequences of the three medications: placebo, 50 mg of tramadol and 100 mg of tramadol. Every patient received 10 doses of each medication for 2 months. Intra-vaginal ejaculatory latency time (IELT) was recorded in seconds initially and for each arm. Successful treatment of PE is defined if IELT exceeded 120 s. Side-effects of medications were reported. ABSTRACT.RESULTS:: Of patients enrolled, 125 (69.4%) continued the study. Patients' age range was 20-55 years with PE complaint of 1 to 10 years duration. Mean IELT was 72 at presentation, 82 for placebo, 150 for tramadol 50 mg, and 272 for tramadol 100 mg (P < 0.001 for all comparisons). PE was successfully treated in only 2.4% of patients with placebo, in contrast to 53.6% and 85.6% with 50 and 100 mg tramadol, respectively (P < 0.001 for all comparisons). On multivariate logistic regression analysis, baseline IELT was the only predictor of successful treatment of PE with both tramadol 50 mg (odds ratio [OR]: 1.05, 95% confidence interval [CI]: 1.03-1.07, P < 0.001) and tramadol 100 mg (OR: 1.07, 95% CI: 1.04-1.11, P < 0.001). Postmicturition dribble annoyed 12.8% of those who received 50 mg tramadol and 33.6% of those who received 100 mg tramadol (P < 0.001). Weak scanty ejaculation was the main complaint in 7.2% versus 21.6% of those using 50 and 100 mg tramadol, respectively (P = 0.002). Two patients discontinued tramadol 100 mg due to side-effects. ABSTRACT.CONCLUSION:: Tramadol hydrochloride exhibits a significant dose-related efficacy and side-effects over placebo for treatment of PE. BODY.INTRODUCTION: Premature ejaculation (PE) is the most common type of ejaculatory dysfunction with an unclear etiology.[1] However, its estimated prevalence has been controversial for decades due to the lack of a strict globalized definition, being reported to reach as high as 20-30% of adult males.[23] The International Society for Sexual Medicine (ISSM) currently defines PE as "ejaculation which always or nearly always occurs prior to or within 1 min of vaginal penetration; together with the inability to delay ejaculation on all/nearly all vaginal penetrations; and negative personal consequences, e.g. distress, bother, frustration and/or the avoidance of sexual intimacy".[4] The condition may be primary or acquired.[1] The problem doesn't only affect the male partner, but it has also a major impact on quality of life of their sexual partners. Several studies revealed that PE can lead to poor sexual satisfaction and increased ejaculation-related distress and inter-personal difficulties.[5678] Until recently, PE has been often attributed to a psychological rather than a physiological problem, and was managed by behavioral modification and psychotherapy. However, these lines are rarely successful on the long-term.[69] Several studies suggest that PE may be related partly to decreased serotonergic activity in the brain.[1] There are several lines of treatment for PE which include: topical agents, sprays, and systemic drugs, e.g. selective serotonin reuptake inhibitors (SSRIs), α-blockers, and phosphodiesterase-5 inhibitors.[11011] Dapoxetine, a short acting SSRIs, is the only currently approved drug for treatment of PE in some countries,[611] but it has not been yet available in our country at the time of the study. Several studies have shown that tramadol HCl oral therapy has yielded promising results in the treatment of PE.[11112131415161718192021222324] It is still not clearly understood how tramadol delays ejaculation; however, several pharmacological studies have proposed some actions as; acting as μ-opioid agonist by the drug or its M1 metabolite,[25] 5-hydroxytryptamine type 2C receptor antagonist,[26] 5-nicotinic acetylcholine receptor antagonist,[27] N-methyl-D-aspartate receptor antagonist,[28] M1/M3 muscarinic receptor antagonist[2930] and an inhibitor of serotonin/norepinephrine uptake.[31] Thus, the exact mechanism remains speculative, and it might be a combination of the fore mentioned mechanisms. The objectives of this work are to assess the dose-related effects of tramadol on the intra-vaginal ejaculatory latency time (IELT), ability to delay ejaculation and enjoyment of sexual intimacy, as well as to evaluate the side-effects and their frequency and tolerability in a group of patients with PE. BODY.SUBJECTS AND METHODS: This is a prospective double-blinded placebo-controlled crossover study that was conducted in the period from June 2010 to July 2012. A total of 180 patients (age range: 20-55 years) with PE for >1 year attending the outpatient clinic in our hospital were enrolled. Sample size calculation was carried out using Epi-infoTM, version 3.5 (CDC, 2008; Atlanta, GA, USA); a calculated sample of 105 or more was needed, with a P < 0.05 and 80% power to detect a difference of 90 s between mean IELT before and after therapy. The study was reviewed and approved by our Institutional Ethical Review Board. All of the consented participants were potent with no history of erectile dysfunction, and had a stable, regular (≥1 sexual attempts per week), single-partner heterosexual relationship for ≥ 1 year. Patients were instructed to stop any relevant medications 1 month before and throughout the study. A detailed history taking (including detailed medical, surgical, and sexual history) and a thorough clinical examination were done for all. Those with diabetes mellitus, hepatic/renal insufficiency, neurological/psychiatric disorder, or drug abuse were excluded. IELT was recorded in seconds by patients initially (for a month before starting the medications); those with mean baseline IELT >120 were excluded. Patients were coded and randomized in a 1:1:1 fashion to receive three different sequences of three medications; the first sequence was: 50 mg tramadol, placebo (multi vitamin tablets) and 100 mg tramadol; the second sequence was 100 mg tramadol, placebo and 50 mg tramadol; and the third sequence was: 100 mg tramadol, 50 mg tramadol and placebo. Every patient received 10 doses of each medication for 2 months. IELT was recorded throughout the 2 months treatment for each arm. The mean IELT at the end of each treatment arm was then calculated. Successful treatment of PE was defined by IELT >120 s together with the ability to control ejaculation and enjoying the sexual intimacy. Side-effects of medications were reported. Patients were instructed to take the medication orally, 2-3 h before intercourse for at least six successive intercourses. Patients used a stop-watch to record the baseline IELT (in seconds) before treatment and in every intercourse during the period of intake of the medications. Patients were instructed to record any encountered side-effects during the period of the study. The following data were collected prospectively and analyzed: patient age, educational level, history of premarital practice of masturbation, duration of marriage in years, frequency of intercourse, duration of PE in years, type of PE (primary or lifelong/secondary or acquired), IELT pre-/post-treatment and side-effects of medications. Statistical analysis and graphical illustrations were performed using IBM® SPSS© statistics V19.0, (SPSS 19 for Windows [SPSS, Inc., Chicago, IL]). Analysis included; the Chi-square test or Fisher's exact test for comparison of the categorical data and the Student's paired-samples t-test (values expressed as mean and standard deviation) or the Mann-Whitney U-test (values expressed as median, inter-quartile range) to compare the noncategorical data. A multivariate logistic regression analysis of all factors associated with PE was done to detect the predictors of successful tramadol therapy. P value was considered as significant when ≤0.05. BODY.RESULTS: Of the 180 initially enrolled patients, 125 (69.4%) continued the study, while 65 were lost to follow-up. The basic patient characteristics are listed in Table 1. Table 1 Demographic and characteristics of 125 patients with PE before and after therapy Mean IELT was 72 at baseline, 82 for placebo, 150 for tramadol 50 mg and 272 for tramadol 100 mg (P < 0.001 for all comparisons). PE was successfully treated in only 2.4% of patients with placebo, in contrast to 53.6% and 85.6% with 50 and 100 mg tramadol, respectively (P < 0.001 for all comparisons). Tramadol therapy was significantly more effective for those with baseline IELT 60-120 [Figure 1]. Figure 1Success rate of premature ejaculation treatment in relation to baseline intra-vaginal ejaculatory latency time among the three therapeutic arms; placebo (P=0.554), tramadol 50 mg (P<0.001) and tramadol 100 mg (P<0.001) On univariate analysis; factors significantly associated with successful tramadol 50 mg treatment of PE were: baseline IELT, primary PE and sexual intercourse more than twice per week. Baseline IELT was the only factor significantly associated with successful resolution of PE using tramadol 100 mg [Table 2]. On multivariate logistic regression analysis, baseline IELT was the only predictor of successful treatment of PE with both tramadol 50 mg (odds ratio [OR]: 1.05, 95% confidence interval [CI]: 1.03-1.07, P < 0.001) and tramadol 100 mg (OR: 1.07, 95% CI: 1.04-1.11, P < 0.001). Table 2 Factors associated with successful treatment of 125 patients with PE using 50 and 100 mg tramadol doses Among the 125 patients included in the study, adverse events were noted in 3.2% with placebo, 21.6% with tramadol 50 mg and 45.6% with tramadol 100 mg (P < 0.001). A summary of the patient-reported reversible side-effects with tramadol 50 mg and tramadol 100 mg doses are shown in Table 3. Two patients discontinued tramadol 100 mg temporarily after the third and fifth dose, respectively due to severe nausea and vomiting. No withdrawal symptoms were noted in any. Table 3 Side-effects noted with tramadol 50 mg and tramadol 100 mg doses in 125 patients with PE BODY.DISCUSSION: Premature ejaculation is a prevalent male sexual dysfunction, which has a significant influence on male sexual practice. Historically, the first report on this condition dates back to 1887 when gross first described a patient complaint of rapid ejaculation. Since then, several treatment lines have been proposed for PE, but none has been the ultimate remedy.[5] In addition to the recent definition of PE by ISSM, another definition of PE that is frequently utilized in literature is that of the Diagnostic and Statistical Manual of Mental Disorders-IV-TR; which did not use IELT.[32] The criteria we used for successful treatment of PE depended on both the fore mentioned definitions and included the IELT as well as ejaculation control and personal satisfaction. Tramadol is a potent centrally acting synthetic opioid analgesic. It was approved by the Food and Drug Administration (FDA) as an analgesic for the United States market with established safety over 30 years of human use.[19] It is suitable for on-demand use as it is completely absorbed after oral intake with peak concentration achieved within 95-110 min and a mean elimination half-life of about 6 h.[24] Its bioavailability is 70% after a single dose and approaching 100% with multiple dosing. It is metabolized in the liver and eliminated mainly in urine (90%) and in feces (10%).[33] Being an opioid, tramadol has a potential risk of drug dependence. Such drug abuse risk is lower with the on-demand use and required continuous administration of 400 mg for >3 months. The rate of tramadol addiction was reported by US FDA as low and almost seen in patients with previous substance abuses.[343536] There has been an increasing trend toward the use of tramadol to treat PE with promising results.[11112131415161718192021222324] The main possible two modes of action for treatment of PE are; binding to μ-opioid receptors and weak inhibition of reuptake of norepinephrine and serotonin.[19] In the current study, the aim was to evaluate both the effects and side-effects of oral tramadol HCl in two different (50 and 100 mg) doses versus placebo. All the patients included in this work have reported an evident ejaculation-related bother as well as a significant intercourse-related stress, and lack of sexual intimacy with a baseline IELT of <2 min. This cut-off limit was used by seven studies addressing the role of tramadol in PE, including ours [Table 4]. There is only one report using IELT cut-off of 1 min (as stated in the PE definition of ISSM).[18] Regardless of the IELT, PE is dependent on 2 other important criteria, namely the ejaculatory control and patient satisfaction, which should be targeted as well. Table 4 Outcomes of tramadol therapy for of PE in the literature Wu et al. conducted a comprehensive meta-analysis of literature (including seven publications) on this topic and reported a 3 min increase of IELT with tramadol HCl over placebo (mean difference 2.77 min; 95% CI: 1.12-4.47; P < 0.001).[14] Such finding is similar to our results with 100 mg tramadol doses. The reported outcome of tramadol therapy in PE is summarized in Table 4. This table includes 10 previously published studies in addition to ours. Among the seven studies of on-demand tramadol versus placebo; one study had similar results of on-demand tramadol for PE treatment when compared with continuous use.[15] Two studies reported tramadol versus paroxetine; showing no differences between the tramadol and paroxetine groups in IELT (mean difference –0.44; 95% CI: –5.07-4.18; P = 0.85).[142122] Another study compared on-demand tramadol with three other on-demand PE potential therapies (sildenafil, paroxetine, and local lidocaine gel); tramadol significantly prolonged IELT values, while sildenafil was significantly associated with better sexual satisfaction than the other drugs.[17] No study compared dapoxetine with tramadol. A reported clinical trial of on-demand dapoxetine showed promising results with moderate efficacy in the treatment of PE with relatively few side-effects (when compared to tramadol and other SSRIs); 1.7% (for 30 mg dose) and 5.1% (for 60 mg dose).[611] The number of participants in our study was relatively small, as they represent only the patients who accepted to participate and were fitting to the inclusion criteria of the study. However, it is still comparable or even larger than patient numbers reported in several single-center studies that discussed the same point.[182223] We made sure that all patients involved did not have any diseases nor were taking any medications that would modify the effect of therapy. Moreover, we tested both placebo and tramadol HCl (50/100 mg) on the same group of patients in a double blind way to avoid inter-personal as well as subjective variations. In both doses of tramadol (available in or country), it has proven its quantitative and subjective efficacy in the treatment of PE that is not attributed to a placebo-effect. We did not use a validated questionnaire neither for enrollment nor for assessment of the degree of satisfaction with treatment results as most of our patients were illiterate/of low education level. Many questionnaires for assessment of patient-reported outcome were used in the literature including; ejaculation control (AEC) and sexual satisfaction scores, Premature Ejaculation Profile, International Index of Erectile Function scores and Arabic Index of Premature Ejaculation score.[15161718192021222324] We believe that our definition of PE is a reliable objective as well as subjective measure of outcome impressions. The regression analysis has shown that the therapeutic effect was stable with no confounding effect noticed with any of age, duration of marriage, premarital masturbation history, occupational nature, educational level, and duration of PE. For those with base-line IELT <60 s, tramadol seems to be less effective. Despite the high frequency of some side-effects of tramadol (up to 37% in our study), the adverse events were minor, reversible and generally well-tolerated by the patients. This was indicated by the small percentage (1.6%) of patients who discontinued tramadol 100 mg temporarily due to side-effects. In a meta-analysis, tramadol had a significant more adverse events when compared with placebo (OR: 2.89; 95% CI: 1.88-4.43; P < 0.0001).[14] The reported side-effects of tramadol include; ED (1%), vertigo, dizziness, headache, drowsiness, and the common cold (<1% for each).[19] Other studies showed the predominance of nausea, sleepiness, and headache.[182324] Adverse events affected a wide range of patient ranging from 12.4% to 100%; mainly related to higher doses (>50 mg) and longer duration of therapy (>12 weeks) as well as personal/racial variations [Table 4]. The more palatable; tramadol orally disintegrating tablet had generally a lower rate of side-effects despite high doses (62 mg and 89 mg). No withdrawal symptoms were noted with on-demand tramadol use.[19] Both the therapeutic effects and side-effects of tramadol for treatment of PE were dose/duration dependent as obvious from previous results [Table 4]. Although in our study, we did a logistic regression model to predict successful therapy of PE, we have some limitations including; relatively small number of cases and lack of a validated questionnaire for assessment of patient/partner satisfaction. In conclusion, our study revealed that tramadol hydrochloride exhibits a significant dose-related advantage over placebo in the treatment of PE. Tramadol increased IELT as well as improve ejaculatory control and personal satisfaction. The only predictor of successful treatment of PE was the baseline IELT. On-demand tramadol 50 mg significantly improved PE with minor side-effects. For those with baseline IELT <60 s, tramadol 100 mg is usually needed. Despite the diversity of the side-effects, their impact on the patients can be fairly referred to as minor, tolerable, and reversible. The potential risk of drug dependence is the limiting factor for routine use of tramadol in PE therapy. Accordingly, larger-scale and longer-term studies are required to investigate the tolerability and safety of tramadol. In addition, future studies are needed to compare the safety and efficacy of tramadol HCl against other therapeutic lines namely; dapoxetine.

TITLE: Effect of Training with Whole Body Vibration on the Sitting Balance of Stroke Patients ABSTRACT: [Purpose] The purpose of this study was to determine the effects of task-oriented training with whole body vibration (WBV) on the sitting balance of stroke patients. [Subjects] The subjects were 30 stroke patients who were randomly divided into experimental (n1=15) and control (n2=15) groups. [Methods] Subjects in both groups received general training five times per week. Subjects in the experimental group practiced an additional task-oriented training program with WBV, which was performed for 15 minutes, five times per week, for four weeks. The center of pressure (COP) path length and average velocity were used to assess subjects static sitting balance, and the Modified Functional Reach Test (MFRT) was used to assess their dynamic sitting balance. The paired t-test was performed to test the significance of differences between before and after the intervention. The independent t-test was conducted to test the significance of differences between the groups. [Results] Following the intervention, the experimental group showed a significant change in MFRT. [Conclusion] The results of this study suggest that task-oriented training with WBV is feasible and efficacious for stroke patients. BODY.INTRODUCTION: Impaired sitting balance is a common problem for stroke patients1), yet a sitting position is essential for safe execution of a variety of movements2). Many studies have shown that sitting balance influences functional outcome after a stroke1). Recovery of sitting balance is therefore a therapeutic goal for most stroke patients3). Several interventions have been devised to improve balance, including task-oriented training4) and treadmill training5). Dean and Shepherd demonstrated that task-oriented training was effective at improving reach while sitting6). Recently, whole-body vibration (WBV) exercise has been developed as a new modality for physical therapy7). Previous studies have suggested that WBV exercise increases muscle strength and improves muscular performance and balance8, 9), and the positive effects of WBV on muscle performance should help to improve balance10, 11). WBV acts through repetitive sensorimotor stimulation and therapies with WBV have been conducted for elderly patients as well as patients with cerebral palsy, multiple sclerosis, and stroke12,13,14,15). Some authors have reported WBV training combined with other physical therapies improves balance16,17,18), and WBV was shown to positively influence the postural control and mobility of chronic hemiparetic stroke patients11). Recently, studies have emphasized the importance of sitting balance for stroke patients and have recommended various balance training methods for its improvement. However, physical therapies for improved sitting balance are underpinned by little empirical evidence19). Some studies have focused on the use of WBV, but no study has been conducted to verify the effects of WBV for stroke patients in the sitting position. Therefore, we investigated the effects of task-oriented training with WBV on the sitting balance of stroke patients. We hypothesized that a group of stroke patients performing task-oriented training with WBV would show greater improvement of sitting balance than a control group that performing task-oriented training alone. BODY.SUBJECTS AND METHODS: The subjects were chronic stroke inpatients of the D Rehabilitation hospital. The inclusion criteria were history and clinical presentation (hemiparesis) of stroke (>6 month post-event); ability to sit independently for at least 10 minutes; no participation in any balance training program during the previous six months; no orthopedic problems, such as a fracture, deformity, or severe osteoarthritis; and sufficient cognitive ability to participate in the training: Korean version of Mini-Mental State Examination (MMSE-K) scores of 21 or higher. The exclusion criteria were comorbidity or disability other than stroke, and an uncontrolled health condition for which vibration is contraindicated. Participation in the study was voluntary and the subjects fully understood the contents of this study. After providing an explanation of the study purpose, as well as the experimental method and processes, written informed consent to participation in the study was obtained from all the subjects. The study was approved by the Daejeon University institutional review board. Static sitting balance was evaluated using analysis of center of pressure (COP). Subjects sat, with their feet unsupported, on a Wii Balance Board (Nintendo, Kyoto, Japan) and were asked to keep their arms folded across their chests. Their thighs were kept parallel, with 75% of their length supported on the Wii Balance Board. Data were acquired at 100 Hz, and the mean value of three measurements collected over 30 seconds was used. COP total path length and average velocity were the outcome measures used in this study. Higher values indicate lower balance ability20). Dynamic sitting balance was assesed using the Modified Functional Reach Test (MFRT). This was performed using a level yardstick mounted on a wall at the height of each subject's acromion of the nonparetic side, while sitting on a chair with no back or arm rests. The subjects were seated with their hips, knees, and ankles positioned at 90° of flexion, with their feet positioned flat on the floor. Three measurements were made under each of the following conditions: (1) sitting with the nonparetic side near the wall and leaning forward, (2) sitting with the back to the wall and leaning toward the nonparetic side, and (3) sitting with the back to the wall and leaning toward the paretic side. The subjects were instructed to lean as far as possible in each direction without rotating or touching the wall. The furthest position of the fifth finger was marked on the yardstick. If the patient could not raise the paretic arm, the distance covered by the acromion during leaning was used. Thirty people fulfilled the inclusion criteria and voluntarily agreed to participate in this study. The participants were randomly assigned to the experimental group (n1 = 15) or the control group (n2 = 15). Both groups received conventional therapy. The intervention comprised 4 weeks of inpatient treatment. Both groups performed one session (15 minutes) a day (5 days/week) of task-oriented training in the sitting position. The experimental group received WBV during task-oriented training (Galileo Pro; Novotec Medical GmbH, Germany). The control group performed only task-oriented training. The WBV frequency (15–22 Hz) and amplitude (0–5.8 mm) were adjusted relative to subjects' physical abilities10). Participants were instructed not to hold onto the table during training. The four exercise tasks were (1) sitting alone at a table and correcting body alignment; (2) reaching in different directions for objects located beyond arm's length using the nonparetic side; (3) reaching in different directions for objects located beyond arm's length using the paretic side; and (4) a bilateral reaching task, such as throwing a ball, lifting a box, and inserting a ring. Each exercise session was 15 minutes in duration, and subjects practiced each of the four tasks for 3 minutes with a 1-minute rest in between. Investigators supervised each session and were responsible for ensuring that the amount and intensity of the exercise at each exercise station was graded to each subject's level of functioning. All collected data were analyzed using SPSS version 18.0. The independent t-test was used to compare differences between group means and changes in values, and the paired t-test was used to test differences in continuous variables within groups. Differences between categorical variables were analyzed using the χ2 test. Statistical significance was accepted for values of p<0.05. BODY.RESULTS: All patients completed the intervention and assessments. There were no significant differences in gender, paretic side, age, weight, height or duration of onset between the groups (Table 1Table 1.General characteristics of subjectsExperimental group(n1=15)Control group(n2=15)GenderMale/Female9/67/8Paretic sideRight/Left6/95/10Age (year)62.8±9.0*65.1±15.7Weight (kg)63.3±6.258.0±9.0Height (cm)162.7±6.2161.1±6.5Duration (month)13.0±5.412.6±5.7*Mean±SD). Differences in static sitting balance are presented in Table 2Table 2.Comparison of COP results between pre- and post-testExperimental group(n1=15)Control group(n2=15)VA (cm/s)Pre3.0±0.2*3.0±0.3Post2.9±0.23.0±0.3Change−0.6±0.10.1±0.1§TL (cm)Pre89.5±6.989.4±9.3Post87.8±6.991.1±8.7Change−1.7±3.21.6±4.1§VA: Velocity Average of COP, TL: Total path Length of COP. *Mean±SD. §p<0.05. There were no significant differences in the average velocity and total path length of COP sway between the groups. Differences in dynamic sitting balance are presented in Table 3Table 3.Comparison of MFRT results between pre- and post-testExperimental group(n1=15)Control group(n2=15)MFRT-A (cm)Pre26.0±9.9*21.0±11.4Post32.1±8.023.5±10.7§Change6.1±5.92.4±4.2MFRT-N (cm)Pre10.9±5.413.6±4.9Post16.6±5.715.5±6.2Change5.7±4.11.9±4.8§MFRT-P (cm)Pre9.9±4.410.7±5.6Post13.2±3.912.1±5.6Change3.4±3.41.3±4.2MFRT-A: Modified Functional Reach Test-Anterior reach. MFRT-N: Modified Functional Reach Test-Non-paretic reach. MFRT-P: Modified Functional Reach Test-Paretic reach. *Mean±SD. §p<0.05. After intervention, anterior, non-paretic and paretic reach were significantly higher in the experimental group (p<0.05). After the intervention, anterior reach was significantly higher (p<0.05) in the control group. There were no significant differences in non-paretic and paretic reach in the control group. Differences in total length of COP between pre- and post-intervention differed significantly between the two groups (p<0.05). BODY.DISCUSSION: In this study we investigated the effect of task-oriented training with WBV on the sitting balance of stroke patients. Average velocity and total path length of COP sway were used to evaluate the static balance of the sitting position. Force platforms have previously been used to investigate the balance control of unsupported sitting of post-stroke individuals through analysis of the COP sway21, 22). Previous research reported that a leg exercise with WBV group and a leg exercise group without WBV showed similar improvements in the average velocity of COP in a standing position23) with no significant difference between them. These results were similar to our present results, as we found no significant differences in average velocity or total path length of COP sway between the groups. It is possible that the training was not sufficiently intense or long enough to elicit improvements in the subjects; it is also possible that the sensitivity of the assessment method was insufficient. In the current study, the MFRT was used to evaluate improvements in dynamic balance in the sitting position. The average anterior and lateral reach distances of adults between 60 and 70 years old are 346 mm and 206 mm, respectively24). In our study, the anterior, nonparetic, and paretic reach of both groups were below the normal averages at pretest, indicating that both groups had impaired sitting balance. However, at posttest, the experimental group showed significant improvements (p<0.05) in the anterior, nonparetic, and paretic reach, demonstrating that task-oriented training with WBV in a sitting position is a useful intervention for improving the dynamic sitting balance of stroke patients. Vibration is a useful method for stimulating proprioception and is capable of long-lasting postural improvement25). Other effects of vibration include modification of correcting movements and increased postural sway. The applications of vibration and predictability of stimuli can influence the physiologic effects26). The control group showed significant improvements (p<0.05) only in anterior reach. This demonstrates that task-oriented training is a useful intervention for improving anterior reach while sitting. In the experimental group, the horizontal sinusoidal wave of WBV effectively improved side reach. Our study suggests that task-oriented training with WBV in the sitting position has beneficial effects on some aspects of sitting balance of chronic stroke patients. We anticipate that this training method will be used in physical therapy at stroke patient care centers as it is an effective form of training for balance functions. Further research is needed with larger numbers of subjects to confirm and generalize our present findings.

TITLE: The Effect of Baduanjin on Promoting the Physical Fitness and Health of Adults ABSTRACT: The purpose of study was to assess the efficacy of a 16-week Baduanjin qigong training intervention in promoting physical fitness and health for adults. An experimental design was adopted, and subjects were assigned randomly into an experimental group (n = 55) and a control group (n = 55). In the intervention group comprised of adults, there were no significant variations in blood glucose, blood lipid, blood pressure, heart rate variability, and vital capacity indices. The body weight and body mass index (BMI) dropped in the intervention group. Compared with the control group, the skinfold thicknesses decreased at lower corner of scapula, triceps brachii, and abdomen, with a statistical significance (P < 0.001; P = 0.005; P = 0.003). By comparing the physical fitness indices, it was found that the increase of the results of sit-and-reach test in the intervention group had statistical significance (P = 0.001). In conclusion, it was found by our trial that Baduanjin exercise could significantly improve the physical flexibility and subcutaneous adipose accumulation in the healthy adults. BODY.1. INTRODUCTION: Baduanjin is a qigong which has more than one thousand years of history in China. It is a typical exercise to promote health and the Chinese Health Qigong Association has recommended its generalized application in the community [1]. Baduanjin exercise studied in this paper is compiled by the Chinese Health Qigong Association. This exercise is based on the common rules of Baduanjin exercise, combined with the holistic view and the theory of Qi in traditional Chinese medicine [2, 3]. When practicing Baduanjin exercise, the body maintains a steady gravity center. With the lumbar spine as the axis, the movement of the four limbs is driven. The muscle tension and relaxation are alternating at different parts of the body. In practicing, the mind, body, and breath are required to be smooth and unstrained [3, 4]. Mind in qigong refers to one's mental state and normal consciousness and bodily movements guided by the mind and thoughts. The two form an interactive and interpromotional integrity, characterized by harmony and symmetry shown in all and between every two movements. Baduanjin is noted for its smooth comfortable postures and its movements are performed with profound inner strength. With a concentrated mind followed by a vigorous body, it naturally combines firmness with gentleness, and exercises through the interplay of empty and full [3]. The control of these elements follows a certain sequence. The continuity and mastery of the movement required in practicing Baduanjin exercise cannot be fully achieved unless after a certain period of training. Therefore, the practicing of Baduanjin exercise requires persistence, gradual improvement, and reasonable arrangement of time [3]. As China's traditional form of sport, Baduanjin exercise has gained a wide popularity in China [1, 5]. However, the health promoting value of Baduanjin exercise has not been fully recognized worldwide as that of Taijiquan [6–8]. The positive effects of Baduanjin exercise as a traditional Chinese qigong are yet to be confirmed sufficiently through the randomized controlled trials [4]. Relevant studies at home and abroad are quite limited. Highlighting the movement rhythm and a good control of the movement intensity, Baduanjin can be used as an auxiliary therapy for knee joint arthritis [1, 9]. Due to the requirement for the coordination between the mind and the body in Baduanjin exercise, psychological assessment has been conducted after the intervention [10]. Baduanjin exercise is widely accepted between middle-aged and elderly populations with abnormal blood fats or associated metabolic diseases. For this reason, the effect of Baduanjin exercise in the intervention for abnormal blood fats has been extensively studied [5, 11–13]. Baduanjin is a safe aerobic exercise which features a movement intensity and format in line with the theories of kinetics and physiology. It is different from other types of aerobic exercise in that the practitioners are required to reach the coordination between the mind and the body [1]. A greater emphasis is laid on exercising the body and cultivating the disposition. So far, few studies have been devoted to the health promoting effect of Baduanjin exercise among healthy adults. Most studies concerning the health promoting effect among patients with chronic diseases are limited to the observation of psychological indices and blood lipid indices [5, 10]. But when evaluating a traditional form of qigong in China from the holistic view, the benefits of Baduanjin exercise cannot be fully reflected by a single index. According to the characteristics of Baduanjin exercise, we designed a pilot randomized controlled trial, in which the effects of Baduanjin exercise on the physical fitness and health were analyzed. The tested indices included the indices of psychology, blood lipids, body shape, physical functions, and physical fitness. The trial was designed with the intention of fully reflecting the health promoting effect of Baduanjin exercise on healthy adults, who practiced Baduanjin for 16 weeks. BODY.2. MATERIALS AND METHODS.2.1. PARTICIPANTS: From October 2012 to February 2013, the subjects were recruited from the surrounding areas of Wuhan Sports University. The subjects were aged from 20 to 59 years and were local residents. They agreed to take part in the tests designed by the Institute and practice Baduanjin for 16 weeks. The subjects were physically healthy, with no cardiovascular diseases, diabetes, or abnormal glucose tolerance or other acute or chronic diseases that might affect the sports activity. These subjects had no regular physical exercises of moderate intensity. This study was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. Ethical approval was obtained from the Ethics Committee of the China Institute of Sport Science. The researcher explained the study purpose, procedure, and right of free withdrawal to all participants willing to participate in this study. Informed consent was also obtained. BODY.2. MATERIALS AND METHODS.2.2. TRIAL DESIGN: In this pilot and randomized controlled trial, several evaluation indices characterizing different aspects of physical fitness and health were selected. As no previous study is available, the sample size was estimated based on the main effect indicators and the measurement data were collected in the preliminary experiment of fifteen volunteers before the trial (Table 1). When Type I error was 5% (α = 0.05), Type II error was 80% (β = 0.20), respectively, to detect a 15% mean difference at end of intervention. The sample size required was calculated according to the formula n 1 = n 2 = 2(Z α/2 + Z β)2 δ 2/σ 2 (Table 1). Therefore, the intervention group and the control group had 55 subjects, respectively, with the dropout rate controlled at 20%. Randomization was performed by computer-generated random allocation sequence by simple randomization and undertaken by a research assistant, from China Institute of Sport Science, not involved in recruitment to ensure allocation concealment. The random assignment occurred after completion of baseline questionnaires and assessment. The search assistant who collected and entered study data remained blinded to group allocation throughout the study. BODY.2. MATERIALS AND METHODS.2.3. INTERVENTION GROUP: The subjects in the intervention group undertook the learning of Baduanjin exercise two weeks prior to the intervention. The training was under the guidance of professional coach. The learning status of the subjects was evaluated before the intervention began. The 55 subjects that could practice the whole Baduanjin exercise were enrolled in the intervention program: the subjects practiced Baduanjin exercise 3 times or above each week, 30–60 minutes each time. The subjects were required to complete the program in the fixed site and in a collective manner, with special personnel recording the attendance and providing guidance. BODY.2. MATERIALS AND METHODS.2.4. DESCRIPTIONS OF THE BADUANJIN ROUTINES: The whole set of Baduanjin exercise consists of 9 postures: (1) ready position, (2) holding the hands high with palms up to regulate the internal organs, (3) posing like an archer shooting on left and right sides, (4) holding one arm aloft to regulate the functions of the spleen and stomach, (5) looking backwards to prevent sickness and strain, (6) swinging the head and lowering the body to relieve stress, (7) moving the hands down the back and legs and touching the feet to strengthen the kidneys, (8) thrusting the fists and making the eyes glare to enhance strength, and (9) raising and lowering the heels to cure diseases [3]. BODY.2. MATERIALS AND METHODS.2.5. CONTROL GROUP: The subjects in the control group were instructed not to take part in any exercise classes or regular physical exercise during the study period. They were informed to maintain their original lifestyle in the intervention period. Sixteen weeks later, all participants in the control group got the 2-week Baduanjin exercise training under the guidance of professional coach. BODY.2. MATERIALS AND METHODS.2.6. STUDY MEASURES: The measurements and survey were performed twice for all subjects, as the baseline and end line survey, respectively. Before the subjects entered into the cohort, they were already informed of the contents of measurement and signed the informed consent. The research assistants in Wuhan Institute of Physical Education who collected and entered study data remained blinded to group allocation throughout the study. Measurement of four blood lipid indices was as follows. The blood samples were collected before meal in the morning. The indices measured included serum total cholesterol (TC), serum triglyceride (TG), serum low-density lipoprotein (LDL), and serum high-density lipoprotein (HDL). The detection kits were provided by Roche; the equipment was Automatic Biochemistry Analyzer (7020, Hitachi, Japan). Measurement of body shape was as follows: the indices of body shape measured included body height, weight, chest circumference, waist circumference, hip circumference, skinfold thickness of inferior angle of the scapula, skinfold thickness of abdomen, and skinfold thickness of triceps brachii. Measurement of physical fitness included the following. (1) Sit-and-reach test (SR): the subjects sat on the cushion, with two legs stretching forward; the soles of the feet were pressed together and placed flat against the baffle of the analyzer, with the tiptoes naturally apart. During the test, the subjects had hands closed together, with palm reaching downwards. The knee joints stretched straight, and the body bent forward. The tip of the middle finger pushed the vernier on the analyzer forward. (2) Eye-closed and single-legged standing test (ES): the subjects were required to close the eyes and lift up one foot. When the supporting foot moved or the lifted foot touched the ground, the test was over, and the time of single-legged standing was recorded. (3) Measurement method of aerobic endurance: the increasing exercise load method was used. The subjects were required to maintain a 60 rpm rotational speed on the bicycle ergometer (ergoselect 100, Ergoline, Germany). Both the initial load and increasing rate were 25 W for males and 20 W for females. The load of each level lasted for 2 min. After 2 min, the load of next level was applied without rest. The load was increased continuously at this rate until the subjects were exhausted. The gas analyzer (MetaMax 3B, Cortex Biophysik, Germany) was used in the whole process for acquisition of signals, including maximal oxygen uptake (VO2max), maximal heart rate (HRmax), and maximal ventilation (VEmax). Measurement of physical functions was as follows: the indices measured included blood pressure, heart rate variability (HRV), vital capacity, and time vital capacity. In the vital capacity measurement, the one-time maximum expiratory volume of the subjects was measured. The time vital capacity was measured by portable lung function meter (CSTF-FH, ZhongTiTongFang, China). The parameters measured in spirometry were vital capacity (VC), forced vital capacity (FVC), forced expiratory volume (FEV) at timed intervals of 0.5, 1.0 (FEV1), 2.0, and 3.0 seconds, peak expiratory flow (PEF), and forced expiratory time (PET). The heart rate variability was measured by Polar RS800cx (Polar, Finland) during the 10-minute recording. The following time domain parameters of HRV were mean RR intervals (Mean R-R). Frequency domain variables included total power, very low (VLF: 0–0.04 Hz), low (LF: 0.04–0.15 Hz), and high (HF: 0.15–0.40 Hz) frequencies. The values for LF and HF were presented as normalized units. Normalization was achieved by dividing the spectral power of LF or HF by the total power minus the VLF. The LF/HF ratio was achieved by dividing the LF by the HF in normalized units. BODY.2. MATERIALS AND METHODS.2.7. STUDY QUESTIONNAIRE: All participants completed a questionnaire about general information about lifestyle and health. The Zung self-rating depression scale (SDS) to measure depression was used at the baseline and end line survey [14, 15]. SDS has 20 items rating the four common characteristics of depression and anxiety. The scores were counted up and multiplied by 1.25 to reach a standardized score. BODY.2. MATERIALS AND METHODS.2.8. DATA ANALYSIS: Statistical analysis was performed using SPSS 10.0 (SPSS Inc., Chicago, IL, USA). Independent-sample t-test was used to test posttest-pretest scores between the experimental and control groups. A two-sided test was used in this study. In all cases, values of P < 0.05 were considered statistically significant. BODY.3. RESULTS: Figure 1 shows the flow of participants through the study. Because ten subjects with suspected chronic diseases were excluded from this trial, the sample consisted of 110 participants (36 men and 74 women) aged 20–59 years, mean age 34.2 years (SD 14.6). The participants in the two groups had similar baseline characteristics (included in Table 2). The 110 subjects were physically healthy, with no acute or chronic disease that might affect the sports activity, and had no regular physical exercises of moderate intensity. Four participants quitted the intervention because they disliked the regular and collective manner. Three participants quitted the intervention without any reasons. Two participants quitted the intervention because of the family vacation. These resulted in completion rates of 100% for the control group and 83.6% for the intervention group in the physical measures and questionnaire, respectively. BODY.3. RESULTS.3.1. INTERVENTION ADHERENCE AND PARTICIPANT RETENTION, INTERVENTION ENJOYMENT/SATISFACTION, AND ADVERSE EVENTS: Of 55 subjects initially recruited into the intervention group, 46 subjects regularly participated in the 16-week Baduanjin exercise training program and the baseline and end line surveys. The loss rate was 16.4%. In addition to Baduanjin exercise training 3 times each week, 30–60 minutes each time, the subjects in the intervention group did not take part in other regular physical exercises. The number of trainings attended ranged from 30 to 48. Five people (10.9%) attended at least 30 of the trainings. Forty-one people (89.1%) attended all 48 trainings. The reasons given for nonattendance included illness (n = 3), going away on holidays (n = 1), and lack of time (n = 1). No subject presented any discomfort related to the practicing of Baduanjin exercise during the intervention period. The satisfaction of subjects towards Baduanjin exercise in the intervention group was 100%. This suggested that Baduanjin exercise had a high suitability as an exercise intervention among the population. BODY.3. RESULTS.3.2. EFFECT OF INTERVENTION ON OUTCOME MEASURES: In the intervention group comprised of adults, there were no significant variations in blood glucose, blood lipid, blood pressure, heart rate variability, and vital capacity indices (Tables 3 and 5). Compared with the control group, the body weight and body mass index (BMI) dropped in the intervention group, but the difference was of no statistical significance. However, compared with the control group, the skinfold thicknesses at three typical positions (lower corner of scapula, triceps brachii, and abdomen) decreased, with a statistical significance (Table 4). By comparing the physical fitness indices of the subjects, it was found that the results of sit-and-reach test in the intervention group were increased significantly. However, the control group showed no such changes. The increase of the results of sit-and-reach test in the intervention group had statistical significance (Table 6). There were no other obvious changes in other physical fitness indices. By comparing the results of SDS scores, there was no significant difference in the average scores between the intervention group and control group (Table 7). BODY.4. DISCUSSION: The physical flexibility was evaluated by sit-and-reach test. It was found that the practicing of Baduanjin exercise could significantly improve the physical flexibility of the subjects. The improvement of the results of sit-and-reach test reflects the effect of Baduanjin exercise in promoting the motion of shoulder joint and sacroiliac joint. Other studies have also confirmed the improvement of knee joint by Baduanjin exercise [1, 9]. Physical flexibility is closely related to health. The increase of physical flexibility can improve the physical coordination ability, physical power and velocity, and the physical skills. It is crucial for preventing the athletic injuries [16–18]. Therefore, Baduanjin exercise has the benefit of contributing to the physical flexibility, which is also one of the aims in the design of Baduanjin exercise [3]. Compared with other gymnastic exercises, Baduanjin exercise highlights the unification of physical movement and the mind, with great attention paid to the connotation of physical exercise. In this study, there was no significant difference in the scores of SDS between the intervention group and the control group. One reason for non-significant findings can be explained that the vast majority of participants are healthy. But in 2 subjects who were assessed as having mild depression before the intervention (initial SDS score higher than 50) [19, 20], the initial SDS scores significantly decreased from 61 to 25 and from 54 to 40, respectively. However, the effect of Baduanjin exercise in alleviating depression needs to be further verified. Existing studies have shown that intervention based on Baduanjin exercise could improve the sleeping quality of elderly subjects [10]. It is obvious that the research on the psychological impacts of Baduanjin exercise on the subjects needs to be furthered. The moderate intensity exercises are beneficial for the improvement of body weight, BMI, and body composition [21–24]. In this study, the decrease in the skinfold thicknesses of the lower corner of scapula, biceps brachii, and abdomen was of statistical significance compared with the control group. However, the changes in body weight, BMI, and waist circumference were of no statistical significance compared with the control group. These results may indicate that although Baduanjin exercise reduces the accumulation of subcutaneous adipose, it is not ideal in controlling the abdominal obesity. The previous researches have shown that Baduanjin exercise could improve the blood lipids of the subjects [5]. However, our research indicated that Baduanjin exercise did not significantly improve the blood lipids in healthy adults who practiced Baduanjin exercise. It is undeniable that the measurement results are affected by the variations in the subjects, intervention time and period, the choice of seasons for practicing and whether the subjects' lifestyle is controlled [24, 25]. There were no significant variations in blood pressure, heart rate variability, vital capacity, time vital capacity, maximum oxygen uptake, maximum heart rate, and maximum ventilation among the subjects. It is indicated that the practicing of 16-week Baduanjin exercise can hardly improve the heart functions, lung functions, and cardiopulmonary endurance. One reason is that Baduanjin exercise as a safe aerobic exercise may be a moderate intensity exercise [26, 27]. Another reason is that the intervention period in this study was only 16 weeks, which is too short to demonstrate the protective effect of Baduanjin exercise to cardiopulmonary functions, if any [28]. We acknowledge that this study has several limitations. Firstly, the small sample size makes it difficult to draw conclusions about wider implications of the results. However, this was designed to be a pilot trial to assess protective effect of Baduanjin exercise. Secondly, the high rates of adherence may have been biased as the Baduanjin trainings were offered free of charge and cost may be a common barrier to exercise participation. Finally, the intervention was limited to only 16 weeks of Baduanjin, which may not be enough to demonstrate all the protective effects of Baduanjin exercise. Future studies should include a longer duration of intervention. BODY.5. CONCLUSION: It was found by our trial that Baduanjin exercise could significantly improve the physical flexibility and subcutaneous adipose accumulation in the healthy adults. Baduanjin exercise is a form of sports highlighting physical flexibility and mind-body coordination. Given the limitations of the present study, the psychological impacts of Baduanjin exercise should be further confirmed.

TITLE: Weekly taxane–anthracycline combination regimen versus tri-weekly anthracycline-based regimen for the treatment of locally advanced breast cancer: a randomized controlled trial ABSTRACT.BACKGROUND: Extensive studies have confirmed the efficacy of taxanes in combination with anthracycline-based chemotherapy on breast cancer. However, few studies have assessed the efficacy of weekly taxane–anthracycline regimens on locally advanced breast cancer. This study was to compare the efficacy and safety of a weekly taxane–anthracycline regimen with those of tri-weekly anthracycline-based regimen in patients with locally advanced breast cancer. ABSTRACT.METHODS: Patients with locally advanced breast cancer were randomized to receive 4–6 cycles of neoadjuvant chemotherapy with tri-weekly 5-fluorouracil–epirubicin–cyclophosphamide (FEC) regimen or weekly paclitaxel–epirubicin (PE) regimen. The primary endpoint was the pathologic complete response (pCR) rate. Other endpoints included the clinical tumor response, breast-conserving surgery rate, and adverse events. ABSTRACT.RESULTS: Between March 2010 and September 2013, 293 patients were randomized to the FEC (n = 151) and PE (n = 142) arms. The overall clinical response rate was significantly higher in the PE arm than in the FEC arm (76.06% vs. 59.95%, P = 0.001). Consistently, the post-chemotherapy pathologic T and N stages were significantly lower in the PE arm than in the FEC arm (P < 0.001). However, the pCR rate was similar in the two arms (10.61% vs. 12.31%, P = 0.665). Overall, 36 (27.27%) patients in the FEC arm and 6 (35.28%) in the PE arm were qualified for breast-conserving surgery. Most adverse events were comparable in both arms, with more severe neutropenia in the PE arm than in the FEC arm (11.97% vs. 5.96%, P = 0.031). ABSTRACT.CONCLUSIONS: In patients with locally advanced breast cancer, weekly PE was not superior to FEC in terms of pCR. However, weekly PE has a higher response rate and superior down-staging effects. On this account, the PE regimen may be considered an alternative option for locally advanced breast cancer. Long-term follow-up data are needed to confirm the efficacy of this regimen on locally advanced breast cancer. Trial registration Chinese clinical trial registry, ChiCTR-TRC-10001043, September 21, 2014 BODY.BACKGROUND: Locally advanced breast cancer is a heterogeneous entity that includes advanced primary tumors, extensive nodal involvement, and inflammatory breast cancer [1, 2]. Despite the progress in understanding tumor biology and the development of targeted therapy, locally advanced breast cancer remains a major clinical challenge with an unfavorable prognosis [1]. Neoadjuvant chemotherapy (NACT) is a standard treatment of locally advanced breast cancer [3, 4]. Women who achieved a pathologic complete response (pCR) during NACT had prolonged survival compared with those who did not achieved pCR [5]. Anthracycline-based regimens are the most effective chemotherapy for breast cancer [6]. Anthracycline-based regimens, such as tri-weekly 5-fluorouracil–epirubicin–cyclophosphamide (FEC) regimen, is widely recommend by guidelines and used in clinical practice. The addition of taxanes to anthracycline-based regimens has been shown to enhance antitumor activity with increased pCR and breast-conserving surgery (BCS) rate as well as prolonged survival [7, 8]. In addition, the Eastern Cooperative Oncology Group (ECOG) 1199 trial demonstrated a significant disease-free survival benefit of weekly paclitaxel [9, 10]. Theoretically, the ideal chemotherapy regimen should be safe, effective, and simple. For these reasons, weekly paclitaxel–epirubicin (PE) regimen is attractive. In addition, considering the mild myelosuppressive effect of paclitaxel, weekly PE could be a convenient outpatient chemotherapy regimen. Till now, extensive studies have evaluated the therapeutic value of taxanes in combination with anthracycline-based chemotherapy in breast cancer; however, limited data are available to evaluate its efficacy on locally advanced breast cancer, especially in Chinese women. In this prospective randomized controlled trial, we compared the safety and efficacy of a weekly PE regimen with those of the tri-weekly FEC regimen in Chinese women with locally advanced breast cancer. BODY.PATIENTS AND METHODS.PARTICIPANT ENROLLMENT: This prospective randomized controlled trial was approved by the Ethics Committee of West China Hospital of Sichuan University. Written informed consent was obtained from all participants. The study was registered with the Chinese Clinical Trial Register on September 21, 2014 (Registration number: ChiCTR-TRC-10001043). Women aged 18–70 years old with locally advanced breast cancer confirmed by core needle biopsy were eligible for our study. Locally advanced breast cancer was classified as clinical stage IIB or III according to the American Joint Committee on Cancer staging system. Before randomization, baseline chest radiography, abdominal computed tomography (CT) or magnetic resonance imaging, and bone scintigraphy were performed to exclude distant metastases. Other eligibility criteria were an ECOG performance status of 0–1; normal cardiac function; no history or evidence of abnormal hematologic, renal, or hepatic function; and no history of other neoplasm (except non-melanoma skin cancer or curatively treated carcinoma in situ of the cervix). Patients were excluded if they were pregnant; had received prior breast cancer surgery or systemic therapy; had uncontrolled concurrent illness such as serious viral, bacterial, or fungal infections, peptic ulcers or diabetes, or autoimmune diseases; had a history of severe hypersensitivity reactions to chemotherapeutic regimens; or had any other illness deemed by the physician to affect chemotherapy tolerability. BODY.PATIENTS AND METHODS.TREATMENT: With simple randomization, the participants were randomly assigned to the PE arm or the FEC arm. In the PE arm, intravenous infusion of epirubicin 30–40 mg/m2 and paclitaxel 70–80 mg/m2 were administered concurrently on days 1, 8, and 15 of every 4-week cycle. In the FEC arm, intravenous infusion of 5-fluorouracil 500 mg/m2, epirubicin 100 mg/m2, and cyclophosphamide 500 mg/m2 were administered on day 1 of every 3-week cycle. Antimimetic drugs were administered prophylactically 30 min before the chemotherapeutic regimen was administered. During NACT, granulocyte colony-stimulating factor (G-CSF) support was required if the neutrocyte count dropped to <1.0 × 109/L. Surgery was undertaken within 1–2 weeks of NACT completion. According to the tumor characteristics and patient preference, women underwent BCS or mastectomy. Those considered eligible for BCS had a single tumor <3 cm in diameter, with the distance between the tumor edge and nipple being ≥3 cm, had no diffuse lesion or skin involvement, and were not contraindicated for radiotherapy. All patients who underwent BCS also underwent postoperative radiotherapy. All these patients underwent axillary lymph node dissection for nodal assessment. All study visits were completed at the Breast Cancer Center of West China Hospital. At the beginning of each cycle, history taking, physical examination, and hematologic assessment were conducted to evaluate safety. The NACT schedule was delayed if the left ventricle ejection fraction (LVEF) decreased by 15% or if the patient showed symptoms of congestive heart failure, a severe hypersensitive reaction, or other adverse events during treatment. In the PE arm, if the patients had severe neutropenia (neutrophil count <1.0 × 109/L), febrile neutropenia (grade 2 and above), or peripheral neuropathy (grade 2 and above), the dose of epirubicin and paclitaxel was reduced by 15%. Participants were withdrawn if they had disease progression or developed severe adverse events (e.g., grade 3 or 4 non-hematologic toxicity), or at their request. BODY.PATIENTS AND METHODS.EFFICACY ASSESSMENTS: Physical examination and imaging data (ultrasonography for tumor response assessment and CT scan for metastasis monitoring) were carefully recorded for clinical assessments before treatment, every two cycles during NACT, and before surgery. The tumor response was assessed by experienced oncologists and was classified as clinical complete response (cCR), partial response (cPR), stable disease (cSD), or progressive disease (cPD). In particular, the clinical tumor response was defined as the achievement of cCR and cPR. Any controversy was solved by discussion with a third oncologist. Tumor responses were used to dictate management strategies. Those showing a cCR, defined as the disappearance of the breast tumor and enlarged nodes on clinical assessments, could undergo surgery. Those showing a cPR, defined as a reduction of ≥30% in the three largest perpendicular tumor diameters, could complete at least four NACT cycles and then undergo surgery. Those with cSD, defined as a tumor reduction of <30%, or those with cPD, defined as an increase of ≥20% in the target tumor diameter or the emergence of a new tumor, could switch NACT regimens or undergo surgery as desired. Postoperative pathologic assessments were conducted by pathologists at the Pathology Department of West China Hospital. A pCR was defined as the complete disappearance of the invasive tumor in the breast and lymph nodes. Residual ductal carcinoma in situ (DCIS) alone was also classified as pCR. Before the assessments and data analysis, the two groups were renamed as group 1 or 2 without detailed information on the NACT provided. Therefore, the surgeons who assessed the suitability for BCS, the pathologists who assessed the postoperative specimens, and the statisticians who performed the analysis were all blinded. BODY.PATIENTS AND METHODS.SAFETY ASSESSMENTS: All adverse events were recorded and graded according to the National Cancer Institute Common Toxicity Criteria (version 2.0). All women who underwent at least 1 cycle of chemotherapy were included in the safety analysis. Only grade 3–4 adverse events were analyzed. BODY.PATIENTS AND METHODS.STATISTICAL ANALYSES: The sample size was estimated to detect a pCR rate difference of 25% for the PE arm and 10% for the FEC arm. The assumed dropout rate was 10%. A sample size of 218 participants was sufficient to provide an 80% power to detect a pCR improvement of 15% in each arm with a type I error rate of 0.05. All data were analyzed based on the intent-to-treat principle at randomization. Descriptive data were used to analyze patient characteristics. Quantitative data were compared using an independent sample t test, and qualitative data were compared using the Chi square test. Ranked data were compared using a non-parametric test. Statistical tests were considered significant with a two-sided P value of <0.05. All data were analyzed using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). BODY.RESULTS.PATIENT CHARACTERISTICS: The results of randomization and treatment assignment are shown in Fig. 1. Between March 2010 and September 2013, 300 patients were enrolled, but 7 of them withdrew prior to treatment. Of the remaining 293 patients, 151 were assigned to the FEC arm, and 142 were assigned to the PE arm. The baseline patient characteristics of the two groups were evenly matched and are shown in Table 1. The median ages were 47 (range 27–69) years for the FEC arm and 47 (range 24–68) years for the PE arm. During chemotherapy, 16 patients (10 in the FEC arm and 6 in the PE arm) had declined further treatment; 15 (9 in the FEC arm and 6 in the PE arm) were lost to follow-up; and 34 (22 in the FEC arm and 12 in the PE arm) had switched chemotherapy regimens due to unsatisfactory outcomes or toxicities.Fig. 1Study flow chart for comparison of weekly PE and tri-weekly FEC regimens in treating locally advanced breast cancer. FEC tri-weekly 5-fluorouracil–epirubicin–cyclophosphamide regimen, PE weekly paclitaxel–epirubicin regimen, NACT neoadjuvant chemotherapy. #All participants who switched chemotherapy regimens had undergone surgery except one in the PE arm that was lost to follow-up Table 1Baseline characteristics of all enrolled patients with locally advanced breast cancerCharacteristicFEC arm [cases (%)]PE arm [cases (%)] P valueTotal151142Age (years)0.430 ≤355 (3.31)8 (5.63) >35146 (96.69)134 (94.37)Menopausal status0.263 Premenopausal31 (20.53)22 (15.49) Postmenopausal120 (79.47)120 (84.51)Clinical tumor stage0.075 T2106 (70.20)86 (60.56) T343 (28.48)52 (36.62) T42 (1.32)4 (2.82)Clinical nodal status0.497 Involved130 (86.09)126 (88.73) Not involved21 (13.91)16 (11.27)ER/PR status0.533 Positive96 (63.58)89 (62.68) Negative47 (31.13)52 (36.62) Missing8 (5.30)1 (0.70)HER2 (IHC staining)0.189 0/1+64 (42.38)66 (46.48) 2+31 (20.53)26 (18.31) 3+45 (29.80)47 (33.10)Missing11 (7.28)3 (2.11) PE weekly paclitaxel–epirubicin regimen, FEC tri-weekly 5-fluorouracil–epirubicin–cyclophosphamide regimen, ER estrogen receptor, PR progesterone receptor, HER2 epidermal growth factor receptor-2, IHC immunohistochemistry BODY.RESULTS.CLINICAL RESPONSE: During chemotherapy, three assessments were performed to grade the clinical response to these two regimens (Table 2). The results of the last assessment for individual participants are shown in Fig. 2. The clinical response rates were significantly higher in the PE arm than in the FEC arm (76.55% vs. 56.95%, P = 0.001). Those in the PE arm achieved higher cPR and lower cSD rates than those in the FEC arm (cPR rate: 70.92% vs. 54.30%, P = 0.006; cSD rate: 14.79% vs. 30.46% P = 0.002). Two patients in the PE arm developed distant metastasis after 4 cycles of NACT.Table 2Clinical responses of patients with locally advanced breast cancer to neoadjuvant chemotherapy (FEC regimen vs. PE regimen) during each assessmentsGroupFirst assessment [cases (%)]Second assessment [cases (%)]Third assessment [cases (%)]FEC arm15113268 cCR0 (0.00)1 (0.76)3 (4.41) cPR70 (46.36)76 (57.58)51 (75.00) cSD67(44.37)45 (34.09)13 (19.12) cPD6 (3.97)7 (5.30)1 (1.47) Missing8 (5.30)3 (2.27)0 (1.47)PE arm14212149 cCR1 (0.70)6 (4.96)5 (10.20) cPR97 (68.31)92 (76.03)41 (83.67) cSD35 (24.65)20 (16.53)2 (4.08) cPD3 (2.11)3 (2.48)1 (2.04) Missing6 (4.23)0 (0.00)0 (0.00) FEC tri-weekly 5-fluorouracil–epirubicin–cyclophosphamide regimen, PE weekly paclitaxel–epirubicin regimen, cCR clinical complete response, cPR clinical partial response, cSD clinical stable disease, cPD clinical progressive disease Fig. 2Final assessment of clinical responses of patients with locally advanced breast cancer to neoadjuvant chemotherapy (FEC regimen vs. PE regimen). FEC tri-weekly 5-fluorouracil–epirubicin–cyclophosphamide regimen, PE weekly paclitaxel–epirubicin regimen, cCR clinical complete response, cPR clinical partial response, cSD clinical stable disease, cPD clinical progressive disease. *P < 0.05 For each participant, the tumor was restaged at the last assessment. As shown in Table 3, both regimens exhibited excellent down-staging effects (both P < 0.001), with the PE regimen exhibiting a superior down-staging effect compared with the FEC regimen (P = 0.026).Table 3Clinical down-staging effects of the FEC and PE regimens on locally advanced breast cancerT stageFEC arm [cases (%)] P valuePE arm [cases (%)] P valueBaselineAfter NACTBaselineAfter NACTTotal151151<0.001142142<0.00100 (0.00)5 (3.31)0 (0.00)12 (8.45)10 (0.00)46 (30.46)0 (0.00)62 (43.66)2106 (70.20)77 (50.99)86 (60.65)55 (38.73)343 (28.48)9 (6.62)52 (36.62)5 (3.52)42 (1.32)5 (2.65)4 (2.82)2 (1.41)Missing0 (0.00)9a (5.96)0 (0.00)6b (4.23) FEC tri-weekly 5-fluorouracil–epirubicin–cyclophosphamide regimen, PE weekly paclitaxel–epirubicin regimen aOf the 9 patients, 2 had migrated to other cities and were not restaged, 5 declined further treatment and were not restaged, and 2 were lost to follow-up bOf the 6 patients, 5 declined further treatment and were not restaged, and 1 was lost to follow-up BODY.RESULTS.SURGERY AND PATHOLOGIC RESPONSE: Among the 293 patients, 262 underwent surgery (132 in the FEC arm and 130 in the PE arm). Among them, 219 patients (114 in the FEC arm and 105 in the PE arm) had completed at least 4 cycles of NACT; 33 (22 in the FEC arm and 11 in the PE arm) had switched chemotherapy regimens. Surgery information is shown in Table 4. No significant difference was observed in the BCS rate. Theoretically, 36 (27.27%) patients in the FEC arm and 6 (35.38%) in the PE arm were candidates for BCS (P = 0.157). However, only 3 (2.27%) in the FEC arm and 4 (3.08%) in the PE arm underwent BCS (P = 0.721).Table 4Surgical breast and lymph node management for the FEC and PE armsSurgery typeFEC arm [cases (%)]PE arm [cases (%)] P valueTotal132130The breast0.456 Breast-conserving surgery3 (2.27)4 (3.08) Mastectomy75 (56.82)66 (50.77) Modified radical mastectomy54 (40.91)60 (46.15)Lymph nodes0.633 No surgerya 0 (0.00)1 (0.77) SLNBa 1 (0.76)2 (1.54) Level I–II node dissection28 (21.21)21 (16.15) Level I–III node dissection86 (65.15)89 (68.46) Level I–III + supraclavicular node dissection17 (12.88)17 (13.08) FEC tri-weekly 5-fluorouracil–epirubicin–cyclophosphamide regimen, PE weekly paclitaxel–epirubicin regimen, SLNB sentinel lymph node biopsy aRequired by participants For pathologic response assessments, all the 262 patients were included in the intention-to-treat analysis. Postoperative pathologic staging indicated that the weekly PE regimen significantly alleviated tumor burden in patients with locally advanced breast cancer as compared with the tri-weekly FEC regimen (P = 0.001) (Table 5). However, no significant differences between the FEC and PE arms were observed in the overall pCR (pCR in both the breast and lymph nodes) rates (10.61% vs. 12.31%, P = 0.665), the breast pCR rates (13.64% vs. 16.92%, P = 0.460), or regional lymph node pCR rates (34.85% vs. 39.23%, P = 0.463).Table 5Postoperative pathologic staging in patients who underwent surgeryPathologic stageFEC arm [cases (%)]PE arm [cases (%)] P valueTotal132130pT stage0.001 011 (8.33)15 (11.53) DCIS7 (5.30)7 (5.38) 140 (30.30)62 (47.69) 257 (43.18)41 (31.54) 32 (1.52)2 (1.54) 415 (11.36)3 (2.31)pN stage0.001 046 (34.85)51 (39.23) 139 (29.55)39 (30.00) 225 (18.94)18 (13.85) 322 (16.67)22 (16.92) FEC tri-weekly 5-fluorouracil–epirubicin–cyclophosphamide regimen, PE weekly paclitaxel–epirubicin regimen, DCIS ductal carcinoma in situ BODY.RESULTS.SAFETY: For the safety analysis, we evaluated only grade 3–4 adverse events, which are listed in Table 6. Both the FEC and PE regimens were well tolerated, and all adverse events were manageable. The frequency of neutropenia was higher in the PE arm than in the FEC arm (P = 0.031). Four patients (2 in each arm) had grade 4 neutropenia and were treated by repeated G-CSF administration. Three patients (2 in the FEC arm and 1 in the PE arms) had liver damage and required breast cancer surgery after 3 or 4 cycles of NACT. One patient had grade 4 bone marrow suppression after 1 cycle of PE and thus switched to the FEC regimen. There were no cardiac events or treatment-related deaths during the study period. Other adverse events were mild and slightly affected patient quality of life during NACT.Table 6Frequency of grade 3–4 adverse events in the FEC and PE armsAdverse eventFEC arm [cases (%)]PE arm [cases (%)] P valueHematologic Neutropenia9 (5.96)17 (11.97)0.031 Anemia6 (4.00)10 (7.04)0.248 Thrombocytopenia5 (3.31)7 (4.93)0.485Non-hematologic Nausea4 (2.64)4 (2.82)0.458 Vomiting2 (1.32)3 (2.11)0.603 Diarrhea2 (1.32)4 (2.82)0.951 Constipation3 (1.99)4 (2.82)0.267 Hair loss24 (15.79)28 (19.72)0.583 Dermatitis8 (5.30)11 (7.75)0.395 Febrile1 (0.66)1 (0.70)0.965 Fatigue7 (4.64)10 (7.04)0.379 Hand-foot syndrome6 (3.97)5 (3.52)0.913 Allergy0 (0.00)1 (0.70)0.143 FEC tri-weekly 5-fluorouracil–epirubicin–cyclophosphamide regimen, PE weekly paclitaxel–epirubicin regimen BODY.DISCUSSION: Our results showed that the weekly PE regimen was not superior to the tri-weekly FEC regimen in treating locally advanced breast cancer in terms of pCR. However, in the NACT setting, the weekly PE regimen showed significant value in the clinical tumor response and down-staging effect. In China, delayed detection and a lack of awareness of breast cancer have led to a high prevalence of locally advanced breast cancer at the initial diagnosis [11, 12]. Locally advanced breast cancer with large tumor lesions and more node involvement is related with a lower pCR rate as compared with early stage or operable breast cancer [13, 14]. In the present study, the overall pCR rate (12.31%) in the PE arm was comparable to those reported previously for taxane–anthracycline-based regimens (13.3%–18%) [15, 16]. Specifically, the results were similar to those of the ABCSG-14 trial, which administered 6 cycles of epirubicin 75 mg/m2 plus docetaxel 75 mg/m2, resulting in overall and breast pCR rates of 15.9% and 18.6%, respectively [17]. However, the ABCSG-14 trial included patients with diseases at any tumor stage, whereas the present study focused on those with locally advanced breast cancer who may have a poorer prognosis. Additionally, the predominant population in the present study was positive for hormone receptor, which was a predictive factor for a low response to NACT [18, 19]. In the present study, the pCR rate was higher in the PE arm than in the FEC arm, although the difference was not significant. However, we must note that more patients in the FEC arm switched regimens during the study than those in the PE arm (22 vs. 12). When performing the intention-to-treat analysis, the high regimen switch rate in the FEC arm was a confounder and an indirect indicator of poor disease control. Although our findings do not support that the weekly PE regimen is superior than the tri-weekly FEC regimen in terms of the pCR rate, the PE regimen elicited a superior tumor response and down-staging effect in terms of the clinical and pathologic evaluations. Our results suggest that the weekly PE regimen can improve disease control and reduce the extent of surgical resection. The increased chance of BCS is an important benefit of NACT. However, BCS might be difficult for women with locally advanced breast cancer, especially Chinese women. In the present study, the theoretical BCS rates were 27.27% in the FEC arm and 35.38% in the PE arm, which were lower than that reported by Amat et al. (overall BCS rate of 72.37%) [20]. These disparities could be caused by the inclusion of women with breast cancer at different stages in their study. The relatively small breast volume in Chinese women may also contribute to this phenomenon. In the present study, many patients who were qualified for BCS declined the surgery. Different attitudes towards breast cancer in eastern and western countries may explain the lower acceptance rate of BCS among Chinese women. In addition, the economic burden of postoperative radiotherapy and long-term follow-up might contribute to the preference of mastectomy over BCS [21–23]. In the present study, the adverse events were comparable in both arms, with more severe neutropenia in the PE arm, which could be successfully treated using G-CSF in our study and other studies [24, 25]. There are some limitations in the present study. Most epidermal growth factor receptor-2 (HER2)-positive patients in our study could not afford HER2-targeting therapy and refused further HER2 status testing. With this lack of data, we could not carry out subgroup analyses to identify any subpopulation that was more likely to benefit from the weekly PE regimen. Because there were no significant differences in terms of the pCR rate at the time of surgery, long-term follow-up data will be reported to further assess the efficacy of the weekly PE regimen on locally advanced breast cancer. BODY.CONCLUSIONS: This prospective, randomized study suggests that the weekly PE regimen is not superior to the tri-weekly FEC regimen in treating locally advanced breast cancer in terms of pCR. However, the weekly PE regimen is well tolerated and has a superior clinical tumor response in Chinese women with locally advanced breast cancer. Long-term outcomes are needed to confirm the efficacy of weekly taxanes in combination with an anthracycline-based regimen.

TITLE: Single-blind randomized clinical trial to evaluate clinical and radiological outcomes after one year of immediate versus delayed implant placement supporting full-arch prostheses ABSTRACT: Purpose: To evaluate and compare peri-implant health, marginal bone loss and success of immediate and delayed implant placement for rehabilitation with full-arch fixed prostheses. Material and Methods: The present study was a prospective, randomized, single-blind, clinical preliminary trial. Patients were randomized into two treatment groups. In Group A implants were placed immediately post-extraction and in Group B six months after extraction. The following control time-points were established: one week, six months and twelve months after loading. Measurements were taken of peri-implant crevicular fluid volume, plaque index, gingival retraction, keratinized mucosa, probing depth, modified gingival index and presence of mucositis. Implant success rates were evaluated for the two groups. The study sample included fifteen patients (nine women and six men) with a mean average age of 63.7 years. One hundred and forty-four implants were placed: 76 placed in healed sites and 68 placed immediately. Results: At the moment of prosthetic loading, keratinized mucosa width and probing depth were higher in immediate implants than delayed implants, with statistically significant differences. However, after six and twelve months, differences between groups had disappeared. Bone loss was 0.54 ± 0.39 mm for immediate implants and 0.66 ± 0.25 mm for delayed implants (p=0.201). No implants failed in either group. Conclusions: The present study with a short follow-up and a small sample yielded no statistically significant differences in implant success and peri-implant marginal bone loss between immediate and delayed implants with fixed full-arch prostheses. Peri-implant health showed no statistically significant differences for any of the studied parameters (crevicular fluid volume, plaque index, gingival retraction, keratinized mucosa, probing depth, modified gingival index and presence of mucositis) at the twelve-month follow-up. Key words:Immediate implants, delayed implants, peri-implant health, success rate. BODY.INTRODUCTION: The immediate insertion of implants in post-extraction sockets is a treatment modality with high success rates (1). Chen et al. (2) conducted a literature review of success rates and clinical outcomes associated with immediate, early and delayed implant placement, finding similar success rates among the different procedures. Nevertheless, immediate placement has certain advantages over delayed implant insertion: the reduction in treatment time and the avoidance of second surgery (3). It is thought that the long-term success of dental implant treatment depends on many factors, including periodical maintenance and follow-up examinations (4). However, few long-term controlled follow-up studies of implants that include periodontal pa-rameters have been reported. Salvi et al. (5) conducted a literature review and concluded that the parameters that may be applied for assessing the state of peri-implant health and the severity of peri-implant disease include: plaque accumulation, probing depth, bleeding on probing, keratinized mucosa width and crevicular fluid volume. According to a recent literature review (6), only two randomized studies (7,8) have compared one-piece implants placed immediately following extractions with the same placed in healed sites. No controlled studies have been found that evaluate the influence on peri-implant health of placing implants immediately or after allowing socket healing in patients rehabilitated with fixed full-arch prostheses. In this way, the aim of this prospective controlled clinical trial was to evaluate and compare peri-implant health, marginal bone loss and success of immediate and delayed implants rehabilitated with full-arch fixed prostheses. BODY.MATERIAL AND METHODS: * Study Population The present study was a prospective, randomized, single-blind, clinical preliminary trial carried out at the Oral Surgery Unit of the University of Valencia between December 2009 and February 2011 on patients requiring implant-supported fixed full-arch prosthetic rehabilitations. Stratification was performed considering the arch to be treated (maxilla/mandible). Random group assignment was performed by a professional statistician using pre-defined randomization tables. A balanced random permuted-block approach was used to prepare the randomization tables in order to avoid unequal balance between the two treatment groups. Patients received eight implants in the maxilla and/or six implants in the mandible to support fixed full-arch prosthetic rehabilitations. Group A patients received implants immediately after extraction; any non-immediate implants in Group A were excluded from analysis. In Group B, all implants were placed in healed sites; the necessary dental extractions were performed during the six months preceding implant placement. This research was performed following recommendations made in the Consort Statement for reporting randomized clinical trials and the principles of the Declaration of Helsinki regarding research on humans; accordingly, all patients provided written informed consent to take part in the trial prior to surgery. The study design was approved by the ethical board of the University of Valencia (Ref. H1335344377076). * Selection Criteria Inclusion criteria for this study were: - Age > 18 years - Full mouth plaque score and full mouth bleeding score < 25 %. - Partially edentulous maxilla with indications for the extraction of all remaining teeth. - Final rehabilitation with fixed full-arch implant-supported prosthesis. - Sufficient bone height and width to place six to eight implants with a minimum length of 10 mm and minimum diameter of 3.8 mm without performing bone grafting procedures (sinus lifting, block bone grafts or GBR); coverage of peri-implant defects and/or gap filling with autologous bone or tricalcium ß-phosphate did not prevent inclusion in the study. - Patients receiving six or more implants with insertion torque > 35 Ncm. - For immediate post-extraction implants, > 4 mm of apical bone were required to ensure the necessary primary stability. - Signature of informed consent form. - Minimum follow-up of 12 months after implant loading. * Exclusion criteria were: - Sites with acute infection - Implants placed with insertion torque < 35 Ncm were excluded from both groups. - Medical conditions contraindicating implant surgery. - Pregnant and lactating patients. - Smokers. - Patients with a history of bisphosphonate therapy. - Patients receiving chemo or radiotherapy of head and neck. - Severe bruxism. - Poor oral hygiene or non-collaborative patients. - Incomplete data gathering or failure to attend scheduled control appointments. * Surgical Procedure All surgeries were performed under local anesthesia (4% articaine with 1:100.000 adrenalin [Inibsa®, Lliça de Vall, Barcelona, Spain]). In Group B, non-immediate implants were placed following the standard surgical procedure. Periodontal treatment was delivered to Group A patients in order to control inflammation before performing the extractions. In Group A, anterior maxillary dental immediate implants were placed palatally, while for upper molars and premolars with two roots, implants were placed in the palatine root. In all maxillary cases, drills and osteotomes were used in combination to prepare implant beds. In the mandibular posterior area, implants were placed in the interradicular septum, whenever possible. Implants used in the present study were Kohno SP® (Sweden&Martina, Due Carrare, Italy) with Dual Engineered Surface (DES®). All patients were treated following a one-step procedure. After implant placement and suturing, each patient received 500mg of amoxicillin (Clamoxyl, GlaxoSmithKline, Madrid, Spain) to be taken three times daily for seven days, 600mg ibuprofen (Bexistar, Laboratorio Bacino, Barcelona, Spain) to be taken as needed and a 0.12% chlorhexidine mouthwash (GUM, John O Butler/Sunstar, Chicago, IL, U.S.A.) for use twice daily for two weeks. Gentle brushing with a chlorhexidine tooth-paste was also recommended. Sutures were removed eight to ten days after surgery. Prosthetic loading was carried out after twelve weeks fol-lowing implant placement in the maxilla and after ten weeks in the mandible. * Data Compilation and Follow-up A previously established, standard protocol was used to compile the following data for all patients: patient age (at implant place-ment), sex, implant length and diameter. Control visits were conducted by a trained clinician, blinded to group assignation, at the following time points: one week after prosthetic placement (time point 1); six months after loading (time point 2); and twelve months after loading (time point 3). At each time point the following data and clinical parameters were registered: • Crevicular Fluid Volume (CFV): each sample was taken using the technique described by Offenbacher et al. (9). • Gingival Retraction: was determined as being either present or absent and where present was measured in millimeters from the midfacial mucosa level to the restorative crown margin (10). • Plaque Index (PI): the modified PI according to Mombelli et al. (11) was used: score 0 = no plaque; score 1 = plaque only detected with probe; score 2 = plaque visible to the naked eye; score 3 = abundant plaque. • Probing Depth (PD): a periodontal probe (Click-Probe®, Kerr, Bioggio, Switzerland) was used to measure PD. • Keratinized Mucosa (KM): utilizing the same probe, this was measured in millimeters from the mucogingival junction to the most coronal point of the keratinized mucosa at the center of the prosthetic restoration. • Modified Gingival Index (mGI): the modified gingival index according to Mombelli et al. (11) was used: score 0 = no bleeding; score 1 = isolated bleeding spots; score 2 = confluent blood; score 3 = profuse bleeding. • Mucositis: an implant was considered to have mucositis when reversible inflammation of the peri-implant mucosa without bone loss existed. The most important diagnostic factor for this pathology was bleeding on probing with a maximum force of 0.25N (12). The definition of implant success was based on the clinical and radiographic criteria defined by Buser et al. (13): 1) absence of clinically detectable implant mobility; 2) absence of pain or any subjective sensation; 3) absence of recurrent peri-implant infec-tion; 4) absence of ongoing radiolucency around the implant after six and twelve months of loading. Intraoral radiographs were made at prosthetic loading (baseline) (Fig. 1) and at the one-year control (Fig. 2) visit using the XMIND intraoral system (Groupe Satelec-Pierre Rolland, Merignac, France) and an RVG intraoral digital receptor (Dürr Dental, Bietigheim-Bissingen, Germany) with the aid of Rinn XCP (Dentsply Rinn, Elgin, IL, U.S.A.) to achieve parallelism. If the bone level around the study implants was not clearly visible a new radiograph was made. The distance from the implant abutment connection to the peri-implant marginal bone level was measured to the nearest 0.1 mm both mesially and distally. Bone loss was calculated from the change in bone level between the baseline and the one-year control radiograph. Figure 1Radiographic assessment at the 3-month follow-up. Figure 2Radiographic assessment at the 12-month follow-up. * Statistical Analysis Clinical Parameter descriptive data were analyzed. Parametric test assumptions were checked and whenever there was doubt that these had been fulfilled, the corresponding non-parametric test was applied. All statistical analysis was performed using SPSS 15.0 for Windows (SPSS Inc, Chicago, IL, U.S.A.). Statistical significance was taken as 5% (p<0.05). BODY.RESULTS: Patient Population Of the 16 patients enrolled in this study, eight belonged to Group B (five maxillary and six mandibular) and eight to Group A (six maxillary and five mandibular). One patient in Group B, who had received six implants in the mandible, was excluded due to failure to attend control visits. The final study sample comprised 15 patients (six women and nine men) with a mean age of 63.7 years ( Table 1). Table 1 Patient and implant distribution per group. Group B patients received 76 implants and Group A patients 68; ten non-immediate implants from Group A were excluded. In this way, a total of 144 implants were included in the study: 76 placed in healed sites and 68 placed immediately. Table 1 details the sample of patients and implants. Clinical Data A higher mean peri-implant crevicular fluid volume (CFV) was observed in immediate implants (Group A) versus non-immediate implants (Group B) at all study time points, in both maxilla and mandible and overall; while overall mean CFV did not show statistically significant differences between groups, there were significant differences between groups in maxillary CFV at time points 2 and 3 ( Table 2). Table 2 Clinical parameters and radiographic results (marginal bone levels) at each follow-up time point after implant Placement. Although plaque levels increased along the different time points, no statistically significant differences were identified between groups (p≥0.05 - Mann-Whitney U-test) ( Table 2). Gingival retraction and probing depth increased in both groups over time; no significant differences were observed between the two groups at any of the time points. Keratinized mucosa width was seen to decrease in both groups over time; the group of immediate implants had more keratinized mucosa throughout the study, although differences were only statistically significant at time point 1 ( Table 2). Modified gingival index was slightly higher among immediate implants (Group A) although no significant differences between groups were identified ( Table 2). There were no implant failures in either group. The overall implant success rate after the 12-month follow-up was 100% in both groups. All the implants fulfilled the success criteria defined by Buser et al. (13) at the 12-month follow-up. The difference between the average bone loss for immediate implants (Group A: 0.54 ± 0.39 mm) and for those placed in healed sites (Group B: 0.66 ± 0.25 mm) was not statistically significant (p=0.201). BODY.DISCUSSION: The purpose of this study was to evaluate and compare peri-implant health, marginal bone loss and success of immediate and delayed implants rehabilitated with full-arch fixed prostheses. According to a recent literature review (6), to date there have only been a very limited number of randomized clinical trials that compare immediate and delayed implants. No controlled studies have been found evaluating the influence on peri-implant health of placing implants immediately following tooth extraction or after allowing socket healing in patients rehabilitated with fixed full-arch prostheses. Randomized assignment of the selected patients should provide the high-quality evidence for surgical protocols (immediate implant and non-immediate implant placement) that is lacking in the literature. In spite of the reduced sample size - 15 patients and 144 implants (although with a statistical power of 95%) - the present study aimed to add to the available evidence for evaluating and comparing peri-implant health, marginal bone loss and success of immediate and delayed implants rehabilitated with full-arch fixed prostheses; the sample consisted of 15 consecutive patients selected by means of strict, uniform criteria and treated by the same team of professionals using exactly the same procedures. Investigation of the biochemical parameters in gingival or peri-implant CFV will determine the current activity of the disease, the patient's susceptibility and possible destruction in the future. So far, numerous studies have focused on CFV analysis in the aim of identifying potential host markers which might provide diagnosis of disease activity and/or prognosis of future disease (14,15). Several studies have shown that CFV volume increases significantly in the presence of inflammatory conditions (16,17) and increased CFV volume is a useful marker of inflammation of periimplant tissue as well as gingival tissue (16). In the present study, higher mean peri-implant CFVs were observed in immediate implants at all study time points, in both maxillae and mandibles and overall. Berglundh et al. (18) describe a model for investigation of the different phases of wound healing involved in the processes resulting in osseointegration, concluding that osseointegration represents a dynamic process both during its establishment and its maintenance. In the establishment phase, there is a delicate interplay between bone resorption in contact regions (between the titanium body and mineralized bone) and bone formation in 'contact-free' areas. During the maintenance phase, osseointegration is secured through continuous remodeling and adaptation to function. The increase in peri-implant CFV could be due to greater neutrophil and macrophage activity, which participate in the initial phases of osteointegration. In a recent literature review (19), after reviewing 171 articles, 13 prospective clinical studies on single immediate implant treatment were selected. Midfacial recession was described in 0-64% of the cases. Only one of these studies identified a high risk of advanced midfacial recession (>10%) (20). In the present study, retraction at the 12-month follow-up for Group A immediate implants was 17.6% (0.43±0.56mm) compared to 8.7% (0.15±0.26mm) for Group B implants placed at healed sites. These results coincide with the findings obtained in other studies (21,22), who obtained values ranging between 0.41 and 0.55mm for immediately placed implants. With respect to plaque index, this increased over the different time points, but without statistically significant difference in either group. These results are slightly higher than those reported by Visser et al. (23), who obtained a mean of 0.4 ± 0.8 for non-immediate implants, while in the present study patients with non-immediate implants were rehabilitated with overdentures. Nishimura et al. (4) and Chung et al. (24) studied immediate and non-immediate implants, obtaining mean probing depths of 2.3 ± 0.5 mm and 2.86 ± 0.08 mm respectively at the 12-month follow-up; most implants presented peri-implant pockets with depths of less than 3mm. These results are consistent with those obtained in this study: 1.59 ± 0.52 mm in implants at healed sites and 1.39 ± 0.44 mm for immediate implants. Similar results were also reported by Botticelli et al. (10), who found a mean probing depth of 2.4 mm for immediate implants and Boynuegri et al. (15) who found 1.71 mm for implants at healed sites. While Wennström et al. (25) have reported that keratinized mucosa does not significantly influence oral hygiene status and soft tissue health, Chung et al. (24) have found that the absence of adequate keratinized mucosa in dental implants, especially in posterior implants, was associated with higher plaque accumulation and gingival inflammation but not with increased annual bone loss. In this study, changes in the width of keratinized mucosa were studied in immediate and non-immediate implants; in both groups the width of keratinized mucosa decreased after 12 months but no significant differences were found between groups. According to Boynuegri et al. (26) an adequate band of keratinized mucosa was related to less plaque accumulation and mucosal inflammation as well as pro-inflammatory mediators such as IL-1β and TNFα present in CFV. However, the present study findings did not establish this relationship. This could be due to the dynamics of the osseointegration process both during its establishment and its maintenance and to greater neutrophil and macrophage activity in immediately placed implants (18). With regard to the bleeding index, Lachmann et al. (27), studying non-immediate implants, obtained a mean of 0.4 bleeding on probing with a range from 0 to 2; this value cannot be compared with results obtained in the present study since the present study used a different evaluation system, the Mombelli modified Gingival Index (range 0 to 3). At 12 months after prosthetic loading, the average mGI was 1.12 ± 0.93 for immediate and 0.60 ± 0.83 for non-immediate implants. Several authors (2) have studied the survival rates of immediate and non-immediate implants, finding no statistically significant differences between the survival rates of immediate and non-immediate implants. These results are consistent with the present study's findings. The difference between the average bone loss for immediate implants (Group B: 0.54 ± 0.39 mm) and for those placed in healed sites (Group A: 0.66 ± 0.25 mm) was not statistically significant. Crespi et al. (28) evaluated changes in the bone around 40 immediate implants, 20 immediately restored and 20 loaded after three months. The authors reported a success rate of 100% in both groups, and radiographic results were similar. A review by den Hartog et al. (29) of single implant restorations in the esthetic zone, found no statistically significant differences in clinical trials comparing immediate, early and delayed implant placement. In contrast, for Atieh et al. (30) there is an added risk in the immediate restoration of immediate implants placed in the esthetic region compared to those placed in healed sites. However, according to these authors this approach may offer advantages with respect to favorable changes in marginal bone levels, the maintenance of soft tissues around the implant, and better esthetic results. Lindeboom et al. (7) compared peri-implant bone loss around immediate and non-immediate implants and found lower losses around immediate implants although differences were non-significant. Botticelli et al. (10) treated 18 patients with 21 immediate implants and after a follow-up of five years found stable bone levels and even gains around some implants. The present study with a short follow-up and a small sample yielded no statistically significant differences in implant success and peri-implant marginal bone loss between immediate and non-immediate implants supporting fixed full-arch prostheses. No statistically significant differences were found in peri-implant health for any of the parameters studied (crevicular fluid volume, plaque index, gingival retraction, keratinized mucosa, probing depth, modified gingival index and presence of mucositis) to the twelve months follow-up. Further studies with longer follow-up times and larger samples are required to better evaluate the influence of immediate and non-immediate implants on peri-implant health.

TITLE: Effect of The Swimmer’s Head Position on Passive Drag ABSTRACT: The aim of this study was to investigate the effect of the head position on passive drag with a towing-line experiment in a swimming pool. The tests were performed on ten male swimmers with regional level swimming skills and at least 10 years of competitive swimming experience. They were towed underwater (at a depth of 60 cm) at three speeds (1.5, 1.7 and 1.9 m/s) and in two body positions (arms above the swimmer's head and arms alongside the body). These two body positions were repeated while the swimmer's head was positioned in three different ways: head-up, head-middle and head-down in relation to the body's horizontal alignment. The results showed a reduction of 4–5.2% in the average passive drag at all speeds when the head was down or aligned to the swimmer's arms alongside the body, in comparison to the head-up position. A major significant decrease of 10.4–10.9% (p < 0.05) was shown when the head was down or aligned at the swimmer's arms above the swimmer's head. The passive drag tended to decrease significantly by a mean of 17.6% (p < 0.001) for all speeds examined with the arms alongside the body position rather than with the arms above the head position. The swimmer's head location may play an important role in reducing hydrodynamic resistance during passive underwater gliding. BODY.INTRODUCTION: In human swimming, the total drag is determined by the resistance forces acting opposite to the direction of travel, and the intensity of these forces is related to the speed. The term "passive drag" relates to the hydrodynamic resistance forces that occur when a swimmer remains in a stable position and is not moving any part of the body. The sums of all drag forces during swimming can be expressed by the passive hydrodynamic resistance in addition to the supplementary drag created by the movement of the swimmer's body and limbs (Gatta et al., 2015). Several attempts have been made to quantify the total drag on swimmers. Passive drag measurements use the same theoretical approach (body in opposition to the water flow), while total drag studies employ systematically different methodologies (Havriluk, 2007). To date, this variability has led to controversial results in the scientific literature about the measurement of active drag; therefore, consistency is not a feature of active drag values (Zamparo et al., 2010). When the total drag is estimated based on data for passive drag and the frontal area is in opposition to the swimmer's direction, the values are close to those of active drag, as measured by most authors (Zamparo et al., 2009). Although the swimmer is in a stable prone position for a short time during a swimming competition, the majority of the starts and turns are performed in glide swimming, which is defined as a rigid streamlined body position used in passive towing (Guimaraes and Hay, 1985). The swimmer keeps a hydrodynamic position for as long as possible in the underwater phases to maintain speed. Guimaraes and Hay (1985) showed that the gliding period was the most important fraction of the swimming start and Vantorre et al. (2014) found that the efficiency of the glide phases was highly correlated with the time of the race and the starting performance. Moreover, as reported by Seifert et al. (2011), approximately 20% of the breaststroke is performed in glide swimming. Similar results by Chollet et al. (2006) pertain to the glide phase in the butterfly stroke. Therefore, a reduction of hydrodynamic resistance during the glide phase seems to be an important factor for swimmers performance. Demonstrating the relevance of this phase, some authors have investigated the changes in body alignment and their correlation with gliding efficiency. Naemi and Sanders (2008) showed that glide efficiency was linked to the swimmer's size and shape. However, to estimate the benefits of a more hydrodynamically stable position, it is necessary to understand the flow characteristics around the swimmer. Pendergast et al. (2006) noted that different flow velocities around the surfaces of the swimmer's body could cause pressure changes. The changing zone of the flow velocity is called the 'boundary layer', and the flow separation and transition from laminar to turbulent depend on uniform velocity and, consequently, a constant pressure. Given that the pressure distribution over the body is the dominant factor of the swimmer's passive drag (Marinho et al., 2009), keeping the boundary layer attached to the swimmer seems to be very important for reducing flow resistance (Polidori et al., 2006). Pendergast et al. (2006) suggested that separation of the boundary layer occurred near the curvatures and circumference of the swimmer's body and that the change of surfaces at the head, back and buttocks could create adverse pressure gradients that would result in an increase of the swimmer's drag. In addition, a previous study showed that a laminar flow in a swimmer's glide profile was disrupted at the head (Mollendorf et al., 2004). It seems that the head position represents the first point of significant shape changes when the swimmer is in a streamline gliding position. Some studies have examined changes in the head position during underwater gliding. Zaidi et al. (2008) evaluated the effect of three head positions (aligned with the body, lifted up or lowered) on hydrodynamic resistance using computational fluid dynamics (CFD) methodology. The authors showed that the head aligned with the axis of the body induced a decrease in the drag of approximately 20% at high swimming velocities. The same results were found by Popa et al. (2011; 2012) who confirmed that the position of the head aligned with the body provided less resistance in underwater glide swimming. As proposed by several authors (Mollendorf et al., 2004; Pendergast et al., 2006; Popa et al., 2011; Zaidi et al., 2008), changes in the head position can affect the hydrodynamic performance of the glide. To our knowledge, only studies with CFD analysis have examined the quality of gliding related to the swimmer's head position. As proposed by Marinho et al. (2009), some restrictions inherent in the use of two- or three-dimensional steady flow models and the assumption that the fluid around the swimmer is laminar or turbulent must be considered when analysing results with CFD. The purpose of our study was to investigate the influence of the head position on passive drag by a direct pool experiment with a swimmer's in-line towing. BODY.MATERIAL AND METHODS.PARTICIPANTS: A total of ten male swimmers (age: 21 ± 2 years; body height: 1.80 ± 0.06 m; body mass: 75.9 ± 6.9 kg) participated in this study after giving their informed consent. All of the swimmers were regional-level and had at least 10 years of competitive swimming experience. The investigation was performed during the winter of 2014, when the swimmers were in the competition period. The study conformed to the standards set by the Declaration of Helsinki, and the procedures were approved by the Bioethics Committee of the University of Bologna. The swimmers were informed about the procedures, potential risks and benefits of the study. BODY.MATERIAL AND METHODS.PASSIVE DRAG MEASUREMENTS: The swimmers' passive drag was measured using an electro-mechanical device (Swim-Spektro, Talamonti Spa, Ascoli Piceno, Italy). A low voltage isokinetic engine anchored static at the edge of the pool measured the force (N) needed for towing the swimmer. Each participant was connected to the machine via a non-elastic wire and was towed at a programmed speed. The distance traversed by the swimmer in passive towing was 20 m in length at a constant depth of 60 cm below the water surface. The control of the depth was conducted by the passage within three rings of 90 cm diameter anchored to the swimming pool bottom. The rings were placed in the path of the towed swimmer at 10, 15 and 20 m after the starting point. A pulley support attached to the starting wall was used to ensure an in-line tow. For further analysis, we considered the data acquired between the first and third rings when the speed was constant. The data acquisition system was linked to a PC and controlled via dedicated software (DB:4, Talamonti Spa, Ascoli Piceno, Italy), and the device was calibrated before each experimental session. BODY.MATERIAL AND METHODS.EXPERIMENTAL PROCEDURES: The 10 swimmers were in the pool for the single test session for average duration of 2.5 hours. The trials were conducted in the morning in a 25 m indoor swimming pool with average water temperature of 28.0 ± 0.5o C. The study protocol was divided into three parts: Anthropometric measurements (body height and mass) were performed after a 15 -min swimming warm-up.Before the data acquisition, the swimmer performed two towing trials in each condition described in the "swimmer's head and body position" section to become familiar with the test.The participant performed the main session of the test procedure. The passive towing protocol was conducted for one swimmer at a time. The order in which the three head position tests were performed was counterbalanced across swimmers, to exclude the effects of sequence noise, as follows: A-B-C (n = 4 swimmers), C-A-B (n = 3 swimmers), and B-C-A (n = 3 swimmers). Five swimmers started this test sequence from the first body position, and five swimmers started from the second body position. Technical suggestions during the trials were deliberately avoided in order to not affect the test participants. BODY.MATERIAL AND METHODS.SWIMMER’S HEAD AND BODY POSITIONS: The basic stable prone position was performed while the subject assumed the best hydrodynamic glide position during towing under the water surface. The lower limbs and feet were held at maximum extension. During passive towing, the swimmer was required to maintain the best hydrodynamic glide position defined by the protocol for the entire trial. Each test began following a maximal inspiration by the participant, who then held his breath during towing. The glide prone position was repeated with the three different swimmer's head positions described below (Figure 1): Head-up (HU): the swimmer looked directly forward, and his ears were above the forearms and fully extended over the head.Head-middle (HM): the swimmer looked downward at 90 degrees to the swimming direction, in a neutral position. The swimmer's ears were covered by the forearms, which were fully extended over the head.Head-down (HD): the swimmer looked backwards, and his ears were below the forearms, which were fully extended over the head. The participant performed each head position trial at the three different speeds that are most used in swimming competition (1.5, 1.7, 1.9 m/s). The three different speeds for each swimmer's head position were repeated in two prone body positions. In the first position, the "long arms" (LA), the swimmer assumed the best hydrodynamic prone position, and the arms were extended over the head with one hand over the other. In the second body position, the "short arms" (SA), the same basic glide position was assumed, with the upper arms positioned along the body and the hands in contact with the sides of the thighs. When the swimmer was in the LA position, he was towed from the device linked to the wrists. In this manner the cable was anchored around the wrist of the swimmer's hand that was positioned above the other, without affecting the streamline position. In the SA position, the wire of the passive drag device turned around the swimmer's trunk at the underarms level. Also for this modality, a connection mode to the swimmer that did not affect the hydrodynamic prone position of the swimmer was chosen (Figure 1). The swimmer's body position during the glide was carefully checked by one operator positioned outside the pool. To assess the test reliability, the passive towing trial was repeated five times for each swimmer's head position, body position and speed, for a total of 90 trials per participant. BODY.MATERIAL AND METHODS.STATISTICAL ANALYSIS: To assess general patterns of the effects, the overall mean and standard deviation of each variable were calculated for all passive drag trials. For all of the variables (the swimmer, head and body positions, speed), the value of the coefficient of variation (CV) did not exceed 15.1%. This result indicates that the mean of the 5 trials was reliable and could be used for further analysis. The data on passive drag were analysed in two ways. First, to evaluate differences among the swimmer's head position, the body position, speed and their interaction, 3-way repeated measures ANOVA was used. Second, to analyse the differences between head positions that were found to be significant with ANOVA, simple pairwise multiple comparison procedures were performed with the Tukey post hoc test. The level of significance was set at p < 0.05, and Cohen's d was reported for pairwise comparisons as a measure of effect size. The statistical analyses were performed with SPSS Statistics Rel. 14.0.0 (SPSS, Chicago, IL, USA). BODY.RESULTS: Mean and standard deviation values of the passive drag (N) are described in Tables 1 and 2. The tables report individual data at the three considered speeds (1.5, 1.7 and 1.9 m/s) for the three head positions (HU, HM and HD) and the two body positions (SA and LA). ANOVA revealed significant differences in the following variables: speed (F(2, 16) = 683,932, p < 0.001), the body position (F(1, 8) = 115,409, p < 0.001), the head position (F(2, 16) = 7,116, p < 0.05), speed ×body position (F(2, 16) = 29,448, p < 0.001) and speed ×head position (F(4, 32) = 2,754, p < 0.05). Passive drag was significantly larger for all of the speeds examined in the SA than in the LA position (p < 0.001). The mean reduction of passive drag was 17.6% in the LA condition. Moreover, the results showed a significant difference for passive drag (p < 0.001) at the three levels of speed (1.5 vs. 1.7, 1.5 vs. 1.9, 1.7 vs. 1.9 m/s). Regarding the swimmer's head position, passive drag was found to be significantly lower in the LA condition (with the arms in front of the head) for the HD and HM than the HU at all speeds (Figure 2). No differences were observed in passive drag between the HD and HM at all speeds. The statistical analysis revealed that in the SA condition (with the arms alongside the body), significant highest values of passive drag were only in the HU rather than in the HD at the two fastest speeds (1.7 and 1.9 m/s). However, the pairwise comparison showed no significant differences for the HM rather than for the HU and HD at all speeds. When the comparison was significant, the effect size of swimming was large (mean Cohen's d = 0.72). Therefore, the results show a reduction of 4–5.2% in average passive drag when the head was down or aligned with the swimmer's arms alongside the body, in comparison to the head-up position. There was a major decrease of 10.4–10.9% in passive drag when the head was down or aligned with the swimmer's arms above the swimmer's head. BODY.DISCUSSION: This study aimed to investigate the effect of the swimmer's head position on passive drag during underwater towing. We considered three head positions, i.e. head-up, head-middle and head-down, with respect to the body's horizontal alignment. Additionally, the passive body positions most used in glide swimming (with upper arms extended above the head or alongside the body) were also investigated. This is the first time that the swimmer's head position has been investigated in a direct pool experiment during passive in-line towing in two stable body positions commonly used in glide swimming. Recent studies (Cortesi et al., 2014; Gatta et al., 2013) have shown that athletic gear worn by swimmers, such as full-body suits and swim caps, may affect the swimmer's body shape and passive drag. It has been previously shown that the shape of the swimmer, determined by the cross-sectional area, is a decisive factor affecting hydrodynamic resistance (Mollendorf et al., 2004; Zamparo et al., 1996). Indeed, passive drag resistance during underwater towing with the trailing arms along the body seems increased by 30–60% compared with the streamline glide position (Bulgakova et al., 2001; Marinho et al., 2009; 2011). However, in our study, increased hydrodynamic resistance due to the LA position was lower by approximately 20%. These different results could be due to the different methodologies used and theoretical assumptions required by different models (for example, for CFD, which was used in previous studies, the zero roughness relates to the swimmer's human skin). Our results have practical applications for swimmers, such as the advantage for the breaststroke start and turn, where more time is spent in the first glide (before the arm pull) than in the second glide position. The LA (streamlined) position seems to smooth the swimmer's anatomical shape especially at the head (Marinho et al., 2011) and when the ears are pressed by the upper arms and shoulders (Zatsiorsky, 2000). In our study, head lifting during streamlined underwater gliding in respect to the middle or down position of the head caused a 10% average increase in passive drag. Previous studies using CFD analysis showed that the head-middle was the optimal position in the underwater glide when the arms were extended at the front (Popa et al., 2011; Zaidi et al., 2008). The authors showed a change of 20% of the swimmer's hydrodynamic resistance when vertical shifts of the head from the position of horizontal alignment were carried out. Despite the validity and accuracy of the approaches used in swimming research (Bixler et al., 2007), the slight differences in passive drag values between previous studies and ours could be due to limitations of the commonly used CFD methodology (Marinho et al., 2009). Several authors have demonstrated that each slight displacement of the head position from horizontal alignment at flow velocities of 1.7–2.0 m/s causes increases in total hydrodynamic resistance from 2 to 40% (Miyashita and Tsunoda, 1978). In the present study, the high drag difference could be due to the methodology of passive drag measurement. Gliding was performed on the surface (Lyttle et al., 1998), and the use of the water tunnel could have produced a less satisfying comparison (Bixler et al., 2007). As confirmed by Zaidi et al. (2008), a lower deficit reduction in the flow velocity around the head occurs when the head is aligned horizontally. In this position, the head is more covered by the arms than in any of the other positions analysed. This phenomenon explains the benefit in hydrodynamic resistance verified in our study, which shows that the aligned head seems to allow the swimmer to perform the best penetration in underwater gliding. In our study, the lower head position, as described in the literature, seems to be the least favourable position. When comparing Figure 1 with the figures shown in another study (Popa et al., 2011; Zaidi et al., 2008), it is clear that the standardisation of the head-down position defined in our protocol shows less displacement from the horizontal alignment. This phenomenon could produce a minor obstacle against the oncoming flow and could thus reduce hydrodynamic resistance. Even correct alignment of the body segments during the glide position with the arms along the sides can affect the swimmer's speed (Gatta et al., 2015; Vilas-Boas et al., 2010). To our knowledge, no author has investigated the relationship between the passive drag with arms alongside the trunk and the swimmer's head position. The present study showed lower reductions in drag when keeping the head in a horizontal position compared to the same speed in gliding with the arms extended over the head. Mollendorf et al. (2004) showed that the laminar flow in a swimmer glide profile was disrupted at the head, most likely due to the head position during underwater streamline gliding. In our opinion, the lower effect of a different head position in gliding with arms along the sides could be explained by smoothing of the flow effect produced by the extended arms over the head. The swimmer's shape influences the ratio of inertial to viscous (friction) forces and affects the point where the boundary-layer flow shifts from laminar to turbulent or separates from the body (Webb, 1975). Raising the arms over the head during gliding could provide, due to the displacement of the head from the aligned position, a major influence on the passive drag, thus changing the flow characteristics around the swimmer. Despite high reliability of the test, flexibility of the human body structure does not ensure streamline alignment as a mannequin simulation. This is certainly the major limitation of this study. Future research with techniques of computational analysis or drag measurements with a towed mannequin should investigate the influence of the head position on passive drag. In conclusion, this study was based on the assumption that the measurement of hydrodynamic resistance when a swimmer was towed and inactive would be a reliable methodology to investigate the swimmer's drag (Havriluk, 2007). Our results suggest that performance improvements could be achieved from changes in the body's position in the water. The swimmer's head location seems to play an important role in reducing hydrodynamic resistance during gliding.

TITLE: Economic evaluation of a group-based exercise program for falls prevention among the older community-dwelling population ABSTRACT.BACKGROUND: Falls among older people are of growing concern globally. Implementing cost-effective strategies for their prevention is of utmost importance given the ageing population and associated potential for increased costs of fall-related injury over the next decades. The purpose of this study was to undertake a cost-utility analysis and secondary cost-effectiveness analysis from a healthcare system perspective, of a group-based exercise program compared to routine care for falls prevention in an older community-dwelling population. ABSTRACT.METHODS: A decision analysis using a decision tree model was based on the results of a previously published randomised controlled trial with a community-dwelling population aged over 70. Measures of falls, fall-related injuries and resource use were directly obtained from trial data and supplemented by literature-based utility measures. A sub-group analysis was performed of women only. Cost estimates are reported in 2010 British Pound Sterling (GBP). ABSTRACT.RESULTS: The ICER of GBP£51,483 per QALY for the base case analysis was well above the accepted cost-effectiveness threshold of GBP£20,000 to £30,000 per QALY, but in a sensitivity analysis with minimised program implementation the incremental cost reached GBP£25,678 per QALY. The ICER value at 95% confidence in the base case analysis was GBP£99,664 per QALY and GBP£50,549 per QALY in the lower cost analysis. Males had a 44% lower injury rate if they fell, compared to females resulting in a more favourable ICER for the women only analysis. For women only the ICER was GBP£22,986 per QALY in the base case and was below the cost-effectiveness threshold for all other variations of program implementation. The ICER value at 95% confidence was GBP£48,212 in the women only base case analysis and GBP£23,645 in the lower cost analysis. The base case incremental cost per fall averted was GBP£652 (GBP£616 for women only). A threshold analysis indicates that this exercise program cannot realistically break even. ABSTRACT.CONCLUSIONS: The results suggest that this exercise program is cost-effective for women only. There is no evidence to support its cost-effectiveness in a group of mixed gender unless the costs of program implementation are minimal. Conservative assumptions may have underestimated the true cost-effectiveness of the program. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12877-015-0028-x) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: Falls pose a major public health concern globally [1]. Approximately 1 in 3 people aged over 65 living in the community fall each year, a rate which increases with age [2]. Falls, fall-related injuries and subsequent fear of falling can have a significant impact on health-related quality of life and function, resulting in loss of independence among older people [3]. The medical costs of fall-related injuries increase rapidly with age and are 2 to 3 times higher for women due to their higher risk of osteoporotic fractures, particularly hip fractures [4]. Developing and implementing cost-effective programs to prevent falls among older people is of utmost importance given the ageing population and associated potential for increased costs of fall-related injury over the next decades. Exercise is currently the only proven falls intervention appropriate for population level delivery [5] and is the falls intervention subject to most economic evaluations [6]. Many previously reported economic evaluations of exercise programs to prevent falls are founded on the home-based "Otago Exercise Program" [7-11]. Whilst this program demonstrated a reduction in falls rate of between 30% and 46% compared to routine care [9-11] it failed to achieve cost-effectiveness or monetary savings apart from in a sub-analysis of people aged over 80 years [9]. Economic evaluations [7,12] based on a systematic review by the Cochrane Collaboration [13] have explored the impact of falls on health-related quality of life. One evaluation included group-based exercise and demonstrated cost-effectiveness for high risk populations only [12]. There are limited published economic evaluations of group-based exercise for falls prevention, a more suitable format for population level delivery. The purpose of this study was to determine cost-effectiveness of one group-based exercise program that has been proven effective in reducing falls [14,15]. BODY.METHODS.STUDY OVERVIEW: The objective of this evaluation was to determine whether a group-based exercise program is cost-effective compared to routine care to prevent falls among the older community-dwelling population. A decision analysis was performed using a decision tree model. The evaluation was conducted from a healthcare system perspective as significant drivers of cost are within the healthcare sector. As health-related quality of life is an important outcome of falls prevention, a cost-utility analysis (CUA) was undertaken as the primary analysis. This analysis also allows broader comparisons to other healthcare programs. Secondary cost-effectiveness analyses (CEA) include the incremental cost per fall, injurious fall, and fracture averted. Measures of falls, fall-related injuries and resource use for the economic evaluation were obtained directly from trial data over an 18 month time horizon corresponding to the duration of follow-up. This was supplemented by literature-based utility measures. All costs were identified from the perspective of the healthcare system and converted from 2010 Australian Dollars to British Pound Sterling (GBP) using 2010 purchasing power parities. All costs and consequences were discounted at a rate of 3% in the base case analysis, as recommended by the Panel on Cost-effectiveness in Health and Medicine [16]. BODY.METHODS.INTERVENTION: This evaluation was based on the results of a previously published randomised controlled trial of the "NoFalls" exercise program [14,15]. The exercise program consisted of a weekly one hour group-based exercise class for 15 weeks, supplemented by daily home exercises. The class consisted of graded exercises to improve flexibility, leg strength and balance [14,17]. The comparator is routine care and activity, considered standard care. Approval for the "NoFalls" trial was obtained from the Monash University's Standing Committee on Ethics in Research Involving Humans. The "NoFalls" trial took place in metropolitan Melbourne, Australia with a community-dwelling population aged over 70 years, recruited from the electoral roll. One thousand one hundred and seven participants were randomised, with 17 withdrawing immediately post-randomisation. Five hundred and forty-one participants were randomly assigned to receive the exercise intervention and 549 to receive no exercise intervention. The original "NoFalls" trial also investigated the effect of home hazard management and vision improvement on falls prevention in a full factorial design. However these interventions on their own failed to show a significant effect so have been excluded from this evaluation. During the 18 month follow-up period 9.5% of participants withdrew and 1.4% deceased, however data was provided for at least 1 month by 98.5% of randomised participants. The study participants at baseline had a mean age of 76.1 years and were 59.8% women. Baseline demographics between groups were similar [14]. Participants in the exercise group fell at a rate 21% lower than those in the routine activity group (IRR:0.79, 95% CI 0.67 to 0.94) [15]. BODY.METHODS.ANALYTICAL FRAMEWORK: A decision tree model was used in the decision analysis to establish potential pathways of participants (Figure 1). A negative binomial regression model was used to calculate the rate of falls in each group and to determine the rate of injury if a fall occurred, using PASW Statistics 18 [18]. Negative binomial regression is recommended for analysis of falls count data as falls are recurrent and data over-dispersed (i.e. variance is greater than the mean) [19,20]. The model involved entering the surveillance period as the offset variable, number of falls as the dependent variable, and the intervention as the categorical variable. Additionally the number sustaining an injury if a fall occurred was substituted as the dependent variable. Interactions with age and gender were also explored. The rates obtained from the analysis were converted to probabilities over 18 months for use in the economic evaluation.Figure 1 Decision tree. All analyses were performed by intention to treat with statistical significance set at p ≤ 0.05. The decision analysis was performed using Microsoft Excel 2007 with Risk Solver Platform V10.0 [21]. A sub-group analysis of women only was performed in keeping with suggestions that women have a higher risk of falls and related injury [1,4]. Results are reported in the form of an incremental cost-effectiveness ratio (ICER), calculated by dividing differences in costs by differences in effects of intervention and comparator. BODY.METHODS.ANALYTICAL FRAMEWORK.SECONDARY CEA: Falls were recorded prospectively for up to 18 months via monthly post-card calendar to optimise accuracy and injuries self-reported via telephone interview. Good agreement has been demonstrated between self-report and medical records, and if anything self-reporting tends to result in under-reporting [22]. A fall was defined as "losing your balance such that your hands, arms, knees, bottom or body touch or hit the ground or floor" [14]. Injuries were classified as a fracture, cut/scrape/bruise, head injury or other injury. Falls data was obtained for 92% of possible surveillance points over 18 months. BODY.METHODS.HEALTH-RELATED QUALITY OF LIFE (HRQOL): Health-related quality of life is measured on a scale of 0 (death) to 1 (perfect health) using a utility instrument which measures individual strength of preference between alternative health states [16]. The EuroQol (EQ-5D) is a valid and reliable instrument widely used to obtain utility weights for fall-related conditions in older populations [23,24]. Utility weights are combined with time spent in different health states to calculate quality-adjusted life years (QALYs) [16]. An utility instrument such as the EQ-5D was not used during the "NoFalls" trial. Therefore utility weights from this instrument have been sourced from published results for similar populations (Table 1).Table 1 Utility value of health states Utility Value Utility applied to 18 month model Source No fall11Fear of falling0.94Iglesias [25] (EQ-5D)Fall0.997Fall – including proportion fear of falling0.99336 (overall)Probability of FOF: Freidman [26]0.98466 (women)Murphy [27]Hip fracture0.73Peasgood [28]Year 10.7(95% CI: 0.64 - 0.77) (EQ-5D)Year 20.8(95% CI: 0.68 - 0.96) (EQ-5D)Shoulder fracture0.940.94National Osteoporosis Foundation [29] (EQ-5D)Wrist fracture0.9560.969 (overall)*Peasgood [28]0.966 (women)(95% CI: 0.86 – 1.00) (EQ-5D)Other fracture0.940.958 (overall)National Osteoporosis Foundation [29] (EQ-5D)0.955 (women)*Disutility of a wrist and other fracture is applied for 12 months and for the remaining 6 months of the 18 month time horizon a proportionate disutility of fear of falling is applied. Literature-based utility estimates for injuries other than fractures are limited, therefore equivalent utility was applied to a fall unless a fracture sustained. Utility was applied evenly to a fracture regardless of management. Although inpatient care may result in higher utility loss this could not be differentiated from the literature. Whilst research continues into the social value of a QALY and how it varies with age [30], a baseline utility of 1 was allocated to a non-faller indicating normal age-specific health. In reality the baseline utility of the elderly is below this value and it has been suggested that using a baseline utility value of 1 provides an overestimate and age/sex norms that exist for instruments such as the EQ-5D should be used instead [31]. This then raises a concern that the value of health benefit such as the QALY depends on when it arises and who receives it [32], making people with higher baseline utility apparently more deserving of a QALY than others. Applying a value of 1 as the baseline utility is more equitable when it comes to making comparisons of programs and their incremental QALY gain. The QALY calculation assumes that falls reduction applies to the 18 month follow-up after which participants return to their pre-treatment falls risk. There is no evidence supporting longer term effects once an exercise program has ceased [33]. Evidence also suggests that following a fracture there is a return to the previous level of utility within 1 to 2 years [28]. The probability of developing fear of falling (FOF) was obtained from studies with similar populations [26,27]. Persistence of FOF is recognised [25] therefore disutility associated with FOF was applied for the 18 month period for those who fell. FOF was applied equally to both groups, although there is evidence to suggest that exercise interventions can reduce FOF in this population [34]. A higher rate of FOF has been associated with more injurious falls [35], but as no clear incidence is available the probability of FOF was applied evenly. Probability of developing FOF was increased in the women only sub-analysis in accordance with the evidence available [26,27]. The probability of developing FOF and utilities applied are conservative, most likely underestimating the true impact of the exercise program. BODY.METHODS.COSTS.PROGRAM IMPLEMENTATION: Program costs included labour, equipment, venue hire, music and consumables (Additional file 1: Table S1). Research protocol driven costs were excluded. The cost of training staff was excluded as their classifications indicate they are already qualified for their role. An Allied Health Assistant (AHA) was designated as group instructor in the base case analysis due to their prevalence in Australian healthcare settings. A fitness instructor could run this program at a cheaper hourly rate and was included in the sensitivity analysis. Labour was valued by hourly wage plus 50% on-costs. For each 1 hour session 1.5 hours labour was allocated, and 5 hours labour per group for screening potential participants. The costs of recruiting each group for this program are unknown. An estimate of advertising was included in the base case analysis. Advertising in a local newspaper is unlikely to be required but is included in a sensitivity analysis. BODY.METHODS.COSTS.COST OF FALLS: Resource utilisation was self-reported via telephone interview following a fall for 93% of reported falls, but could not be verified against healthcare records as permission was not obtained for this trial. General Practitioner consultations, ambulance services, Emergency Department visits and hospital admissions were included as fall-related costs (Additional file 2: Table S2). Outpatient utilisation of Allied Health, investigations, medication and specialists was not available from the trial. As hospital inpatient services are major cost drivers following falls in this age group [9,36,37] exclusion of outpatient services was not expected to alter cost-effectiveness substantially, but was explored in a sensitivity analysis. Consultation with a General Practitioner was recorded but not occasions of service, so this is guided by the literature available [38,39]. It was estimated that on average 3 visits would be required following a fracture and 2 visits following other injuries in the base case analysis. Standardised prices or charges were applied to resources used. Whilst using charges instead of actual costs may not reflect the true opportunity cost of resources, applying standardised charges is more relevant for the healthcare system perspective and improves generalisability. Standardised prices used include Commonwealth Medicare Benefits Schedule (CMBS) [40] fees for General Practitioner consultations, an average of ambulance service costs obtained from the Private Health Insurance Administration Council (PHIAC) [41], Australian Ambulatory Classes [42] for Emergency Department visits, Australian Refined Diagnostic-Related Group (AR-DRG) cost weights [43] per hospital admission, and the Victorian Casemix Rehabilitation and Funding Tree (CRAFT) [44] for rehabilitation costs. BODY.METHODS.SENSITIVITY ANALYSIS: A Monte Carlo Simulation was utilised in a probabilistic sensitivity analysis, and a cost-effectiveness acceptability curve constructed to indicate the probability of cost-effectiveness at any given value of willingness-to-pay [16]. 10,000 trials were run per simulation, with 56 uncertain variables covering the probabilities of each outcome, resource use and utility (Additional file 3: Table S3). The discount rate of costs and consequences was varied to 0 and 5% in the sensitivity analysis to allow broader interpretation of the results. A threshold analysis was conducted to ascertain the falls rate reduction required to reach a cost-effectiveness threshold of GBP£20,000 to £30,000 per QALY [45] or to break even (cost-neutral). In further sensitivity analyses key inputs for program implementation were varied including the use of a fitness instructor, no opportunity cost of the venue or equipment and inclusion of local newspaper advertising. A gross outpatient cost incorporating Allied Health, General Practitioner consultations, specialists and pharmaceuticals was applied to investigate the effect of their exclusion on the ICER. The overall estimate was based on previously published cost estimates of falls [36,37]. The effect of missing falls surveillance data was explored in sensitivity analyses. In the most-likely scenario it was assumed that, in the absence of any intervention, participants with missing data would continue to fall at the baseline falls rate. In a worst-case scenario, it was assumed no change for exercise group participants with missing data, and no falls experienced by routine care group with missing data, and the reverse in a best-case scenario. The 17 participants who were randomised but did not commence the trial were included in this analysis. BODY.RESULTS.EFFECTIVENESS: Altogether there were 1448 falls recorded by the 1090 participants in the trial, 803 resulting in an injury. The rate of falls per year in the exercise group was 0.309 compared to 0.390 in the routine activity group (IRR:0.79, 95% CI 0.67 to 0.94) [15]. The injurious fall incidence rate ratio was 0.85 (95% CI 0.70 to 1.04) [15], just failing to reach statistical significance most likely as the RCT was powered to detect a difference in the falls rate rather than the injurious falls rate. However when a fall occurred there was compelling evidence showing no significant difference in the rate of injury in the routine activity compared to the exercise group (combined group: IRR 0.962, 95% CI: 0.76 to 1.21; women only group: IRR 0.989, 95% CI: 0.75 to 1.23). Subsequently a linear relationship was assumed between falls and injury, falls data pooled and equal transition probabilities applied to both groups after a fall (Additional file 4: Table S4). There was no significant difference in the falls rate reduction in the women only analysis compared to the overall group. However compared to women, men had a 44% lower rate of injury when they fell (IRR 0.56, 95% CI: 0.44 to 0.72). Of all falls 55.5% resulted in injury. Fractures resulted from 2.5% of falls (3.7% for women). Altogether 36 fractures were recorded, 6 of which were hip fractures. 21.7% of injurious falls required medical care, and 3% of injuries required hospital admission. The most common injury sustained was a cut, scrape or bruise (73.2% of all injury), 91% of which did not require medical care. BODY.RESULTS.COSTS: In the base case analysis with an AHA instructor the class cost £52.37 per participant, assuming 15 participants per instructor. In the sensitivity analysis this was varied to £36.09 with AHA instructor excluding venue hire and annual equivalent cost of equipment, £45.52 employing a fitness instructor or £29.24 excluding venue hire and annual equivalent cost of equipment. Non-discounted effects and costs per individual are presented in Table 2. As women are more likely to sustain fall-related injuries the cost of a fall and associated utility loss is higher for women. The estimated cost of each health state (excluding program cost) applied to the decision tree model is presented in Table 3.Table 2 Non-discounted individual costs and effects, "NoFalls" exercise program Group Effects Costs (2010 GBP) QALY Probability fall Probability injurious fall Probability fracture Exercise Program 1.495300.3710.2060.009£32.61 + program costWomen only1.490060.2260.014£46.08 + program cost Routine Activity 1.494380.4430.2460.011£38.94Women only1.488130.2700.016£55.03Table 3 Estimated cost of health states (excluding program cost) Health state Cost (GBP 2010) Hip fracture£6611Shoulder fracture - inpatient care£8224- ambulatory care£46Wrist fracture - inpatient care£3,219- ambulatory care£46Other fracture - inpatient care£4762- ambulatory care£60Cut / scrape / bruise - inpatient care£1645- ambulatory care£39Head injury - inpatient care£2303- ambulatory care£75Other injury - inpatient care£4790- ambulatory care£48 BODY.RESULTS.COST-EFFECTIVENESS: A summary of incremental cost-effectiveness ratios (ICERs) is shown in Table 4. The overall base case incremental cost per QALY is well above the accepted cost-effectiveness threshold. In the best-case scenario with a Fitness Instructor and minimised program implementation costs it is possible for the incremental cost per QALY to fall within the acceptable range. For women only the ICER is more favourable with all analyses falling within or below the cost-effectiveness threshold.Table 4 Summary ICER and sensitivity analysis, "NoFalls" exercise program Cost scenario GBP (2010) Mixed gender Women only Effect: Incremental QALYs 0.0009/0.0019 (women only) Incremental cost per QALY (Value at 95% Confidence) AHA - base case (incremental cost £45.87/£43.31 - women only)£51483 (£99664)£22986 (£48212)AHA - no venue and minimal equipment cost (incremental cost £29.68/£27.13 - women only)£33316 (£65218)£14397 (£30373)Fitness instructor – base case (incremental cost £ 39.06/£36.51 - women only)£43845 (£84399)£19375 (£41002)Fitness instructor – no venue and minimal equipment cost (incremental cost £22.88/£20.32 - women only)£25678 (£50649)£10786 (£23645) Effect: Incremental falls averted 0.0703 (mixed gender and women only) Incremental cost per fall averted AHA - base case£652£616AHA - no venue and minimal equipment cost£422£386Fitness instructor – base case£556£519Fitness instructor – no venue and minimal equipment cost£331£289 Effect: Incremental injurious falls averted 0.039/0.043 (women only) Incremental cost per injurious fall averted AHA - base case£1176£1011AHA - no venue and minimal equipment cost£761£633Fitness instructor – base case£1,002£853Fitness instructor – no venue and minimal equipment cost£596£475 Effect: Incremental fractures averted 0.0017/0.0026 (women only) Incremental cost per fracture averted AHA - base case£26236£16581AHA - no venue and minimal equipment cost£16978£10385Fitness instructor – base case£22343£13976Fitness instructor – no venue and minimal equipment cost£13294£7780 BODY.RESULTS.SENSITIVITY ANALYSIS: Incorporating advertising costs has an impact on the ICER, but does not significantly alter cost-effectiveness. Increasing costs associated with ambulatory care according to published cost estimates had minimal effect on the ICER. This indicates that excluding outpatient services such as Allied Health, specialists and pharmaceuticals has not substantially altered results (Additional file 5: Table S5). The cost-effectiveness acceptability curves (CEAC) for the incremental cost per QALY are presented in Figures 2 and 3. The probability of reaching the accepted cost-effectiveness threshold of GBP£20,000 to £30,000 per QALY is extremely low for the overall base case utilising an AHA or Fitness Instructor, but much more encouraging for women only.Figure 2 CEAC - probability of cost-effectiveness at given value of willingness to pay, "NoFalls" Exercise Program (markers at GBP£20,000 and £30,000 per QALY). AHA: Base Case. AHA: No Venue and minimal equipment cost. Fitness Instructor: Base Case. Fitness Instructor: No venue and minimal equipment cost.Figure 3 CEAC - women only analysis, "NoFalls" Exercise Program (markers at GBP£20,000 and £30,000 per QALY). AHA: Base Case. AHA: No Venue and minimal equipment cost. Fitness Instructor: Base Case. Fitness Instructor: No venue and minimal equipment cost. BODY.RESULTS.SENSITIVITY ANALYSIS.THRESHOLD ANALYSIS: To fall within the cost-effectiveness threshold in the overall base case, the exercise program required a falls rate reduction of between 32% and 42%, assuming injury distribution remains constant. In the base case scenario employing a fitness instructor the falls rate reduction required is 28% to 37%. There is virtually no chance of the intervention breaking even or being cost-neutral, with a reduction in falls rate of over 80% required (61% for women only) in the best-case scenario. BODY.RESULTS.SENSITIVITY ANALYSIS.MISSING DATA SENSITIVITY ANALYSIS: The falls rate reduction remains statistically significant under all scenarios explored in the missing data analysis (Additional file 6: Table S6). When the baseline falls rate is applied to all missing data there is little impact on the ICER. Apart from some of the best-case analyses, the overall ICER generally remains well above the cost-effectiveness threshold. Under most conditions the ICER remains within or below the cost-effectiveness threshold for women only analyses. As the injury data was pooled and applied equally to both groups missing data may weaken the results if fall-related injuries have been underestimated but would not cause bias. BODY.DISCUSSION: Based on this evaluation there is little evidence to suggest that a group-based exercise program is more cost-effective than routine care to prevent falls among the older community-dwelling population. However there is evidence to support the program if offered to women only. This is driven by the higher probability of women sustaining a fracture in a fall resulting in higher costs and disutility, as well as the higher probability of women developing fear of falling and its associated disutility following a fall. Direct comparison of this economic evaluation to other studies is difficult due to methodological differences in perspective, time frame and cost inclusions, as well as varied contexts within overseas health systems. Nevertheless a systematic review of economic evaluations of falls prevention interventions [6] identified three cost saving interventions in subgroups of high falls risk participants. These were a targeted multi-factorial intervention in the USA [46], the home-based Otago exercise program for people aged over 80 years in New Zealand [9] and a home safety program targeting those who had previously fallen and were discharged from hospital in Australia [47]. Another UK based study investigating the cost-effectiveness of cataract surgery for falls prevention in women reported an incremental cost per QALY well below the cost-effectiveness threshold when modelled over a lifetime [48]. An Australian based economic evaluation by Church et al. [12] utilising the effectiveness data from a systematic review by the Cochrane Collaboration [13] identified that group-based exercise was only cost-effective in a high risk population. The overall falls rate reduction of 22% (IRR: 0.78, 95% CI 0.71-0.87) utilised for group-based exercise is consistent with the falls rate reduction of 21% found in the "NoFalls" trial (IRR:0.79, 95% CI 0.67 to 0.94) [15]. Despite methodological differences the results of this evaluation support the findings of Church et al. in that group-based exercise programs in the general population would not be considered cost-effective. However, group-based exercise in the study by Church et al. and in variations of program implementation in this evaluation are approaching cost-effectiveness. Both evaluations indicate that in order to be cost-effective group-based exercise programs need to target sections of the older community-dwelling population. Although the percentage of falls resulting in injury from this trial was consistent with previous reports [49,50] the proportion of fractures following a fall was lower than others reporting between 6% and 13% [35,49,50]. This may be due to differences in study populations, missing data or variations in definitions and methods used to record injurious falls [51], and could result in an underestimation of the cost of falls and therefore cost-effectiveness. The "NoFalls" trial was also powered to detect a difference in the rate of falls and had insufficient power to detect differences in less frequent injuries such as fractures. However it seems a reasonable assumption that a 15 week exercise program would not alter the fracture rate if a fall occurred. The large variation in the value of the ICER observed in the probabilistic sensitivity analysis was contributed to by the small numbers of more serious injuries such as fractures and infrequency of inpatient care. There are some limitations which should be considered. There were differences between the study population and the general community-dwelling older population which may limit the generalisability of the results to people who are Australian born, aged 70–84 and of good to excellent health [14]. The lack of culturally diverse study populations is not unusual in falls prevention literature [2]. This economic evaluation was not planned for the "NoFalls" trial so data collection was not optimal. Sourcing utility values from the literature may have introduced some inaccuracy as these were derived from other study populations, although matched as best possible. Literature based utility estimates for fall-related injuries are also limited. Mortality, lifetime costs of falls such as nursing home placement or other longer term injury complications, and societal costs to families and friends providing support following falls have not been included. Conservative assumptions regarding utilities, time horizons and costs applied to this model potentially result in underestimation of true cost-effectiveness. The cost-utility analysis is a strength of this evaluation as it allows broader comparison to other healthcare programs thereby facilitating decision making. In addition, fear of falling was incorporated as an important consequence of falling known to have a significant impact on the health-related quality of life of the older population. This study makes a contribution to the limited literature on trial based economic evaluations of group-based exercise programs for falls prevention. Whilst the limitations of the data available from the "NoFalls" trial have been acknowledged, this study has less potential for inaccuracies than those fully reliant on assumptions based on published literature and expert opinion. Further research is required with larger sample sizes to enable more accurate observation of less frequent endpoints, over longer time frames to capture the full impact of fall-related injuries and fear of falling on utility. Measuring and reporting injurious falls using standardised methodology will also further enhance the accuracy and comparability of future research [51]. Standardising methodology and improving the accuracy of results will better inform budgetary decision-making. BODY.CONCLUSION: Falls and fall-related injury significantly impact on the health-related quality of life of older people and pose a considerable burden on the healthcare system. Although group-based exercise programs have proven effectiveness in reducing the falls rate in the older community-dwelling population, this economic evaluation provides little evidence to support its cost-effectiveness in a group of mixed gender. However the evidence does suggest that a group-based exercise program is cost-effective in an older female community-dwelling population. Group-based exercise programs aimed at falls prevention in the older community-dwelling population are more likely to provide value for money when targeted at women only due to their higher likelihood of fall-related injury and fear of falling. Group-based exercise programs are potentially a valuable component of broader inter-sectoral strategies for falls prevention and improved health in the community and warrant consideration as part of an Active Ageing policy.

TITLE: Does teaching non-technical skills to medical students improve those skills and simulated patient outcome? ABSTRACT.OBJECTIVES: The purpose of this study is to evaluate the effects of a tailor-made, non-technical skills seminar on medical student's behaviour, attitudes, and performance during simulated patient treatment. ABSTRACT.METHODS: Seventy-seven students were randomized to either a non-technical skills seminar (NTS group, n=43) or a medical seminar (control group, n=34). The human patient simulation was used as an evaluation tool. Before the seminars, all students performed the same simulated emergency scenario to provide baseline measurements. After the seminars, all students were exposed to a second scenario, and behavioural markers for evaluating their non-technical skills were rated. Furthermore, teamwork-relevant attitudes were measured before and after the scenarios, and perceived stress was measured following each simulation. All simulations were also evaluated for various medical endpoints. ABSTRACT.RESULTS: Non-technical skills concerning situation awareness (p<.01, r=0.5) and teamwork (p<.01, r=0.45) improved from simulation I to II in the NTS group. Decision making improved in both groups (NTS: p<.01, r=0.39; control: p<.01, r=0.46). The attitude 'handling errors' improved significantly in the NTS group (p<.05, r=0.34). Perceived stress decreased from simulation I to II in both groups. Medical endpoints and patients ́ outcome did not differ significantly between the groups in simulation II. ABSTRACT.CONCLUSIONS: This study highlights the effectiveness of a single brief seminar on non-technical skills to improve student's non-technical skills. In a next step, to improve student's handling of emergencies and patient outcomes, non-technical skills seminars should be accompanied by exercises and more broadly embedded in the medical school curriculum. BODY.INTRODUCTION: According to an evidence-based estimate, 400,000 preventable patient deaths occur annually in US hospitals.1 In Germany, preventable deaths in hospitals are five times more frequent than deadly traffic accidents, and up to 80% of adverse events in anaesthesiology are attributed to human errors, including team performance breakdown.2 For reliable team performance and to minimise human errors in medicine and other domains such as aviation, non-technical skills (NTS) are essential.3-5 Feedback based on NTS rating systems for evaluating trainee surgeons' NTS and for improving performance, for example, are perceived as highly valuable and useful by trainees and supervisors.6 Accordingly, interventions such as "aeromedical" and "anaesthesia crisis resource management" have been developed to improve teamwork-relevant NTS (e.g., communication, decision making, situation awareness, or adaptation) and to increase team performance and safety in high-responsibility teams (HRTs).5,7-9 NTS comprise an individual's cognitive, attitudinal, and social skills that supplement the individual's task work-related expertise.5 NTS-oriented interventions are instructional strategies for HRTs to a) train the usage of all available resources efficiently (i.e., humans, equipment, and information), b) enhance teamwork and thereby performance, and c) diminish the likelihood of possible human error to mitigate consequences for humans or the environment.10 Study findings have supported the effectiveness of NTS-oriented interventions in augmenting teamwork competencies, e.g., in aviation, military, fire service, or medical teams on their reactions, teamwork safety-relevant attitudes, knowledge, and behaviour.5,10-12 For example, a meta-analysis by Salas and colleagues reported positive effects of NTS interventions on team member's reactions and teamwork safety-relevant attitudes.13 A meta-analysis conducted by O`Connor and colleagues found support for these effects.12 The reported studies demonstrated positive effects of NTS interventions on team member's reactions, teamwork safety-relevant attitudes and behaviour. Medium-sized effects were found concerning safety-relevant knowledge gain. Positive effects of teamwork competencies on team performance have also been demonstrated, with medium to large effect sizes found regarding the positive effects of team process behaviours on clinical performance measures such as task management, surgical complications, operating time, or patient morbidity.14 Thus, NTS interventions support teamwork-relevant competence acquisition, and teamwork competencies positively influence clinical performance. In a novel attempt to unify their curricular targets, German medical faculties have recently implemented the teaching of non-technical skills, and the German Association for Medical Education has recently published a "Learning Objective Catalogue for Patient Safety".15 However, limited data exists regarding the suitability and effectiveness of NTS interventions for medical students. To assess positive seminar effects on teamwork competencies for this target group, the widely used training evaluation hierarchy from Kirkpatrick (1998) is applied.16 This hierarchy categorises training outcomes on four levels. The first level comprises the evaluation of "reactions", such as subjectively perceived enjoyment and perceived usefulness of the NTS seminar. The second level is "learning", and encompasses the participant's attitudinal changes and knowledge gain after an NTS seminar. The third level is "behavioural changes", and refers to the application of acquired knowledge and skills to the job. The fourth level is "results", and refers to the benefits for the organisation, such as successful patient treatment. In light of the above, we investigated the effects of a single short NTS-oriented seminar on medical student's reactions, teamwork, safety-relevant attitudes, and non-technical skills as well as on simulated patient's outcome. Based on studies from various occupational domains that found positive changes in team member's NTS after receiving NTS training, we assume that medical students will also show a positive change concerning their NTS after receiving an NTS seminar, whereas a medical seminar control group will not show this improvement.5,8,10-13 Hypothesis 1: Medical students who receive an NTS seminar will have higher values concerning their NTS after the seminar than before the seminar. Based on previous study findings that demonstrated positive changes in team members' safety-relevant attitudes after receiving NTS training, we also assume that medical students will show a positive change concerning their teamwork safety-related attitudes after receiving an NTS seminar, whereas the medical seminar control group will not show this improvement.9-13 Hypothesis 2: Medical students who receive an NTS seminar will have higher values concerning their teamwork safety-relevant attitudes after the seminar than before the seminar. As positive effects of teamwork competencies and NTS on team performance (e.g. surgical complications, patient morbidity) have been demonstrated, we also assume that teaching NTS will lead to a more successful patient treatment after the NTS seminar, as evidenced by a comparison between the NTS seminar group and the medical seminar control group.14 Hypothesis 3: Medical students who receive an NTS seminar will show a better performance in managing a simulated patient than those who receive a medical seminar. BODY.METHODS: This study explored the effects of an NTS seminar on student's behaviours during simulations as well as medical endpoints and simulated patient's outcome. Additionally, we analysed changes in teamwork-supporting attitudes and perceived stress during both simulation scenarios. Data was collected from questionnaires, observations and the simulator software. BODY.METHODS.STUDY DESIGN AND PARTICIPANTS: This was a randomised, double-blind study with a pre-test-post-test design investigating the usefulness of NTS seminars within medical education. It was randomised because at the beginning of the semester, students were randomly assigned to either an NTS seminar group or a medical seminar group by Dean's department. It was double-blind as the students were unaware of their group allocation. Moreover, the investigators were also unaware of the student's group allocation when they conducted behavioural assessments during the simulations. The design was pre-test-post-test as we used a baseline measurement at the beginning of the study and a follow-up measurement after the NTS seminar. Thus, we were able to analyse effects over time. Based on the comparison between the NTS seminar group and the medical seminar group, we were also able to analyse between-group effects. One hundred and four 4th-year medical students participating in a two-week course on emergencies taught by the Department of Anaesthesiology & Intensive Care Medicine were initially enrolled and randomly allocated to either an NTS seminar group or a control group receiving a traditional medical seminar. Participation in the study was voluntary and had no influence on successful participation in the two-week course. We obtained oral and written informed consent from each participant, and all students received the complete course contents regardless of study participation. Thirteen participants had to be excluded from the analysis as they did not attend one of the simulations, and a further fourteen participants were excluded as they either did not attend one of the seminars or did not complete all questionnaires. Therefore, data from seventy-seven participants were ultimately analysed. The students' mean age was 25.9 years (±3.5 SD, range: 21 to 39) and thirteen students had previous experience working in the field of medicine (eight in nursing and five in emergency services). All participating students completed the same simulated resuscitation scenario at the beginning of the study, which served as baseline measurement in week one. The following week, they attended either a 90-minute seminar entitled "Factors influencing successful teamwork" (NTS group) or a medical seminar unrelated to the non-technical skills to be tested ("concepts for mass-casualty incidents", the control group, CG). On the subsequent two days, students were tested in a second simulation (post-test measurements), an anaphylactic shock scenario. To ensure that no student missed any lectures and no knowledge gaps existed, after study data sampling, students of the control cohort attended the NTS seminar, while the NTS group was instructed in mass-casualty incidents. The experimental design is depicted in Figure 1. In detail, students were welcomed individually in week one and asked to complete a questionnaire measuring teamwork safety-relevant attitudes (simulation scenario I at baseline). Afterwards, each student was accompanied to the simulation room and familiarised with the team (medical assistants performing to predefined standards) and equipment and received standardised scenario information. Each student was observed and videotaped through a one-way transparent window from a second room in order to assess NTS and key medical interventions at baseline. After the simulation scenario, students completed a second questionnaire measuring presence and perceived stress in simulation I. Before leaving, students received a short debriefing regarding their performance. In week two, NTS-group participants attended the NTS seminar (CG attended the medical seminar). At the end of the seminar, each participant filled in the Training Evaluation Inventory for assessing the seminar. On the following days, students participated in the second simulation scenario (post-test measurement) and were observed and videotaped again (same procedure as in simulation I). Subsequently, students completed questionnaires measuring the presence and perceived stress in simulation II and the teamwork safety-relevant attitudes. Again, students received a short debriefing. The study was conducted at the Department of Anaesthesiology & Intensive Care Medicine, University Hospital Essen, University of Duisburg-Essen, Germany and was approved by the University's local institutional ethics committee in Essen in September 2014. BODY.METHODS.TREATMENT: THE NTS SEMINAR DESIGN: In week two, students attended a 90-minute NTS seminar entitled "Factors influencing successful teamwork", led by the same two instructors (two authors) throughout the course. The seminar aimed to sensitise students for teamwork-supporting behaviour and attitudes towards leadership and assertiveness as well as dealing with mistakes and stress.17 Topics covered included situation awareness18 and its impact on teamwork, as well as shared mental models and strategies to improve them (e.g., loud verbalisation during work or debriefings) as prerequisites for team coordination,19 communication as a prerequisite for coordination (e.g., clear pronunciation and reconfirmation),20 and feedback rules. Students were also familiarised with possible obstacles which might impair communication such as perception and selectivity, and discussed rules for successful communication. The final exercise was a type of demonstration-based learning technique considering observational learning processes.21,22 "Observational (or demonstration-based) learning is the process of acquiring knowledge, skills, and attitudes (KSAs) through viewing examples of performance".21 The exercise required the students to point out successful and less successful behaviours regarding reliable teamwork based on a video demonstrating teamwork in a patient-handover situation between hospital staff and a helicopter emergency medical service. The critical cues from the video were subsequently discussed and the previously acquired competencies concerning the teamwork-supporting behaviour and attitudes had to be consolidated. Successful and less successful behaviours were chosen in the demonstration, as positive and negative models support the generalization of the targeted behaviours.23 BODY.METHODS.DATA COLLECTION METHODS.THE SIMULATIONS: A human patient simulator was used for assessing the student's behaviour. It was placed in a mock patient wardroom next to a bed-ridden "roommate" (manikin). With its integrated physiology software, this simulator allows for realistic scenarios adjusted to medical actions such as endotracheal intubation, defibrillation, or drug administration. Patient's comments, sounds, and vital signs were loaded wirelessly while running the scenario. All medication and equipment required for managing the simulated patient were provided. The scenarios were recorded using an audio-visual recording system, which captures physiological data including event logs, waveform displays, and annotations. Scenarios were developed and programmed by three of the authors with a strong background in handling emergencies. One author operated the simulator, the recording system and technical facilities. Figure 1Outline and timeline of experimental set-up In simulation I (10 minutes duration, pre-test measurement), a cardiovascular risk patient with sudden unconsciousness and ventricular fibrillation was portrayed and cardiopulmonary resuscitation (CPR) according to ERC guidelines was required. The simulator was programmed for a return of spontaneous circulation after the 4th defibrillation attempt if correct CPR had been performed and adrenaline and amiodarone had been administered. In simulation II (10 minutes duration, post-test measurement) a young patient recovering from surgery for radial fracture was portrayed. Here, an anaphylactic reaction to an antibiotic infusion was presented, which progressed to anaphylactic shock if not treated early with adrenaline. Specifically, the simulation was escalated every 3 minutes to increasing symptoms (stage 1: coughing, itching; stage 2: worsening to tachycardia and hypotension; stage 3: overt shock). Treatment with adrenaline was necessary to improve the simulated patient's condition. BODY.METHODS.DATA COLLECTION METHODS.NON-TECHNICAL SKILLS: The Anaesthesia Non-Technical Skills (ANTS) observation system, including the four categories situation awareness, task management, team working, and decision making, was applied to assess the students' NTS during simulations I and II.24,25 The modified instrument for assessing Danish anaesthesiologist's NTS (ANTSdk) was not applied, as the authors stress the cultural specificity of this instrument. Denmark is considered a feminine society by Hofstede, whereas Germany is not.26 Each category consists of 3-5 elements characterised by various behavioural markers for good and poor performance (see Appendix A). The behavioural markers were adapted to the simulation scenarios. Two observers rated every student's behaviour. Due to the substantial number of behavioural markers, each observer rated half of them. All elements and categories were rated on a four-point scale from 0 to 3, i.e., from poor (performance endangered or potentially endangered patient safety, serious remediation required) to good (performance of consistently high standard, enhancing patient safety; could be used as a positive example for others).25 Element ratings were averaged, resulting in ratings for each category. BODY.METHODS.DATA COLLECTION METHODS.MEDICAL SKILLS: The completion of key treatments and their timing was recorded for each scenario (see Appendix B), e.g. "call for assistance", "application of oxygen", "time to first chest compression", "time to adrenaline administration", and "time to defibrillation". The relevant outcomes were "return of spontaneous circulation" (yes/no) and "resolution of anaphylaxis (yes/no)" in simulations I (CPR) and II, respectively. BODY.METHODS.DATA COLLECTION METHODS.TEAMWORK SAFETY-RELEVANT ATTITUDES: A 20-item questionnaire was applied before simulation I and after simulation II with a five-point Likert scale ranging from 0 (total disagreement) to 4 (total agreement). The questionnaire was tailored to the target cohort and based on established instruments.9,27-29 It covers eight of the most frequently investigated safety-relevant attitudes, i.e. command roles and responsibilities (four items, e.g., 'Team members should not question the decisions or actions of senior staff', α=.72), speaking up (two items, e.g., 'I inform other team members when my workload is too high', α=.56), debriefing (two items, e.g., 'A regular debriefing of procedures and decisions after an emergency care is an important part of teamwork', α=.42), feedback and critique (two items, e.g., 'Disagreements in the team are appropriately resolved, i.e., it is not 'who' is right but what is best for the patient', α=.21), realistic appraisal of stress (three items, e.g., 'Personal problems can adversely affect my performance', α=.76), denial of stress (three items, e.g., 'A professional doctor can hide personal problems during the whole emergency care', α=.71), handling errors (two items, e.g., 'I am more likely to make errors in tense or hostile situations', α=.69), and teamwork (two items, e.g., 'I enjoy working in a team', α=.40). BODY.METHODS.DATA COLLECTION METHODS.PRESENCE WITHIN THE SIMULATION SCENARIO: The instrument "Presence for lab-based microworld research" (PLBMR) was used to measure the immersion in the two scenarios.30 The questionnaire uses a six-point Likert scale from 0 (is not true) to 5 (is fully true). An example item is 'I felt like I was part of the simulation context' (Cronbach's α=.83). BODY.METHODS.DATA COLLECTION METHODS.PERCEIVED STRESS: Five items were developed to measure perceived stress (five-point Likert scale from 0 (total disagreement) to 4 (total agreement), e.g. 'In retrospect, during the simulation scenario I worked up a sweat', Cronbach's α=.85). BODY.METHODS.DATA COLLECTION METHODS.TRAINING EVALUATION INVENTORY: The 17-item Training Evaluation Inventory (TEI) was applied to assess the student's reactions and subjectively rated learning success after the NTS seminar (five-point Likert scale from 1 to 5).31 It covers seminar outcomes based on the reaction level, which included reported enjoyment (three items, e.g., 'I enjoyed learning', α=.8), perceived difficulty (three items, e.g., 'I understood all technical terms', α=.69), and perceived usefulness (four items, e.g., 'The training is useful for my profession', α=.93).10,32-33 Additionally, the learning level of Kirkpatrick's levels of evaluation is assessed.16 Learning is divided into knowledge (three items, subjectively rated learning success, e.g., 'I think my knowledge has been expanded in the long term', α=.66) and attitudes (three items, e.g., 'I would recommend this training to my colleagues', α=.87). BODY.METHODS.DATA ANALYSIS: To ensure that the NTS group and control group did not differ before the seminar intervention, t-tests for unpaired samples were performed to detect significant differences in demographic or study variables. The prerequisites concerning normal distribution and homogeneity of variance (Levene's test) for the scales and groups were met. To analyse changes over time and between groups regarding NTS and attitudes, univariate analyses of variance with repeated measures were conducted. To analyse differences between groups concerning simulated patient outcome, continuous values were analysed by t-tests. If the assumption of normality was not met, the Mann-Whitney U test was conducted. Categorical values (yes/no) were compared using a Chi-squared test. Analyses were performed using SPSS, Version 23.0, and statistical significance was assumed using an a priori alpha error less than 0.05. BODY.RESULTS: Before presenting the results of the hypothesis-testing, descriptive data of study variables are reported as well as the level of significance, to demonstrate that the study results are not systematically influenced by previous differences between groups. The NTS seminar group and the control group were comparable, as no significant differences in age, semester, presence, stress, attitudes, and NTS at baseline (simulation I) were revealed, as depicted in Appendix C. BODY.RESULTS.TREATMENT CHECKS: Presence and perceived stress during simulations: Group means and standard deviations during simulation I (pre) and II (post) as well as the average over both groups (total) are displayed in Table 1. Both groups showed a moderate presence during both simulations (pre and post), without significant differences between groups (p=.11), and the presence remained stable over time, as no changes from pre to post were detected (p=.94). Both groups also showed a moderate level of stress during simulations I and II, without significant differences between groups (p=.10). However, perceived stress changed over time (F(1,75) = 10.11, p<.01, η2p=0.12): T-tests for dependent samples revealed a significant decrease from pre to post for the NTS group (t(42) = 2.19, p<.05, r=0.32) and the control group (t(33) = 2.26, p<.05, r=0.37), with a small to medium effect size. Subjectively perceived outcomes of the NTS seminar were assessed by applying the Training Evaluation Inventory scales regarding reaction and learning. The students assessed their enjoyment (M=3.90, SD=0.85), their perceived usefulness of the seminar (M=3.98, SD=0.94) and their acquired knowledge (M=3.75, SD=0.67) rather positively and considered the seminar to be easy to follow (M=1.37, SD=0.42). Only the attitudes towards the contents of the seminar (M=2.58, SD=0.71) were evaluated as moderate to negative. Overall, the seminar was positively evaluated. Table 1 Mean values and standard deviations regarding presence and perceived stress concerning simulation I (pre) and II (post) Groups   Presence   Stress   M SD   M SD Total pre 3.41 ±1.02   2.82 ±0.49 post 3.40 ±1.06   2.58 ±0.56 NTS group pre 3.55 ±1.02   2.75 ±0.55 post 3.54 ±1.07   2.51 ±0.62 Control group pre 3.23 ±1.00   2.90 ±0.39 post 3.22 ±1.05   2.68 ±0.46 Note: Scale ranges from 0 to 5 for the presence and from 0 to 4 for stress. BODY.RESULTS.TESTING HYPOTHESIS 1: IMPACT OF NTS SEMINAR INTERVENTION ON NON-TECHNICAL SKILLS DURING SIMULATIONS: The results address whether the NTS of the NTS seminar group improved from simulation I (before seminar intervention, pre) to simulation II (post), compared to the control group. Mean values and standard deviations for all four NTS categories for both groups and both performance assessments (pre and post) are displayed in Table 2. Regarding situation awareness, the main effect for time of measurement was significant, with situation awareness improving significantly from simulation I to II (F(1,75) = 12.83, p<.01, η2p=0.51). Situation awareness improved only in the NTS group (t(42) = -3.74, p<.01, r=0.50). Team working improved significantly from pre- to post-test (F(1,75) = 11.19, p<.01, η2p=0.13). The parameter estimation for simulation II (post) showed a significant difference between the NTS and control group (p<.05). T-tests for dependent samples showed a significant improvement from pre to post only for the NTS group (t(42) = -3.27, p<.01, r=0.45). Regarding task management, no significant effects were found. Regarding decision making, the main effect for time of measurement was significant, with decision making improving significantly from pre to post (F(1,75) = 16.48, p<.01, η2p=0.18). Results of t-tests for dependent samples showed that both the NTS group (t(42) = -2.75, p<.01, r=0.39) and the control group (t(33) = -3.00, p<.01, r=0.46) improved significantly. Thus, hypothesis 1 was predominantly supported, as three NTS changed significantly from simulation I to simulation II within the NTS group. BODY.RESULTS.TESTING HYPOTHESIS 2: IMPACT OF NTS SEMINAR INTERVENTION ON TEAMWORK SAFETY-RELEVANT ATTITUDES: Data for 'command roles and responsibilities', 'speaking up', 'debriefing', 'feedback and critique', 'realistic appraisal of stress', and 'denial of stress' showed no significant effects between or within groups over time (.06<p<.81) (see Table 2). The attitude concerning 'Handling errors' improved significantly within the NTS group from simulation I to II (t(42) = -2.33, p< .05, r = 0.34), and did not improve overtime in the control group (t(33) = -0.90, p=.37, r=0.15). 'Teamwork' decreased significantly over time in the NTS group (t(42) = 2.26, p = .03, r = 0.33), but not in the control group (t(33) = 1.15, p=.26, r=0.20). Means and standard deviations for all attitudes for both groups and both simulations (pre and post) are provided in Table 2. Thus, hypothesis 2 was not supported, as only one significant positive attitude change was detected within the NTS group. Table 2 Non-technical skills (NTS) and attitudes for simulation I (pre) and II (post) Groups Pre/ post NTS Attitudes Means SD Means SD     Situation awareness Command roles and responsibilities NTS group pre 1.17 ±0.50 3.23 ±0.50 post 1.47 ±0.48 3.24 ±0.43 Control group pre 1.18 ±0.43 3.23 ±0.34 post 1.33 ±0.58 3.38 ±0.44     Team working Speaking up NTS group pre 1.37 ±0.57 2.81 ±0.71 post 1.63 ±0.36 2.85 ±0.60 Control group pre 1.31 ±0.53 2.75 ±0.68 post 1.43 ±0.43 2.82 ±0.57     Task management Debriefing NTS group pre 1.40 ±0.65 3.71 ±0.40 post 1.50 ±0.52 3.57 ±0.52 Control group pre 1.30 ±0.56 3.56 ±0.53 post 1.43 ±0.54 3.59 ±0.54     Decision making Feedback and critique NTS group pre 0.90 ±0.58 3.63 ±0.54 post 1.15 ±0.48 3.58 ±0.76 Control group pre 0.85 ±0.58 3.59 ±0.56 post 1.15 ±0.49 3.76 ±0.50       Realistic appraisal of stress NTS group pre - - 2.76 ±0.86 post - - 2.88 ±0.82 Control group pre - - 2.74 ±0.59 post - - 2.54 ±0.84         Denial of stress * NTS group pre - - 1.92 ±0.99 post - - 1.78 ±0.95 Control group pre - - 1.81 ±0.78 post - - 1.79 ±0.91         Handling errors NTS group pre - - 2.49 ±0.87 post - - 2.70 ±0.69 Control group pre - - 2.76 ±0.63 post - - 2.87 ±0.56         Teamwork NTS group pre - - 2.70 ±0.63 post - - 2.51 ±0.69 Control group pre - - 2.71 ±0.64 post - - 2.60 ±0.69 Note: Scale for assessing NTS ranges from 0 to 3; and scale for assessing attitudes ranges from 0 to 4; *Low values indicate a positive attitude. BODY.RESULTS.TESTING HYPOTHESIS 3: IMPACT OF NTS SEMINAR INTERVENTION ON SIMULATED PATIENT OUTCOME: Student`s management skills during a resuscitation scenario I (before the seminars) and an anaphylactic shock scenario II (after the seminars) were assessed using videotapes of 154 simulations (77 students experiencing both scenarios) using predefined variables and endpoints, e.g., "time until calling a chief resident or a resuscitation team" or "time until return of spontaneous circulation". All results (either mean values and standard deviations or percentage, as well as levels of significance) are presented in Table 3. The groups were comparable at baseline (scenario I), as no significant differences between the groups were detected. A comparison of results for simulation II did not reveal a significant intervention effect on simulated medical outcomes. Hypothesis 3 was therefore not supported. Neither the variable "patient ́s condition improved" nor "time until patient ́s condition was improved" differed between groups following the intervention (Table 3). In the anaphylactic shock scenario, 60.5% (26 of 43) and 61.8% (21 of 34) of simulated patients died in the NTS and control group, respectively, indicating that the scenario had been properly calibrated to detect intervention effects if present. BODY.DISCUSSION: This study demonstrates a positive impact of a single NTS seminar on student's NTS since only the student group that had received the NTS seminar improved significantly from simulation I to II. Nevertheless, this did not translate into the students' teamwork safety-relevant attitudes, and no statistically significant benefit was apparent regarding simulated patient's medical outcome. Overall, the students perceived the simulations as quite realistic and not too stressful. The NTS seminar was well accepted, with the students rating their enjoyment and the perceived usefulness very positively. Although they did not express a great deal of liking for the seminar contents, this is not especially important regarding competence acquisition, as the perceived usefulness is the most important predictor of motivation and intention to apply learned knowledge and skills at work.31,32 The students extended their knowledge regarding safety-relevant teamwork competencies, as 75% of the NTS items improved significantly in students who had received the 90-minute NTS seminar, including a demonstration-based learning approach. On decision making, the control group's behaviour also improved significantly from simulation I to II. Since students are not yet experts and still have to acquire technical knowledge, the effects of the NTS seminar on their NTS are even more remarkable. In contrast, no significant influence of the NTS seminar on teamwork safety-relevant attitudes was found between groups or over time. However, the fact that not all assessed items showed significant results is not unusual, and many studies addressing the impact of an NTS seminar on attitude changes have failed to find significant effects.34 Our results show that a change in the student's behaviour, the NTS, occurred without concurrent changes in their attitudes. Thus, Kirkpatrick's assumption of the hierarchy of the four levels of evaluation is again disproved.35 In this study, we also tried to assess whether the NTS seminar was able to affect simulated patient's medical outcomes due to better coordination and leadership behaviour during teamwork, the fourth level in Kirkpatrick's model of evaluation. While simulated patient outcomes (e.g. variables such as "patient ́s condition improved" or "time until patient ́s condition was improved") did not differ between groups in simulation II and were, therefore, unaffected by the NTS seminar intervention, we observed deficits regarding medical treatment in both groups. About 60% of simulated patients died in both groups. This lack of effect might be due to the students` limited previous practical training to manage emergencies. Alternatively, the medical tasks required to treat simulated patients in our scenarios successfully might have been too complex for the students, although 4th-year medical students should have already acquired the proper theoretical knowledge. Since the theoretical knowledge did not seem to have been transferred to practice, the students' limited abilities may have masked any effect of the acquired NTS on simulated patient treatment. Table 3 Results of comparison between NTS group and control group regarding performance of treatment steps in both simulation scenarios Resuscitation scenario I Treatment steps NTS Group Control Group Significance Call for help (yes/no)   Yes: 51.20% No: 48.80% Yes: 35.30% No: 64.70% χ 2 (1) = 1.94, p = .16 Return of spontaneous circulation (yes/no) Yes: 39.50% No: 60.50% Yes: 32.40% No: 67.60% χ 2 (1) = 0.42, p = .52 Time until emergency call(s) 303.00 ± 39.90 335.80 ± 41.20 U = 120.00, z = -0.43, p = .68 Time until first defibrillation(s) 184.80 ± 8.60 178.40 ± 11.20 U = 698.50, z = .72, p = .47 Time until return of spontaneous circulation(s) 435.00 ± 26.80 466.00 ± 21.50 t (26) = 0.83, p = .41 Anaphylactic shock scenario II Treatment steps NTS Group Control Group Significance Call for help (yes/no) Yes: 46.50% No: 53.50% Yes: 23.50% No: 76.50 % χ 2 (1) = 4.33, p = .04 Provided oxygen therapy via non-rebreathing mask (yes/no) Yes: 93.00% No: 7.00% Yes: 94.10% No: 5.90% χ 2 (1) = 0.04, p = .85 Adrenaline administration (yes/no) Yes: 48.80% No: 51.20% Yes: 64.70% No: 35.30% χ 2 (1) = 1.94, p = .16 Antihistamine administration (yes/no) Yes: 69.80% No: 30.20% Yes: 67.60% No: 32.40% χ 2 (1) = 0.04, p = .84 Initiation of blood volume expansion therapy (yes/no) Yes: 55.80% No: 44.10% Yes: 67.60% No: 32.40% χ 2 (1) = 1.12, p = .29 Patient´s condition improved (yes/no) Yes: 39.50% No: 60.50% Yes: 38.20% No: 61.80% χ 2 (1) = 0.01, p = .91 Time until emergency call(s) 318.40 ± 34.40 356.90 ± 49.90 t (27) = 0.60, p = .55 Time until oxygen administration(s) 152.30 ± 17.20 153.50 ± 18.90 U = 623.50, z = 0.41, p = .69 Time until adrenaline administration(s) 322.50 ± 20.80 336.60 ± 27.30 t (41) = 0.41, p = .69 Time until antihistamine administration(s) 247.20 ± 18.70 189.60 ± 13.90 U = 470.50, z = 2.26, p = .02 Time until initiation of blood volume expansion therapy(s) 219.00 ± 21.00 285.00 ± 21.00 U = 167.50, z = -2.31, p = .02 Time until patient´s condition improved(s) 273.10 ± 13.70 241.80 ± 14.30 t (28) = -1.56, p = .13 Although no significant impact on patient outcomes was observed under the experimental conditions chosen, our results are nevertheless meaningful, as such analyses are often missing in non-technical skills training research.5 Possibly, if simulation training were extended and broadly integrated into the student's medical curricula, NTS seminars might be more effective. As the NTS seminar, with duration of 90 minutes, was a rather short intervention, large effects might not be expected. Furthermore, it was a knowledge-based NTS seminar with demonstrated examples, and its learning goals were to enhance the student's knowledge about teamwork-relevant skills. From training research, it is well known that knowledge-based interventions do not, or only minimally, influence behaviour compared to demonstration-based or practice-based interventions.36 It is therefore recommended that future studies analyse NTS seminars of longer duration (possibly over two or more days) combined with practice sessions to achieve both behavioural learning goals and a transfer concerning patient treatment. Such simulations should be used not only to assess the student's behaviour but also as practical training sessions including debriefings, as simulation-based learning enhances the educational curriculum results such as clinical competency.37 A particular asset of our study regarding internal validity lies in its randomised, blinded, and pre-test-post-test design as well as in the inclusion of an NTS seminar group and a control group for analysing between-group effects.38 Unfortunately, this approach is often lacking in studies addressing seminar or training effects.34,39,40 BODY.DISCUSSION.FUTURE RESEARCH: Our findings suggest that studies which include simulation sessions as practice sessions added to a single seminar design are desirable. This would enable an analysis of the number of practice sessions required to affect behaviour and patient treatment. It would also be interesting to analyse whether several simulation sessions including debriefings are superior to an NTS seminar on patient outcome. Such a study design might show that practical session and debriefed problem-based learning are required rather than theoretical inputs such as seminars. If this is the case, an adapted medical school curriculum might only contain well-designed and debriefed simulation sessions but no knowledge-based seminars. The simulation sessions, and possibly the particular scenarios, functioned as stressors for the students. Since stress might enhance the retrieval of learned knowledge, but too much stress might decrease memory capacity,41,42 future studies should determine suitable simulation scenarios for the respective student cohorts so that the requirements do not overwhelm the student's abilities. Furthermore, it might be desirable to measure cortisol concentrations as a more objective biological indicator of the stress responses. Finally, it seems prudent to analyse effects of a more extensive NTS training on the performance of medical students when completing tasks of which they have better mastery. In this respect, a study on medical students in their internship year would appear to be most appropriate. BODY.CONCLUSIONS: In summary, although seminar-based NTS training demonstrated many positive sequelae, such training without practice appears to be only partially effective, as the NTS seminar showed no statistically significant improvement in simulated patient's outcome. However, if such NTS courses are to be embedded into medical schools' standard curricula to improve student's handling of emergencies, a curricular design that combines practical simulation sessions with debriefings along with seminar sessions appears to be crucial. BODY.CONCLUSIONS.CONFLICTS OF INTEREST: The authors declare that they have no conflict of interest.

TITLE: Comparison of ABSTRACT.BACKGROUND: Orgasmic disorder can create a feeling of deprivation and failure and provide mental problems, incompatibility and marital discord. This study aimed to compare the effects of Elaeagnus angustifolia flower extract and sildenafil citrate on female orgasmic disorder in women in 2013. ABSTRACT.METHODS: In this randomized clinical trial, 125 women between 18-40 years old who suffered from orgasmic disorder were divided into three E. angustifolia, sildenafil citrate and control groups. The data were gathered using Female Sexual Function Index and through measurement of TSH and prolactin. The first intervention group had to consume 4.5 gr E. angustifolia extract in two divided doses for 35 days and the second one had to use 50 mg sildenafil citrate tablets for 4 weeks one hour before their sexual relationship. However, the control group had to consume the placebo. The data were analyzed using paired t-test, one-way ANOVA, and Bonferroni posthoc test and p<0.05 was considered significant. ABSTRACT.RESULTS: The frequency of orgasmic disorder before the intervention was 41.5%, 40.5%, and 57.1% in E. angustifolia, sildenafil citrate, and control groups, respectively (p=0.23). However, these measures were respectively 29.3%, 16.7%, and 50% after the intervention (p=0.004). A significant difference between the two groups regarding sexual satisfaction after the intervention (p=0.003) compared to the beginning of the study (p=0.356). Besides, the highest reduction of changes after the intervention (58.82%) was observed in the sildenafil citrate group. ABSTRACT.CONCLUSION: Both E. angustifolia extract and sildenafil citrate were effective in reduction of the frequency of orgasmic disorder in women. BODY.INTRODUCTION: Female sexual dysfunction is a serious, common, multifactorial general health problem which is usually neglected in the general population while affecting the women's quality of life to a great extent (1). Lack of sexual health and security will result in anger, excessive rage, depression, drug abuse, lack of physical and psychological capability for parenting and child care, lack of sufficient skills for having a healthy emotional relationship, inability to flourish in the society, infanticide and even death (2). In one study conducted in Hong Kong in 2007, Zhang interviewed 1510 Chinese women who were between 19 and 49 years old and showed that 37.9% of the participants suffered from at least one sexual disorder (3). One other study in Egypt showed the prevalence of sexual dysfunction to be 76.9% among the studied women, with low sexual desire being the most prevalent one (66.4%). Besides, as the women's age increased, the prevalence of sexual disorders increased, as well (4). One study on Kurdish women in Iran indicated that 77% of the studied participants had one sexual disorder (5). Another study in Iran also revealed that 10.5% of women had never experienced orgasm (6). In the study that Safarinejad (2006) conducted on 20-60 year old women in Iran, the prevalence of low sexual desire, lack of sexual arousal, lack of orgasm, and pain during intercourse was reported as 35%, 30%, 37%, and 26%, respectively (7). Up to now, most studies regarding the pharmacological treatments for sexual dysfunction have been conducted on males and female sexual dysfunction has been less taken into account (8–10). Recently, pharmacological treatments have been considered for female sexual dysfunction mainly focusing on improvement of androgen deficiency, increase of blood flow to the genital area and stimulation of the central nervous system (11). Estrogen, progesterone, and testosterone are the main effective hormones in females' sexual response (12). In addition, androgen deficiency is one of the main causes of female sexual dysfunction (13). Thus, these compounds have been used for hormone therapy in some studies. Nonetheless, testosterone has been less welcomed due to its side-effects, including hirsutism and acne (9, 14). According to some researches, sildenafil citrate is effective in treatment of orgasm as well as arousal disorders and increase of females' sexual experiences (15–18). Sildenafil citrate is among the phosphodiesterase type 5 (PDE5) inhibitors which improve female sexual dysfunction through increasing blood flow to corpus cavernosum of clitoris, vagina and labia minor (15–17, 19–25). However, in rare cases, its consumption might be accompanied by complications, such as mild headache, urinary tract infection and nausea (14). Complementary and Alternative Medication (CAM) is also used in treatment of sexual disorders (26). Ginseng which is a Chinese traditional medication is one of the effective herbal medicines in treatment of sexual disorders (27–28). Other common medications include Yohimibine and Ginkgo biloba. However, Yohimibine has been shown to be more effective in treatment of sexual dysfunction in males as compared to females (29–31). In folk medicines, Elaeagnus angustifolia (Elaeagnaceae) is a Mediterranean medicine representative of the family Elaeagnaceae, commonly named oleaster, which is cultivated in north American, Eurasia as far southwards as Malaysia, Australia, northern regions of Asia to the Himalayas and Europe and for centuries in the desert and sub desert regions of western Asia including Iran (32–35). A previous study showed that E. angustifolia flower and its derivatives had antibacterial and antifungal properties. It also led to cutaneous wound healing by increasing re-epithelialization and collagen deposition (31). E. angustifolia flower is one of the herbal medications which, according to traditional medicine, is hot and dry, aromatic, and can stimulate sexual activity especially in young girls and women (36). It is also believed to have antioxidant effects (37). Due to its analgesic and anti-inflammatory effects, E. angustifolia extract is used in oils for external application (38). Moreover, one study showed E. angustifolia ointment to be effective in treatment of some skin disorders (39). Other studies have also confirmed that the fruit core and leaves of this plant had analgesic and anti-inflammatory effects (40–43). Furthermore, one study evaluated the effect of aqueous and alcoholic extracts of E. angustifolia on intestinal smooth muscle relaxation in mice. It indicated that the presence of flavonoids in the fruit was effective in intestinal smooth muscle relaxation. Therefore, the researcher suggests that this plant is a suitable candidate for treatment of muscle-skeletal disorders (40) due to its muscle relaxant effect. On the other hand, E. angustifolia has been found to contain flavonoid components and some flavones, such as chrysin, (5, 7-dihydroxyflavone) which have a partial agonistic effect on benzodiazepine receptors. Central benzodiazepine receptor (BDZ-R) agonists have been known to induce multiple effects including anxiolytic, myorelaxant, anticovulsant, hypnotic and amnestic effects (44–46). These two folk indications, anti-rheumatoid, anti-tetanus activities (40), anxiolytic, myorelaxant effects acting on central benzodiazepine receptors of flavonoid found in E. angustifolia, may make this plant a suitable candidate for inducing muscle relaxant activity for sexual dysfunction. Up to now, several studies have been conducted on female sexual dysfunction in Iran. However, most of these studies have focused on the prevalence and types of sexual disorders and have paid less attention to the treatment dimensions, including training, consultation and pharmacotherapy. Hence, the present study aimed to compare the effects of E. angustifolia flower extract and sildenafil citrate on female orgasmic disorder in the women referring to selected gynecology clinics affiliated to Shiraz University of Medical Sciences, Shiraz, Iran. BODY.METHODS: This study was a randomized clinical trial. Based on the study objectives and the previous studies conducted on the issue, considering error rate of 5%, power of 80%, minimum mean difference of 0.6, and variance of 0.92. A sample size of 126-subjects was selected for the study. Overall, out of the 140 qualified women entered into the study, 125 ones (41 in the E. angustifolia group, 42 in the sildenafil citrate group, and 42 in the control group) completed the study. The study population included 18-40 year old women suffering from sexual dysfunction. The health centers were selected through stratified sampling and convenience sampling was used to select the women at each center. Then, the subjects were divided into E. angustifolia flower, sildenafil citrate, and control groups using stratified block randomization. The inclusion criteria of the study were obtaining scores more than 22 in Female Sexual Function Index (FSFI), having normal menstruation, not being pregnant, not breastfeeding, without the history of heart attack, hypertension, and cardiovascular diseases, not consuming the medications which are effective in sexual function such as common antidepressants, not suffering from dyspareunia and vaginismus, not being menopausal, with the history of different types of headaches such as migraine, not using hormone drugs particularly oral contraceptive pills and lack of drug or alcohol abuse. On the other hand, the exclusion criteria of the study were showing allergic reactions to medicines and not being able to continue consuming the drugs for any reason. The data were collected through demographic information form, FSFI, and measurement of TSH and prolactin. FSFI which contains 19 items evaluates female sexual function in 6 domains of sexual desire, arousal, lubrication, orgasm, satisfaction and pain. The women were required to answer the questions according to their sexual desire and function during the past 4 weeks. In general, the scores ≤28 were considered as sexual dysfunction. Nevertheless, since assessment of pain (6 points) was omitted from the present study, score of 22 was considered instead of 28. The reliability and validity of the Persian version of FSFI were determined. The reliability of the whole questionnaire and the subscales was confirmed by Cronbach's alpha ≥0.70. Moreover, investigation of the validity of the Persian version of this questionnaire indicated a significant difference between the total mean score and the mean scores of the subscales in the two groups (p≤0.001) (47–48). After obtaining written informed consents, TSH and prolactin were assessed in all the participants in order to reject thyroid and prolactin disorders which are the secondary causes of sexual dysfunction. In this study, the samples were selected through convenience sampling and were randomly allocated into two intervention groups and a control group. The first intervention group had to consume 4.5 gr of E. angustifolia extract in two divided doses (2 capsules every 12 hr) for 35 days. The E. angustifolia capsule dosage was determined based on other studies conducted on the issue (49–50) and a pharmacognosy advisor. In the second group, the participants had to consume 50 mg sildenafil citrate tablets for 4 weeks. The dosage of sildenafil citrate was determined based on the previous studies most of which have used 50 and 100 g tablets (16, 51–53). E. angustifolia capsules were made in the Pharmacology Department under the supervision of a professional counselor. Moreover, the placebo was prepared from starch in similar packages to E. angustifolia capsules. The subjects had to consume their medications one hour before their sexual relationships. They were also required to continue using the medications during their menstrual cycles. The control group received the placebo which they were required to consume for 35 days (2 tablets every 12 hours). The participants were followed up twice a week through Short Message Sending (SMS) service and once a week through phone contact. After the intervention, the women completed FSFI. Finally, the data were entered into the SPSS statistical software (version 18) and analyzed using paired t-test, one-way ANOVA, and Bonferroni post-hoc test. Besides, p<0.05 was considered statistically significant. BODY.METHODS.ETHICAL CONSIDERATIONS: This research project was approved by the local Ethics Committee of Shiraz University of Medical Sciences and written informed consents were obtained from all the participants. In addition, the control group was provided with the results after completion of the study. BODY.RESULTS: The mean age of the study women was 32.67± 5.05 years. Most of the subjects (67.2%) were between 30 and 40 years old. Besides, the age of marriage in 44% and 4% of the subjects were below 20 and above 30 years old, respectively. In addition, the mean length of marriage was 21.59± 4.61 years. Moreover, most of the participants (43.2%) had academic degrees and 33.6% had high school and diploma degrees. The results revealed no significant difference among the three groups regarding TSH (p=0.448) and prolactin levels (p=0.179) before the intervention. Also, no significant difference was found among the three groups concerning orgasmic disorder before the intervention (p=0.23). However, a significant difference was observed among E. angustifolia, sildenafil citrate, and control groups in this regard after the intervention (p=0.004) (Table 1). Table 1 Comparison of frequency of sexual dysfunction (orgasm disorders) before and after intervention between the intervention and control groups Areas of sexual dysfunction (orgasm) E. angustifolia flower capsule group Sildenafil citrate group Control group Total p-value Number (%) Number (%) Number (%) Number (%) Before intervention 17 (41.5) 17 (40.5) 24 (57.1) 58 (46.4) 0.23 After intervention 12 (29.3) 7 (16.7) 21 (50) 40 (32) 0.004 p-value 0.267 0.006 0.549 -- According to the results of McNemar test, no significant difference was found in the control group before and after the intervention (57.1% vs. 50%; p>0.05). Also, no significant difference was observed in E. angustifolia group before and after the intervention (41.5% vs. 29.3%; p=0.267). On the other hand, a significant difference was found in the sildenafil citrate group before and after the intervention (40.5% vs. 29.3%; p=0.006) (Table 1). Overall, E. angustifolia, sildenafil citrate, and control groups respectively showed 29.41%, 58.82%, and 12.5% reduction of sexual dysfunction score as compared to the condition before the intervention. The findings of the current study indicated a significant difference between the EA group and the control group (p=0.001) and between the sildenafil citrate group and the control group (p<0.001) regarding the mean score of orgasm (Table 2). Table 2 Comparison of average score of sexual function (in orgasm and sexual satisfaction area) before and after intervention between the tests and control groups Areas of sexual function E. angustifolia Sildenafil citrate Control p-value Orgasm  Before intervention 3.31±1 3.48±0.94 3.18±1.19 0.418  After intervention 4.03±1.01 4.15±0.89 3.23±1.36 <0.001  p-value <0.001 <0.001 0.715 -- Sexual satisfaction  Before intervention 3.82±1.31 4.25±0.8 3.62±1.37 0.051  After intervention 4.75±1.2 4.73±0.78 3.74±1.65 <0.001  p-value <0.001 <0.001 0.515 -- The study results revealed no significant difference among the three groups concerning sexual satisfaction before the intervention (p=0.356). However, a significant difference was observed among the three groups in this regard after the intervention (p=0.03) (Table 3). According to the results of McNemar test, a significant difference was observed in E. angustifolia group before and after the intervention (p<0.001). On the other hand, no significant difference was found in the sildenafil citrate group before and after the intervention (p=0.06). Table 3 Comparison of frequency of sexual dysfunction (sexual satisfaction area) before and after intervention between the intervention and control groups Areas of sexual function (sexual satisfaction) E. angustifolia Sildenafil citrate Control Total p-value Number (%) Number (%) Number (%) Number (%) Before intervention 21 (51.2) 15 (35.7) 19 (45.2) 55 (44) 0.356 After intervention 9 (22) 8 (19) 18 (42.9) 35 (28) 0.03 p-value <0.001 0.065 0.999 -- -- Overall, E. angustifolia and sildenafil citrate groups respectively showed 57.14% and 46.66% reduction sexual dysfunction score as compared to the condition before the intervention. Also, a significant difference was observed between the EA group and the control group (p<0.001) and between the sildenafil citrate and control groups (p<0.001) regarding the mean score of sexual satisfaction (Table 2). BODY.DISCUSSION: In this study, EA, sildenafil citrate, and control groups respectively showed 29.41%, 58.82%, and 12.5% reduction sexual dysfunction score as compared to the condition before the intervention. This difference was statistically significant in the sildenafil citrate group (p=0.006), but not in E. angustifolia group (p=0.267). Nonetheless, a significant difference was observed between the two intervention groups and the control group regarding the mean score of orgasmic disorder (p<0.001). Caroso (2002) performed a study on 68 women between 19 and 38 years old who had no symptoms of sexual dysfunction in order to assess the effectiveness of sildenafil citrate. The participants had to consume sildenafil citrate and placebo for 4 weeks and were entered into the study based on Personal Experiences Questionnaire (PEQ). The study results showed that sildenafil citrate improved arousal (p<0.01), orgasm (p<0.001) and sexual satisfaction (p<0.001) compared to the placebo group (22). Furthermore, Cavalcanti et al. (2008) carried out a research on 22 menopausal women who suffered from orgasmic disorder. The participants received either 50 mg sildenafil citrate (11 subjects) or placebo (11 subjects) in a single dose for 15 days. The results revealed a considerable increase in the vaginal blood flow in the sildenafil citrate group as compared to the placebo group (p<0.05) (18). These results were all in agreement with those of the present study. In contrast, Dasgupta et al. (2004) investigated the effectiveness of sildenafil citrate in treatment of the women suffering from sexual dysfunction due to Multiple Sclerosis (MS). The study was conducted on 19 women with both sexual dysfunction and MS and the results showed that sildenafil citrate did not improve their sexual dysfunction (p>0.441) (54). Similarly, Wylie et al. (55) reported that sildenafil citrate had no positive effects on women's sexual disability score, which is in contrast to the findings of the current study. Sildenafil citrate is among the PDE5 inhibitors which by preventing cyclic guanosin monophosphate (CGMP) catabolism, leads to an increase in Nitric Oxide (NO) production, stimulation of NO release and increase in vaginal blood flow. This, in turn, results in an increase in sexual desire, orgasm, tissue hyperpolarization and relaxation, increase in blood flow, hyperemia and swelling (12), eventually leading to improvement of sexual dysfunction. Up to now, no specific clinical scales have been used for diagnosis of female sexual dysfunction and no medications have been confirmed for its treatment. Yet, two strategies, i.e., androgens and PDE5 inhibitors, are utilized for reduction of sexual disorders in women (56). In the present study, E. angustifolia extract decreased the sexual dysfunction score as compared to the condition before the intervention and the control group; however, the differences were not statistically significant. To date, no studies have been conducted on the effectiveness of E. angustifolia flower in sexual disorders. Therefore, the study results were compared with those of the studies performed on other herbal medications used for sexual disorders. Waynberg and Brewer (2000) aimed to find an alternative for chemical medications used for treatment of female sexual dysfunction and consequently, assessed Muira Puama and Ginkgo in 202 healthy women with low sexual desire. In that study, different aspects of the participants' sexual life were investigated before and one month after the treatment. After consuming the supplements, the self-assessment questionnaire scores were higher than average in 65% of the participants. In addition, the results indicated a significant improvement in the frequency of sexual desires and fantasies, satisfaction with sexual life, intensity of sexual desires, ability to reach orgasm and intensity of orgasm (p<0.001) (57). Similarly, Ito (2006) reported that ArginMax (an herbal supplement) improved sexual dysfunction particularly regarding sexual desire (51% vs. 8% in the placebo group; p=0.008) (58). E. angustifolia plant contains significant amount of flavonoids, terpenoids and sitosterol (59–62). Research has shown that certain flavonoids and sitosterol have analgesic and anti-inflammatory effects (62) and lead to vascular smooth muscle relaxation (63). Flavonoids are also believed to increase NO eventually resulting in vascular smooth muscle relaxation. Evidence has shown that NO, as an endogenous vascular dilator, plays a critical role in regulation of vascular tone (64, 65) and is the main stimulant of cGMP production in smooth muscles (66). Increase in cGMP leads to an increase in the activity of kinase G protein which leads to vascular relaxation by phosphorylation of different molecules (67). On the other hand, some experts believe that increase of cGMP results in smooth muscle vascular relaxation through other mechanisms (68). For instance, cGMP may lead to relaxation of smooth muscles by decreasing the intracellular calcium concentration (69). The positive effect of E. angustifolia on sexual dysfunction and sexual satisfaction in this study may be justified by the aforementioned mechanisms. The findings of the present study indicated a significant difference between the two intervention groups and the control group concerning the mean score of sexual satisfaction (p<0.001). Other studies have also found a significant relationship between improvement of sexual performance and sexual satisfaction (15, 57). Sexual relationships are among the main issues in marriage. Research has indicated that improvement of the quality of sexual relationships between husband and wife led to great satisfaction. It is, therefore, a decisive factor for marital and sexual satisfaction (2, 70–71). Overall, it seems that identification and treatment of sexual dysfunctions can increase satisfaction and thus can improve the families' strength and survival and the community's health. According to the previous studies, herbal medications, such as ArginMax, Ginseng, Ginkgo biloba, and ethanol extract (72–73) improved sexual dysfunction through an increase in NO production. NO is also among the compounds derived from E. angustifolia flower. In conclusion, sexual dysfunction in women is a serious, complex, multi-factorial problem which might result from biological, psychological, cultural, social and hormonal factors. Since the participants had normal menstrual cycles, hormones, and endocrine parameters, their sexual dysfunction might have occurred due to other factors, including insufficient stimulation and lack of preparation. Phosphodiesterase-type 5 inhibitors (sildenafil citrate) and E. angustifolia flower also enhance vaginal smooth muscles relaxation, artery vasodilatation and swelling in the genital system, thereby improving sexual function. Other factors associated with sexual dysfunction include relational problems, lifestyle, and type of food regimen that were not investigated in the current study. BODY.CONCLUSION: In this study, E. angustifolia, sildenafil citrate, and control groups respectively showed 29.41%, 58.82% and 12.5% reduction of changes as compared to the condition before the intervention. Thus, it seems that sildenafil citrate has been more effective in improvement of sexual dysfunction as compared to E. angustifolia. The findings of the current study can be used for improvement of women's and families' sexual health. Yet, further studies are recommended to be conducted using different doses of E.angustifolia at different times.

TITLE: The mind–body connection in irritable bowel syndrome: A randomised controlled trial of hypnotherapy as a treatment ABSTRACT.BACKGROUND:: Hypnotherapy has been reported as being beneficial in the treatment of irritable bowel syndrome (IBS). We aimed to test the hypothesis that patients with IBS treated 'holistically' by hypnosis (i.e. by combined psychological and physiological symptom imagery) would have greater improvement in their IBS symptoms than patients treated by hypnosis using standard 'gut-directed' hypnotherapy, and both would be superior to simple relaxation therapy. ABSTRACT.METHODS:: Patients (n = 51) with Rome II criteria were randomised to 'individualised' (holistic) hypnotherapy, standard 'gut-directed' hypnotherapy or relaxation therapy for a period of 11 weeks with two follow-up assessments at 2 weeks and at 3 months after the completion of the trial. The primary outcome was bowel symptom severity scale (BSSS). ABSTRACT.RESULTS:: All the participants in this study improved their IBS symptoms (pain, bloating, constipation and diarrhoea) and physical functioning at the end of the treatment from baseline, but this was not significantly different across the treatment arms. ABSTRACT.CONCLUSION:: Neither 'individualised' nor 'gut-directed' hypnotherapy is superior to relaxation therapy in IBS. BODY.INTRODUCTION: Modern neuroscience has shown that emotions permeate both mind and body and can affect our susceptibility to stress, cognitive function, and vulnerability to particular psychiatric disorders and illnesses. Emotions can also affect the function of our respiratory, immune, cardiovascular, reproductive, endocrine and gastrointestinal (GI) systems (Davidson, 2013). Research in epigenetics further supports the idea that our beliefs and emotions translate into physiological changes in the body (Church, 2009; Lipton, 2008), and this mind–body connection is apparent in sufferers of irritable bowel syndrome (IBS). IBS affects approximately 15 per cent of the general population at any one time and is one of the most common disorders encountered by both gastroenterologists and physicians in primary care (Camilleri and Choi, 1997; Farthing, 1995). The disorder typically affects those of working age and imposes an economic burden on the patient through increased health-care costs and loss of income because of illness (Dean et al., 2005). These patients also often experience a decrease in quality of life (Creed et al., 2000). The aetiology of IBS is as yet unknown, but most researchers agree that a subset of IBS sufferers have visceral hypersensitivity of the gut (Bouchoucha et al., 1999; Camilleri et al., 2001). Other possible mechanisms in predisposed persons that have been proposed include previous infectious gastroenteritis, mast cell infiltration, an imbalance of neurotransmitters including serotonin dysregulation, small intestinal bacterial overgrowth and psychological precipitants including acute life stress and abuse (Gershon, 2004; Gui, 1998; Koloski et al., 2005; Levy et al., 1997; Neal et al., 1997; Pimentel et al., 2000). Hence, IBS is probably a multi-faceted brain–gut disorder resulting in alteration in the regulation of gastrointestinal (GI) motility and/or sensory function (Smith and Morton, 2001; Tortora and Grabowski, 2000). Studies have shown that emotions such as anger, fear, pain and anxiety affect colonic motility more in IBS patients than in healthy controls (Gorard et al., 1996; Welgan et al., 2000), and for IBS, the most frequent co-morbid psychiatric disorders are anxiety, depression and somatoform disorders (Creed, 1992, 1994). This evidence suggests that the pathophysiology of IBS involves the brain–gut axis (Salt and Neimark, 2002), and 'gut-directed' hypnotherapy has been shown to be a successful intervention in breaking abnormal cycles occurring within this axis (Camilleri, 2001; Farhadi et al., 2001). Studies have especially shown the efficacy of hypnosis in the treatment of IBS (Palsson, 1998; Whorwell, 1987, 2006), but these studies only addressed physiological symptoms ('gut-directed' hypnotherapy) and did not take into account psychological symptoms such as anxiety and depression which are part of the IBS symptom picture. The mechanisms responsible for the therapeutic success of hypnotherapy are largely unknown, but research has shown that it may act by modulating visceral sensitivity, motor function and psychological distress (Gruzelier et al., 2001; Houghton et al., 1999; Marchioro et al., 2000). Imagery is a major component of hypnosis, and research has provided numerous examples of the physiological effects imagery has on the body (Graham, 1995). Unlike many other treatment options which separate the mind–body by focusing on either the psychological or the physiological aspects of IBS, hypnotherapy potentially addresses both via the brain–gut axis. However, the application of 'gut-directed' hypnosis has not been compared with combined gut and mind–body hypnosis: the latter theoretically might increase the benefit of hypnotherapy as an intervention because psychological distress is so prevalent in patients with IBS (Labus et al., 2007; Park et al., 2008). We aimed to evaluate the efficacy of hypnosis and imagery in the treatment of IBS. More specifically, we aimed to compare the use of standard 'gut-related' imagery used in previous trials which addressed the patients' physiological problems only (Gonsalkorale et al., 2002; Whorwell, 1987, 2006), with mind–body imagery that reflects the patients' complete symptom picture (i.e. imagery which addresses both the psychological and physiological aspects of IBS) in a randomised controlled trial. BODY.METHOD.STUDY PARTICIPANTS: A total of 51 symptomatic volunteers with IBS aged between 17 and 75 years were recruited from medical and naturopathic clinics and were invited to undergo a 1-hour screening session. They were then randomly assigned to one of three groups. Concealed allocation was assured by an assistant placing the names of participants into opaque envelopes and placing them in a locked filing cabinet. A fortnight after the screening session, all three groups began the treatment programme which consisted of five fortnightly treatment sessions (half an hour each) over a period of 9 weeks, with a subsequent follow-up of 2 weeks and 3 months. The flow of patients through the study is summarised in Figure 1. Figure 1.Flow diagram of subjects' progress through the phases of the trial. The Ethics Committee of the University of Sydney approved the trial, and guidelines on patient consent were met. BODY.METHOD.INCLUSION CRITERIA: The participants were deemed suitable for the study after having been diagnosed with IBS by a primary care physician or gastroenterologist. All had to meet the Rome II criteria for IBS (Drossman, 1999; Drossman et al., 2000) (specifically, pain or abdominal discomfort relieved by defecation, and/or onset associated with a change in stool frequency, and/or onset associated with a change in stool form), had failed to respond adequately to conventional medicines, and who had experienced at least 4 days with at least moderate pain over a 2-week period after screening. BODY.METHOD.EXCLUSION CRITERIA: Participants who were not free of organic disease (e.g. those who were diagnosed with coeliac disease, inflammatory bowel disease or diverticulitis as well as IBS) and who did not fit the inclusion criteria were excluded. Current medications were not discontinued. BODY.METHOD.GASTROINTESTINAL SCREENING OF STUDY POPULATION: During the initial screening session, participants completed the validated Talley Irritable Bowel Symptom Questionnaire (IBSQ) (Talley et al., 1995) to establish diagnosis and exclude other differential diagnoses. They also completed the validated Bowel Symptom Severity Scale (BSSS) (Boyce et al., 2000) to establish the severity of their IBS symptoms. BODY.METHOD.INTERVENTIONS: The participants were randomly allocated to one of three groups – two experimental groups (either 'individualised' or standard 'gut-directed' hypnosis) and one control group (relaxation therapy) – by means of random number tables (Boyer, 1968) and were unaware of which treatment group they were assigned to and of other participants in the trial. Also, consultations for other patients were always interspersed between those for any IBS trial participants to minimise contact between subjects. At the end of the trial, participants in the control group were offered two complementary sessions in hypnosis. The five treatment sessions were spaced approximately a fortnight apart over a period of 9 weeks. In the first of the five half-hour sessions, the patient's full history was taken and the functioning of the GI tract was explained. The following procedure was then strictly adhered to for all five sessions of the trial. BODY.METHOD.INTERVENTIONS.‘INDIVIDUALISED’ HYPNOSIS: Hypnosis was induced using a rapid eye catalepsy technique (Elman, 1964) after which the therapist read two prepared scripts – one containing 'gut-directed' imagery (physiological symptoms of IBS), plus a further script containing the patient's individual psychological symptoms as evidenced by the Symptom Checklist 90-R (SCL-90-R) (Derogatis, 1994; Derogatis et al., 1975). At the end of the second session, a CD of the scripts used in this session was given to the patient to be taken home and practised daily. BODY.METHOD.INTERVENTIONS.STANDARD ‘GUT-DIRECTED’ HYPNOSIS: Hypnosis was induced (using a rapid eye catalepsy technique), after which the therapist read the same prepared script containing 'gut-directed' imagery (physiological symptoms of IBS) as for group one. No script containing the patient's psychological symptoms was read. At the end of the second session, a CD of the 'gut-directed' imagery script was given to the patient to be taken home and practised daily. BODY.METHOD.INTERVENTIONS.RELAXATION THERAPY: No hypnosis was induced. The therapist read a prepared script of a progressive relaxation exercise that contained neither the aforementioned 'gut-directed' imagery nor the patient's individual psychological symptoms. At the end of the second session, a CD of the relaxation exercise was given to the patient to be taken home and practised daily. Compliance for home practice was checked by the therapist at each treatment session and was also evidenced by the participants' responses to Question 6 of the Bowel Symptom Scale 3 (BSS3) and Question 7 of the Bowel Symptom Scale 4 (BSS4). BODY.METHOD.INTERVENTIONS.FOLLOW-UP: The Bowel Symptom Severity Scale (BSSS) (Boyce et al., 2000) and the SF-36 Health Survey (Stewart et al., 1988) were completed by each participant, at 2 weeks and again at 3 months after completion of the 9-week treatment period, to assess whether IBS symptoms had significantly changed compared with baseline. No further treatment was given during the follow-up period, nor were participants invited to return for further treatment if symptoms returned. BODY.INSTRUMENTS.IRRITABLE BOWEL SYMPTOM QUESTIONNAIRE (IBSQ): A modified validated version of the previously validated Talley BSQ (Talley et al., 1995) was utilised in order to verify the diagnosis of IBS and to acquire general data on patients participating in the trial. The BSQ addressed aspects such as pain/discomfort, bloating, frequency and type of bowel movement, urgency, and frequency of visits to a doctor or alternative therapist. BODY.INSTRUMENTS.BOWEL SYMPTOM SCALES (BSS1-5): The BSS (Boyce et al., 2003) were used to assess change in IBS symptoms during the course of the five treatment sessions. The first item of all five BSSs consists of five visual analogue scales which refer to each of the principal symptoms of IBS (pain/discomfort, bloating, constipation and diarrhoea), plus an overall symptom severity rating. All five of the BSS also assess stool form and the degree to which IBS symptoms interfered with the patient's life and activities (see Tables 2a to 2d and Figure 2). Figure 2.Bowel Symptom Scales (BSS1-5) – overall symptom severity by treatment groups and control group. BODY.INSTRUMENTS.BOWEL SYMPTOM SEVERITY SCALE (BSSS): The BSSS (Boyce et al., 2000) consists of eight questions relating to possible symptoms the patient may have endured in the 2 weeks between treatment sessions. The symptoms specific in this questionnaire enquire about stool formation, abdominal pain, frequency of bowel motions, bloating, urgency, inability to have a bowel motion and a general feeling of discomfort in the abdomen. Each question also has two sub-questions which asked how distressed the patient had been during this period and how much the specific symptoms had interfered with his or her daily life. Each of the symptoms is given a severity rating between 0 and 4, a higher rating being indicative of greater severity. BODY.INSTRUMENTS.SYMPTOM CHECKLIST 90-R (SCL-90-R): The SCL-90-R (Derogatis, 1994; Derogatis et al., 1975) helps to evaluate a broad range of psychological problems and symptoms of psychopathology and is also useful in measuring the patients' progress or treatment outcomes. It provides an overview of the patients' symptoms and their intensity. The questionnaire consists of 90 items which measure nine primary symptom dimensions: somatisation, obsessive-compulsive disorder, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety, paranoid ideation and psychoticism. For the purpose of this trial, four of the nine primary symptom dimensions (anxiety, depression, interpersonal sensitivity and obsessive-compulsive disorder) which reflected the psychological symptoms ofthe majority of the participants were measured a priori. Those participants in the 'individualised' hypnosis group who scored 10 or higher on any of the four scales were read specific scripts pertaining to their disorder during the treatment sessions. BODY.INSTRUMENTS.SHORT FORM GENERAL HEALTH SURVEY (SF-36): The SF-36 General Health Survey (McHorney et al., 1994) has been applied widely to clinical trials and is capable of discriminating between healthy participants and those with moderate levels of psychiatric illness (Russo et al., 1998). The SF-36 is a 36-item questionnaire consisting of eight health concepts or sub-scales broadly related to quality of life, mental health and social activities. The sensitivity of the SF-36 to change in health status of IBS patients was tested by examining changes in SF-36 sub-scale scores throughout the treatment period and as follow-up 2 weeks and 3 months after the completion of the trial. BODY.INSTRUMENTS.DUKE-UNC FUNCTIONAL SOCIAL SUPPORT QUESTIONNAIRE: The eight-item Duke-UNC Questionnaire (Broadhead et al., 1988) was completed by the participants at the screening session to assess the amount of support patients had and to what extent it influenced their health and well-being. The questionnaire contains questions in two content areas: confident social support, which reflects a confidant relationship where important matters in life such as social contact and personal/work/financial problems are discussed and shared; and affective support, which reflects a more emotional form of support and caring. BODY.INSTRUMENTS.SURVEY OF RECENT LIFE EXPERIENCES (SRLE): Emotional responses to life stress can influence GI function via the brain–gut axis and produce symptoms such as pain and altered bowel function (Lundberg, 2005; Mayer, 2000). The SRLE (Kohn and MacDonald, 1992) which consists of 51 items covering six concepts (social and cultural difficulties, work, time pressure, finances, social acceptability and social victimisation) was completed by the participants at the screening session to determine whether these stresses had had an influence on their IBS symptoms. BODY.INSTRUMENTS.CREDIBILITY SCALE: The Credibility Scale (Borkovec and Nau, 1972) was issued to participants on two occasions throughout the trial (session 2 and session 4) to assess the credibility of treatment as perceived by the participants and to test the success of blinding. The four-item assessment contains questions on how confident the patients are in the treatment they are receiving, how confident they are in recommending the treatment to a friend suffering a similar complaint, how logical the treatment seems to them and how successful they think this form of treatment would be in alleviating other complaints. BODY.STATISTICAL ANALYSIS: For the primary endpoint (BSS), an intention to treat analysis of variance was used to determine the differences among groups at baseline, end of treatment, and follow-up and repeated measures for trend over time were also determined. All p values were two-tailed, unless otherwise indicated, and the alpha level of significance was set at 0.05. Missing scale and item scores were not replaced. Data for all other outcome measures (psychological stress (SRLE)), quality of life (SF-36) and social support (Duke-UNC) are presented as per protocol analysis. It was hypothesised a priori that, with respect to the abdominal pain sub-scale of the SCL-90-R, the control group would not change on average, the standard therapy group would improve by one point and the individualised therapy group by two points. Under these conditions and with an assumed standard deviation (SD) = 2.0, n = 25 participants per study group would have yielded statistical power >0.85 at the 0.05 (two-tailed) level of statistical significance. Due to difficulty with recruitment, n = 17 participants were actually recruited per group, and this yields statistical power of approximately 0.72 under the same conditions. While this power is less than the desired 0.8, it is unlikely to have materially affected the statistical analysis. BODY.RESULTS.BASELINE CHARACTERISTICS: At baseline, the 51 participants in this study were similar across treatment areas (Table 1). All participants presented with psychological problems. Of these, 30 participants presented with depression, 4 with anxiety, 2 with obsessive-compulsive disorder and 1 with interpersonal sensitivity. Of the remainder, 5 had both depression and anxiety, 5 had depression and obsessive-compulsive disorder, and 4 presented with depression and interpersonal sensitivity. Table 1. Demographic and baseline data for subjects randomised to the three treatment groups. Variables Group 1 Group 2 Group 3 Total ( n  = 51) ‘Individualised’ group ( n  = 17) Standard ‘gut-directed’ group ( n  = 17) Relaxation group ( n  = 17) Mean 95% CI Mean 95% CI Mean 95% CI Mean 95% CI Age in year 47.8 38.6–57.1 37.4 31.1–43.6 34.9 27.5–42.4 40.0 35.6–44.5 Female (%) 88.2 – 82.4 – 88.2 – 86.3 – Bowel Symptom Severity Scale  Distress 18.7 14.9–22.5 17.8 15.0–20.6 21.8 17.9–25.6 19.3 17.4–21.3  Frequency 20.0 17.0–23.0 19.3 17.5–21.1 22.0 19.2–24.8 20.4 19.0–21.8  Interference 16.4 12.2–20.6 16.6 13.5–19.6 20.2 16.2–24.1 17.6 15.6–19.7 SCL-R  Total  Depression 19.3 12.8–25.8 15.5 9.9–21.2 17.2 10.9–23.4 17.3 14.0–20.7  Anxiety 8.7 5.8–11.7 9.2 5.1–13.3 10.5 4.8–16.1 9.5 7.1–11.8  Obsessive-Compulsive 11.4 8.0–14.8 11.1 6.9–15.4 11.8 6.7–16.9 11.4 9.1–13.7  Interpersonal sensitivity 10.5 6.0–15.1 8.8 4.9–12.6 8.5 4.9–12.1 9.3 7.1–11.4 SF-36  Physical functioning 75.7 64.0–87.4 92.3 87.3–97.3 79.6 68.8–90.4 82.9 77.4–88.3  Role-physical 53.6 28.3–78.8 63.3 40.6–86.1 55.8 34.3–77.3 57.7 45.4–70.1  Pain 53.0 38.8–67.2 65.3 55.9–74.7 54.7 42.4–67.0 57.9 51.4–64.5  General health 51.6 38.8–64.3 62.7 50.4–75.1 60.0 45.7–73.7 58.1 51.1–65.1  Vitality 45.4 32.4–58.3 49.3 41.7–56.9 35.8 26.3–45.2 43.8 38.2–49.5  Social functioning 55.4 36.2–74.5 73.3 59.5–87.1 59.6 47.2–72.0 63.1 54.6–71.6  Role-emotional 54.8 32.6–76.9 55.6 30.7–80.4 61.5 38.5–84.6 57.1 44.7–69.6  Mental health 56.0 44.9–67.1 64.0 56.1–71.9 61.5 50.2–72.9 60.6 55.2–66.0 CI: confidence interval; SCL-90-R: symptom checklist-90-revised; SF-36: 36-Item Short Form Health Survey. Data are means ± and 95% CI unless otherwise specified. At the commencement of the trial period, the majority of participants (81.6%) suffered abdominal pain most of the time (of these, 59.2% had mild to moderate pain and 40.8% had severe to very severe pain) with 61.2% of participants experiencing pain several times a week or daily. In the 3 months prior to treatment, 98.0% of participants experienced bloating and 78.4% of participants had visible abdominal swelling. Of the 51 participants, 51% werediarrhoea predominant, 44.9% constipation predominant, and 4.1% had mixed diarrhoea and constipation. The majority of participants in this study were women (86.3%), and the majority of sufferers were aged between 20 and 40 years (82.3%). Three participants withdrew from the trial (one each in sessions 1, 2 and 4), and six subjects were excluded from the analysis at the end of the trial due to incomplete or missing records. BODY.RESULTS.RESPONSE TO INTERVENTION: All participants demonstrated improvement in the overall severity of their individual symptoms and functioning at the end of the treatment period (Session 5), with the 'individualised' group (group one) having a numerically better outcome than the other two groups (Tables 2a to 2d). The overall symptom score was not significantly different at Session 5, and improvement in IBS symptoms (Figure 3) and general health outcomes (Figure 4) did not continue during the 3-month follow-up period. Table 2a. Bowel Symptom Scales (BSS1-5) – abdominal pain: mean scores and SD at each session from randomisation. Treatment groups Sessions Group 1 Group 2 Group 3 Total F1 F2 F3 Mean SD Mean SD Mean SD Mean SD Session 1 4.0 2.2 4.4 2.1 5.1 2.8 4.5 2.4 5.54 ** 17.90 ** NS Session 2 3.8 2.8 3.8 2.0 4.9 2.6 4.2 2.5 Session 3 4.1 3.3 5.0 2.8 4.3 3.0 4.5 3.0 Session 4 4.0 2.7 3.4 2.4 4.0 2.5 3.8 2.5 Session 5 2.5 2.2 3.5 2.8 3.2 2.0 3.1 2.3 Total 3.7 2.6 4.0 2.4 4.3 2.6 4.0 2.6 SD : standard deviation; NS: not significant; F1: main effect; F2: linear trend; F3: treatment effect; SF-36: 36-Item Short Form Health Survey. ** p  < 0.01 and * p  < 0.05. Table 2b. Bowel Symptom Scales (BSS1-5) – bloating: mean scores and SD at each session from randomisation. Treatment groups Sessions Group 1 Group 2 Group 3 Total F1 F2 F3 Mean SD Mean SD Mean SD Mean SD Session 1 4.2 2.5 5.4 2.5 6.0 2.4 5.2 2.5 9.23 ** 33.40 ** NS Session 2 4.0 3.0 5.1 2.6 5.3 2.4 4.8 2.7 Session 3 3.8 2.4 5.6 3.0 4.8 3.2 4.7 2.9 Session 4 3.9 2.6 3.9 2.8 3.5 2.6 3.8 2.7 Session 5 2.7 2.5 3.6 3.2 3.7 2.2 3.3 2.6 Total 3.7 2.6 4.7 2.9 4.7 2.7 4.4 2.8 SD : standard deviation; NS: not significant; F1 = main effect; F2 = linear trend; F3 = treatment effect. ** p  < 0.01 and * p  < 0.05. Table 2c. Bowel Symptom Scales (BSS1-5) – constipation: mean scores and SD at each session from randomisation. Treatment groups Sessions Group 1 Group 2 Group 3 Total F1 F2 F3 Mean SD Mean SD Mean SD Mean SD Session 1 3.6 3.0 3.5 2.9 4.9 2.6 4.0 2.9 2.66 * 5.43 * NS Session 2 3.5 3.5 3.8 3.1 4.0 2.9 3.8 3.1 Session 3 3.3 2.8 4.5 3.2 3.2 2.7 3.6 2.9 Session 4 2.8 2.4 3.2 2.5 2.6 2.4 2.9 2.4 Session 5 3.1 2.5 3.2 3.2 3.4 3.0 3.2 2.8 Total 3.3 2.8 3.6 3.0 3.6 2.8 3.5 2.8 SD : standard deviation; NS: not significant; F1: main effect; F2: linear trend; F3: treatment effect. ** p  < 0.01 and * p  < 0.05. Table 2d. Bowel Symptom Scales (BSS1-5) – diarrhoea: mean scores and SD at each session from randomisation. Treatment groups Sessions Group 1 Group 2 Group 3 Total F1 F2 F3 Mean SD Mean SD Mean SD Mean SD Session 1 3.3 2.7 3.1 3.1 2.9 2.6 3.1 2.7 NS NS NS Session 2 3.2 2.7 2.9 2.1 2.5 3.0 2.9 2.6 Session 3 3.0 2.9 3.5 3.1 3.4 2.9 3.3 2.9 Session 4 1.9 2.4 2.8 2.8 3.5 3.1 2.7 2.8 Session 5 2.1 2.5 3.2 2.8 2.5 2.4 2.6 2.6 Total 2.7 2.6 3.1 2.8 3.0 2.7 2.9 2.7 SD: standard deviation; F1: main effect; F2: linear trend; F3: treatment effect. ** p  < 0.01 and * p  < 0.05. Figure 3.Bowel Symptom Severity Outcome – treatment sessions and follow-up – by treatment groups and control groups. Figure 4.Secondary Health Outcome SF-36 – treatment sessions and follow-up – by treatment groups and control group. BODY.RESULTS.PSYCHOLOGICAL STRESS: The bowel symptom severity measures (BSSS) specific to frequency of bowel motions, distress and interference with daily life at the end of treatment sessions, and the SCL-90-R measures, depression, obsessive-compulsive and interpersonal sensitivity at baseline, were significantly correlated (p < .01), but anxiety was unrelated. At the end of treatment sessions, the overall severity of IBS (BSSS) was significantly correlated (p < .05) with all SCL-90-R scores except anxiety (Table 3). Table 3. Intercorrelations among four dimensions of the SCL-90-R at baseline and the SF-36 health concepts at the end of treatment. Physical functioning Role-physical Pain General health perceptions Vitality Social functioning Role-emotional Mental health Depression Obsessive-compulsive Anxiety Physical functioning 1 Role-physical 0.495 (**) 1 Pain 0.431 (**) 0.697 (**) 1 General health perceptions 0.199 0.479 (**) 0.603 (**) 1 Vitality 0.312 (*) 0.559 (**) 0.703 (**) 0.491 (**) 1 Social functioning 0.303 0.681 (**) 0.688 (**) 0.374 (*) 0.633 (**) 1 Role-emotional 0.141 0.657 (**) 0.579 (**) 0.575 (**) 0.538 (**) 0.582 (**) 1 Mental health 0.098 0.248 0.349 (*) 0.462 (**) 0.450 (**) 0.260 0.629 (**) 1 Depression −0.081 −0.329 (*) −0.389 (*) −0.572 (**) −0.429 (**) −0.366 (*) −0.619 (**) −0.521 (**) 1 Obsessive-compulsive 0.058 −0.287 −0.334 (*) −0.408 (**) −0.192 −0.234 −0.601 (**) −0.456 (**) 0.795 (**) 1 Anxiety −0.067 −0.328 (*) −0.193 −0.571 (**) −0.094 −0.150 −0.539 (**) −0.384 (*) 0.695 (**) 0.725 (**) 1 Interpersonal sensitivity 0.044 −0.306 −0.267 −0.467 (**) −0.200 −0.198 −0.484 (**) −0.391 (*) 0.814 (**) 0.741 (**) 0.805 (**) 1 BODY.RESULTS.SOCIAL SUPPORT: At baseline, the confident social support score (Duke-UNC) was negatively correlated with bowel symptom severity scores and with all psychological scores (as per the SCL-90-R) except depression. The correlation between the confident support score and depression was significant (p < .05) (Table 3). BODY.RESULTS.QUALITY OF LIFE: Participants in all three groups demonstrated an improvement in overall severity in their IBS symptoms and a subsequent improvement in their quality of life. There was a highly significant decrease in the level of pain, and also a highly significant improvement in the patients' vitality, social functioning and mental health. There was also significant improvement in physical functioning, general health and the extent to which emotional problems interfered with their work or daily activities from baseline (Figure 4). However, there was no significant treatment effect (Table 4). Table 4. Medical Outcomes Index Mean Changes Over Time: Tests of Within-Subjects Effects, Linear Trend, and Treatment Effect. SF-36 df F p-value Physical Functioning Main effect of scores over time 2.7 .022 * Linear trend of scores to change over time 6.48 0.15 * Treatment effect 2.57 n.s. Role-Physical Main effect of scores over time 5 0.35 n.s. Linear trend of scores to change over time 1 0.66 n.s. Treatment effect 2 0.99 n.s. Pain Main effect of scores over time 5 6.23 0.000 ** Linear trend of scores to change over time 1 23.99 0.000 ** Treatment effect 2 2.15 n.s. General Health Main effect of scores over time 5 2.27 0.049 * Linear trend of scores to change over time 1 6.00 0.019 * Treatment effect 2 0.704 n.s. Vitality Main effect of scores over time 5 3.20 0.008 ** Linear trend of scores to change over time 1 5.68 0.022 * Treatment effect 2 2.20 n.s. Social Functioning Main effect of scores over time 5 5.13 0.000 ** Linear trend of scores to change over time 1 11.77 0.001 ** Treatment effect 2 1.164 n.s. Role-Emotional Main effect of scores over time 5 1.63 0.153 Linear trend of scores to change over time 1 4.45 0.041 * Treatment effect 2 0.586 n.s. Mental Health Main effect of scores over time 5 5.99 0.000 ** Linear trend of scores to change over time 1 15.61 0.000 ** Treatment effect 2 0.488 n.s. ** p <0.01, * p <0.05. BODY.RESULTS.CREDIBILITY SCALE: The participants in all three groups showed that their expectancy for improvement increased from the commencement of their treatment to the end of treatment, although the results did not show statistically significant differences among the three treatment groups (Figure 5). Figure 5.Credibility Scale: mean scores between Session 2 and Session 4 by treatment groups and control group. BODY.DISCUSSION: The study follows on previous research which has shown 'gut-directed' hypnotherapy to be of benefit to patients with IBS (Gonsalkorale et al., 2002; Whorwell, 1987, 2006). The study tested both physiological and specific psychological imagery in the hypnotherapy scripts and compared this technique to standard 'gut-directed' hypnotherapy. Psychological distress, which can trigger or exacerbate symptoms (Jarrett et al., 1998; Koloski et al., 2003), has been shown to be an important component of IBS symptoms and probably should be considered when treatment strategies are designed. To our knowledge, studies on hypnotherapy as a treatment for IBS have not taken this into account (Anbar, 2001; Forbes et al., 2000; Gonsalkorale et al., 2002; Harvey et al., 1989; Palsson, 1998; Whorwell, 1987). By using scripts that specifically target each individual patient's emotional/psychological symptoms (in conjunction with scripts for the physiological aspects of the disease), the therapist in this trial attempted to address the whole patient profile. Based on previous research and consistent with the aims of this study, the present research sought to empirically investigate three main hypotheses. The first hypothesis was that participants who had been diagnosed with IBS would present with not only physiological symptoms but psychological ones as well and, that at the end of the study, participants who underwent 'individualised' hypnotherapy (using imagery which addressed both the psychological/emotional aspects and the physiological symptoms of the syndrome) would have a better outcome in the improvement in their IBS symptoms than participants who underwent standard 'gut-directed' hypnotherapy in which physiological symptoms alone were treated. In this study, participants presented with high baseline scores on psychological ratings. However, while all groups improved from baseline on multiple measures, there was no overall benefit detected for hypnotherapy over relaxation therapy. The second hypothesis we tested was that participants' IBS symptoms would improve during the trial period, and, as a result, their quality of life would subsequently improve. Quality of life did improve in all treatment arms but, again, we detected no differential effect of hypnotherapy. The third hypothesis was that participants who had a support system in place would improve more quickly than those who had not. We found that there was no significant correlation between support and improvement in the overall severity of IBS symptoms. The only significant correlation found was between the confident support score and the depression score. This study had several strengths. The study was randomised and concealed allocation was assured by an assistant placing the names of participants into opaque envelopes and placing them in a locked filing cabinet. IBS and the functioning of the GI tract were explained. Careful attention was given to blinding throughout the trial, and the therapist (the principle author) who administered the therapy was an experienced and qualified hypnotherapist. Other strengths of this study lie in the fact that participants were recruited prospectively; participants with other pre-existing functional GI diseases were carefully excluded; and both validated, standardised questionnaires to define the outcome measure of IBS and strict criteria for diagnosing IBS were utilised. Also, the drop-out rate during the trial was small. There were also limitations in this study. It may not have been sufficiently powered. Due to time limits and the fact that the therapist was the only person recruiting, the sample size was modest. A much larger sample size would have been required to detect such a small effect size difference among the groups, but such a difference would possibly still not have been clinically significant. Another potential limitation was an absence of contact with participants by the therapist during the follow-up period. In previous trials in hypnotherapy (Gonsalkorale et al., 2002; Whorwell, 1987, 2006), participants continued to receive hypnotherapy sessions on a monthly basis during the follow-up period and were asked to telephone if they experienced a relapse so that a further session of hypnotherapy could be arranged. In this study, there was no further contact with the therapist by the participants, adherence to protocols during the follow-up period was not checked and participants were not given further hypnotherapy sessions (either on a regular basis or in case of relapse) to maintain remission. This could be a possible explanation for the lack of improvement in IBS symptoms during the follow-up period. With continued checking of adherence to autohypnosis practice and ongoing hypnotherapy sessions with the therapist, improvement in scores may have increased but, in the authors' view, to do so would not have addressed if any benefit persisted. Research has indicated that EEG wave changes associated with hypnosis can also be triggered by other methods of deep concentration, such as the relaxation response (Jacobs and Friedman, 2004; Williams and Gruzelier, 2001). Previous trials on relaxation training as a treatment for IBS, however, have shown mixed results (Blanchard et al., 1993; Boyce et al., 2003; Spiller, 2005; Van der Veek et al., 2007). Nevertheless, the control group in the trial underwent sessions in relaxation as a treatment for their IBS symptoms, whereas treatment for the other two experimental groups involved sessions in hypnotherapy. Considering that the relaxation response can trigger brain wave changes associated with hypnosis and that hypnotherapy sessions, themselves, involve deep relaxation, participants in the control group could have easily lapsed into hypnosis. This could account for the similarities in treatment outcome and the small effect difference between the groups. Notwithstanding the limitations of this clinical trial and that the findings need further confirmation, this study appears to support a psycho-physiological hypothesis that successful treatment of the psychological aspect is accompanied by improvement in IBS symptoms. Future research needs to continue the investigation of the brain–gut axis in IBS and the role of hypnotherapy which addresses both the psychological and physiological aspects of this disorder, as an effective and viable treatment option. In conclusion, in a randomised, controlled trial of hypnotherapy in IBS, symptoms (pain, bloating, constipation and diarrhoea) and physical functions improved from baseline in each arm. Hypnotherapy, however, was not shown to be superior to relaxation therapy for symptom reduction in this disorder. A possible explanation for this could be that the relaxation response is also associated with hypnosis and participants in the control group could have lapsed into a trance state. This also suggests that reducing anxiety through relaxation may be the key to reducing IBS symptoms in patients, whatever the mechanism may be.

TITLE: Pretreatment metabotype as a predictor of response to sertraline or placebo in depressed outpatients: a proof of concept ABSTRACT: The purpose of this study was to determine whether the baseline metabolic profile (that is, metabotype) of a patient with major depressive disorder (MDD) would define how an individual will respond to treatment. Outpatients with MDD were randomly assigned to sertraline (up to 150 mg per day) (N=43) or placebo (N=46) in a double-blind 4-week trial. Baseline serum samples were profiled using the liquid chromatography electrochemical array; the output was digitized to create a 'digital map' of the entire measurable response for a particular sample. Response was defined as ⩾50% reduction baseline to week 4 in the 17-item Hamilton Rating Scale for Depression total score. Models were built using the one-out method for cross-validation. Multivariate analyses showed that metabolic profiles partially separated responders and non-responders to sertraline or to placebo. For the sertraline models, the overall correct classification rate was 81% whereas it was 72% for the placebo models. Several pathways were implicated in separation of responders and non-responders on sertraline and on placebo including phenylalanine, tryptophan, purine and tocopherol. Dihydroxyphenylacetic acid, tocopherols and serotonin were common metabolites in separating responders and non-responders to both drug and placebo. Pretreatment metabotypes may predict which depressed patients will respond to acute treatment (4 weeks) with sertraline or placebo. Some pathways were informative for both treatments whereas other pathways were unique in predicting response to either sertraline or placebo. Metabolomics may inform the biochemical basis for the early efficacy of sertraline. BODY.INTRODUCTION: Response to current therapies in treating major depression varies considerably, with ∼40% of patients not responding and 60% not remitting after an initial trial of therapy.1, 2 Further, the onset of antidepressant therapeutic action typically does not occur until after a few weeks of treatment, which delays clinicians from knowing whether an antidepressant is going to work for a particular patient. The detailed molecular mechanisms underlying variation in treatment response in depression and the genetic and biochemical basis remain unknown although numerous studies implicate the norepinephrine and serotonin (5-HT) systems.3, 4, 5, 6, 7, 8, 9, 10 The placebo effect adds complexity, as 30–40% of major depressive disorder (MDD) patients respond to placebo through mechanisms that are not yet understood.11, 12, 13 Further insights into the mechanisms of response to placebo vs response to medications are needed. To date, there are no valid biomarkers of depression itself, or of response to medication or placebo. Mapping biochemical pathways that are modified in the presence of the disease alone—or upon treatment of the disease—could provide deeper insights into disease mechanisms, enable the sub-classification of disease and yield valuable biomarkers for monitoring disease progression and response to therapy. This approach could enhance the accuracy of treatment selection and minimize poly-pharmacy and trial and error treatment selection among antidepressant medications. Metabolomics, the study of metabolism at a global 'omics' level, is a new, rapidly growing field with potential to impact clinical practice. The central concept is that an individual's metabolic state reflects the individual's overall health status. It involves the systematic study of the 'metabolome', a repertoire of small molecules present in cells, tissues and body fluids. The identities, concentrations and fluxes of these molecules represents the interactions from gene sequence to gene expression, protein expression and the total cellular environment, an 'environment' that—in the clinical setting—includes drug exposure.14, 15, 16, 17, 18, 19 Metabolomics typically utilizes technologies that aim at simultaneously quantifying thousands of small molecules in a biological sample. This analytical capability can then be joined to sophisticated mathematical analyses to identify metabolic profiles, which could provide valuable data to design (1) prognostic, diagnostic and surrogate markers for disease state; (2) the ability to subclassify disease; (3) biomarkers of drug response phenotypes leading to patient stratification (pharmaco-metabolomics); and (4) information about disease mechanisms. Sophisticated metabolomic analytical platforms, statistics and bioinformatics tools have recently been developed to enable metabolic characterization of initial signatures for several diseases including depression,20, 21, 22 motor neuron disease,23 Parkinson's disease,24 cocaine and opiate addiction,25, 26 schizophrenia,27, 28, 29 rat models of caloric restriction.16 This study obtained metabolic profiles of depressed outpatients using electrochemistry-based metabolomics and considered the following questions: Can baseline metabolic profiles distinguish between patients who do and do not respond to 4-week treatment with sertraline?Can baseline metabolic profiles distinguish between patients who do and do not respond to 4 weeks of treatment with placebo?Can baseline metabolic profiles differentiate patients who respond on sertraline from those who respond on placebo? BODY.PATIENTS AND METHODS.STUDY DESIGN: The 89 patients in this report were a subset of the 165 patients who entered a randomized, double-blind, flexible dosing, placebo-controlled study performed at 12 clinical sites. In the larger study, patients were randomized to treatment (oral administration) with either sertraline or placebo at a ratio of 1:1. Sertraline dosing was started with 50 mg per day at baseline (week 0), with dose increased up to 100 mg per day at week 1 and up to 150 mg per day at week 2, as seen needed by the treating clinician. These 89 patients were older (42 yrs vs 33 yrs, P<0.0001) and included more females (69 vs 57%) than the 76 patients for which we did not have adequate data to include in the analysis. Baseline HRS-D17 scores were similar for the two groups (the mean (s.d.) baseline HRS-D17 for subjects within this analysis was 24.8 (2.9) vs 24.6 (2.8) for the remainder of the trial subjects), indicating no significant difference in terms of pre-treatment severity of disease. Subjects selected for this study were those with serum samples and HRS-D17 scores available at baseline and at 4 weeks (±1 week) after treatment. BODY.PATIENTS AND METHODS.PATIENTS: Study participants were outpatients, 18–65 years of age, from various sites across the United States. Patients had a primary diagnosis of MDD by DSM-IV criteria, with symptoms of depression present for at least 1 month before screening, and a total baseline score >22 on the 17-item Hamilton Rating Scale for Depression (HRSD17)30 at screening. A complete description of the inclusion and exclusion used in this study can be found in Supplementary Table S1. The study protocol was developed in accordance with the principles of the Declaration of Helsinki. All patients provided written informed consent. The study was sponsored and monitored by Pfizer. Each site's IRB approved and oversaw the study. BODY.PATIENTS AND METHODS.ASSESSMENTS: Measures were gathered at baseline, week 1 and week 4 of treatment at the clinic visits. The primary outcome measure was the HRSD17,30 used to assess depressive symptom severity. Assessments of the HRSD17 were obtained at baseline and at week 4. The HRSD score was modeled as dichotomous response variable. A patient was considered a responder to therapy if they showed a ⩾50% reduction if HRSD scores from baseline to 4 weeks. This study included the collection of serum samples drawn from the subset of 89 patients at baseline and week 4. The samples from these 89 patients were profiled using the liquid chromatography electrochemical array (LCECA) platform described below. BODY.PATIENTS AND METHODS.METABOLOMIC PROFILING.ANALYSIS METHOD: Samples were analyzed using a long gradient LCECA method that resolves ca.1500–2000 compounds in levels to ca. 500 pg per ml.31, 32, 33, 34, 35, 36, 37, 38, 39 The method is specific for compounds that will undergo electrochemical oxidation or reduction, and includes multiple compounds from the tyrosine, tryptophan, sulfur amino acid and purine pathways, and markers of oxidative stress and protection. The method employs a 120-min gradient from 0% organic modifier with an ion-pairing agent (pentane sulfonic acid) to a highly organic mobile phase with 80/10/10% methanol/isopropanol/acetonitrile. An array of 16 serial coulometric electrochemical detectors is set at incremental potentials from 0–900 mV, responding to oxidizable compounds such as tocopherol in lower potential sensors and higher oxidation potential compounds such as hypoxanthine in the higher potential channels. BODY.PATIENTS AND METHODS.METABOLOMIC PROFILING.ANALYSIS SEQUENCE AND DATA OUTPUT: At the time of preparation, a pool was created from small aliquots of each sample in the study, which was then treated identically to a sample. All assays were run in sequences that included mixed standard, five samples, pool, five samples, mixed standard, and so on. Run orders of all samples in this study were randomized. The sequences minimized possible analytical artifacts during further data processing. Data were time normalized to a pool at the midpoint of the study, aligning major peaks to 0.5 s and minor peaks to 0.5–2 s. For the purpose of this study, the data were exported in digital format (digital maps), which allows to capture all analytical information for the following data analysis and to avoid possible artifacts introduced by peak-finding algorithms. In this study, resolution was set at 1.5 s and the number of data points (variables, defined as the signal at a given time on a given channel) obtained from one sample, using our current LCECA platform, was 65 000. Values for each sample were then adjusted by averaging between pools to compensate for any response drift over the study period. All rows for which the maximum value was below the noise level of the system were eliminated from subsequent data analysis, leaving ∼14 000 variables. It is important to note that the number of variables in digital maps is not equivalent to the number of analytes, because an individual analyte is represented by more than one variable. Depending on the concentration of analyte and on its separation across EC array, the number of variables characterizing an analyte could be between 10 and 100. Following finding the variables differentiating the groups, the variables were sorted by retention time and channel. This step allowed isolation of 'peak clusters' (that is, all digital map variables characterizing one specific analyte), which, in turn, provides an identification of specific markers. Then the most significant variables in the digital maps were used to identify the location of the actual marker peaks within the chromatograms. BODY.PATIENTS AND METHODS.DATA ANALYSIS.ANALYSIS OF THE DIGITAL MAPS: Before analysis, a relative s.d. was calculated for each variable in order to identify possible outliers.40 Variables with a relative s.d. value >120% were taken out of the data set. A high relative s.d. often reflects sporadic use of other drugs. Thus, these variables were removed primarily to avoid artifacts from differential drug use in responders or non-responders. The remaining variables were log 2 transformed. The primary objective of the modeling was to determine whether the baseline metabolomic profile could predict responder versus non-responder status at week 4, based on percent of HRSD17 score change from Baseline. Digital maps were used to construct partial least square-discriminant analysis (PLS-DA) models for the 43 patients in the sertraline group and the 46 patients in the placebo group. We used the variable influence on the projection (VIP) parameter to identify variables making the most significant contribution in discriminating between responders and non-responders on sertraline therapy and placebo in the PLS-DA models. VIP is a weighted sum of squares of the PLS weight, which indicates the importance of the variable to the whole model. Higher scores reflect a greater contribution to the separation of the groups, in this case responders and non-responders to sertraline or placebo. Cross-validation of these models was performed by omitting the data from one patient before model constructions.40 After the one-out models were built the data sets were reconstructed, including only the variables with VIP values >0.7. These data sets contained ∼4000 variables representing roughly 30 discrete compounds. The one-out models were rebuilt with the reconstructed data set and used to classify the omitted patients as a responder or non-responder. This procedure was repeated until every subject had been kept out once. BODY.RESULTS.SAMPLE CHARACTERISTICS: The sertraline and placebo groups did not differ significantly in age (44±11.4 years vs 40±12.7 years, P=0.12), body mass index (27.76±5.76 vs 29.37±6.45, P=0.22), gender (29/43 (69%) female vs 32/46 (70%) female, P=0.83) or race (34/43 (79%) white vs 34/46 (74%) white, P=0.57). The response rate was slightly higher for sertraline than for placebo, but this difference was not statistically significant (25/43 (58%) vs 18/46 (39%) responders, χ2(1)=1.39, P=0.24). BODY.RESULTS.METABOLOMIC PROFILES AT BASELINE PARTIALLY SEPARATE RESPONDERS AND NON-REPONDERS TREATED WITH SERTRALINE: We used an electrochemistry based metabolomics platform (LCECA) to profile samples at baseline and post treatment with sertraline or placebo. This platform enables quantification of over a thousand compounds that will undergo electrochemical oxidation or reduction, and is particularly suitable for studying neurotransmitter pathways tryptophan and tyrosine as well as sulfur amino acid and purine pathways, and markers of oxidative stress and protection.31, 32, 33, 34, 35, 36, 37, 38, 39 A list of metabolites with known chemical structure that were quantitatively measured using this platform is presented in Table 1. The chemical structure of many metabolites detected by this highly sensitive platform currently remains unknown. The variables from the digital maps were used to construct PLS-DA models for the 43 patients in the sertraline group (see methods section). Figure 1a shows the separation of responders (⩾50% decrease in HRSD17 scores at week 4) and non-responders (<50% decrease in HRSD17 scores at week 4) to sertraline. We used the VIP parameter to identify variables that have the most significant contribution in discriminating between responders and non-responders on sertraline therapy in the PLS-DA model (See Table 2). Some of the metabolites that contribute to separation have known chemical structure and included dihydroxyphenylacetic acid (DOPAC, dopamine pathway), 4-hydroxyphenyllactic acid (4-HPLA, phenylalanine pathway), serotonin (5-HT, tryptophan pathway) and gamma tocopherol (vitamin E pathway). There were several compounds of unknown structure that emerged as significant in contributing to the separation of responders and non-responders. Many of these had VIP values greater than the one associated with DOPAC, which had the highest VIP value of the known compounds. Cross-validation of these models was performed by omitting the data from one patient during the PLS-DA. After this model was built the data set was trimmed, including only the variables with a VIP of greater than 0.7. The trimmed data sets contained ∼4000 variables representing about 30 discrete compounds. The model was rebuilt with the trimmed data set and used to classify the omitted patient as a responder or non-responder. This procedure was repeated until every subject had been kept out once. Using this one-out method we were able to correctly classify 21 of 25 (84%) responders and 14 of 18 (74%) non-responders with an overall classification rate of about 81%. BODY.RESULTS.METABOLIC PROFILES AT BASELINE PARTIALLY SEPARATE RESPONDERS AND NON-REPONDERS TREATED WITH PLACEBO: PLS-DA analysis of the 46 patients in the placebo arm show partial separation of responders and non-responders using binary analysis described above (Figure 1b). Hypoxanthine, xanthine and uric acid (purine pathway); 5-methoxytryptophol (5-MTPOL), serotonin (5-HT), 3-hydroxykynurenine (3-OHKY) and 5-hydroxyindoleactic acid (tryptophan pathway); DOPAC (dopamine pathway); cysteine (sulfur amino acid, one carbon metabolism); and several tocopherols (vitiman E) contributed to the separation of the responders and non-responders on placebo (see Table 2). The highest known VIPs were from compounds that are from the purine pathway (that is, hypoxanthine and uric acid) indicating that this biochemical pathway is of particular importance. It is also important to note that there were several unknown compounds that were among the most influential variables in this model. Cross-validation of these models using the 'one out' method described above enabled correct classification of 8 of 19 (42%) of the responders and 25 of 27 (93%) of the non-responders with an overall classification rate of 72%. BODY.DISCUSSION: It is important to define the metabotype of patients who will not respond to treatment with a particular SSRI, as it can reduce the trial and error approach to treatment and help with selection of an efficacious drug for each patient. Defining who might respond on placebo is also of great importance as those who are very likely to respond on placebo could be excluded from clinical trials to increase the chances for seeing a drug-specific effect. This may also reduce patient's exposure to drugs they might not need. In antidepressant trials, placebo response is typically high and tends to obscure efficacy signals of drugs, while introducing heterogeneity in the observed endpoints such as the HRSD17. Any new tool to reduce this unwanted data variability, based on well-demonstrated data, would greatly benefit clinical trials as well as clinical practice. Analysis of metabolic profiles at baseline using digital maps enabled separation of responders and non-responders on sertraline, and between responders and non-responders on placebo. This approach identified several compounds that contributed to separation of responders and non-responders on sertraline in the PLS-DA model. These included DOPAC, 4-HPLA, 5-HT and gamma tocopherol. In the placebo group we note several metabolites contributed to separation of responders and non-responders. These include: hypoxanthine, xanthine and uric acid (purine pathway); 5-MTPOL, 5-HT, 3-OHKY and 5-hydroxyindoleactic acid (tryptophan pathway); DOPAC (tyrosine), cysteine and tocopherols (vitamin E). Analysis of sertraline and placebo models shows that DOPAC, gamma tocopherol, and serotonin significantly contributed to the separation of responders and non-responders, though the relative strength of DOPAC was different in the two models. There were a number of known compounds that contributed to separation of responders and non-responders that appeared to be unique to either sertraline or placebo. Examples include the importance of purines and 5-MTPOL in response to placebo, but not to sertraline and the role of 4-HPLA in response to sertraline, but not to placebo. Thus, it appears that the metabotype of responders to sertraline only partially overlaps with that of responders to placebo. Models based on variables selected from the total output of the LCECA platform enabled partial classification of responders and non-responders on sertraline based on percent change in HRSD17 score from baseline to week 4. With this relatively small sample size, we were able to classify 21 of 25 (84%) responders and 14 of 18 (78%) non-responders on sertraline with an overall classification rate of about 81%. In the placebo-treated group, we were able to predict better the non-responders 25 of 27 (93%) but prediction of responders was not possible in this pilot study (8 of 19 or 42% of the responders). The overall classification rate in placebo was 72%. This suggests that biological response to place could be more complex, as reflected by metabolomic profiles, compared with the response to drug. It is interesting to note that 5-MTPOL is a methylated product within the methoxyindole pathway in the pineal gland.41, 42, 43 5-MTPOL is produced from serotonin through a series of steps involving monoamine oxidase, aldehyde reductase and hydroxyindole-O-methyltransferase. 5-MTPOL has important functions that parallel melatonin, another methylated product of serotonin produced in pineal gland. Both molecules are implicated in sleep architecture, circadian rhythm and hormonal axis regulation, among other important functions that are often altered in depressed patients. Full analysis of this pathway is underway. 4-HPLA belongs to the phenyl alanine pathway and is interconnected with the tyrosine pathway and catecholamine production. Many of the most influential variables discriminating between responders and non-responders to sertraline and responders and non-responders to placebo were of unknown structure. If these associations are confirmed in larger studies it would be of importance to focus on elucidating the chemical structure of those metabolites as a next step towards a more complete understanding of the biochemistry underpinning response variation to sertraline and placebo. This will entail a larger initiative beyond the scope of the current work. Studies with larger sample sizes will be needed to better predict who will and will not respond to either sertraline or placebo at week 4 and to determine whether they have a common metabolomic profile. This might enable the pre-selection of these patients for a different treatment strategy. Any tools that can help a clinician define early on if a patient is not going to respond to an SSRI would be very useful and can minimize the poly-pharmacy approach in depression treatment. Our findings suggest that response to sertraline and response on placebo are a function of the underlying unique metabotype of the patient at baseline. This inherent metabolomic fingerprint captures a metabolic state as regulated by genome, proteome and environment interactions. This holds promise for enabling sub-classification of depression states and improving clinical decisions from an initial set of biochemical analyses before prescription of a given therapy, much like tumor genotyping for treatment selection. It is important to note that this analysis was performed on a subset of MDD patients and presented inherent limitations. We have focused on an early time point of response and a longitudinal study is needed where one would evaluate earlier and later time points up to 12 weeks. Our interest in the earlier time points was based on our desire to develop biomarkers of early response, or no response. Larger studies would be needed to better define effects of gender and ethnic background on metabolic signatures of response and to enable sub stratification of depression. The presence of outliers might represent subtypes of MDD that will require larger sample sizes to more fully characterize. In an ongoing study of 1200 MDD patients treated with escitalopram citalopram at the Mayo Clinic, metabolomic and genetic data are being used to better define subtypes of depression and metabolic signatures of early response, late response or no response. An additional limitation to our study is that the LCECA platform captures information on only redox active compounds in the tyrosine, tryptophan, purine and sulfur amino acid pathways and several markers of vitamin status and oxidative processes. The integration of data from lipidomics and mass spectrometry-based metabolomics platforms in future studies will be necessary to yield additional response predictive power. In summary, we have shown that the baseline metabolomic profile or 'metabotype' of an MDD patient, as indexed by an electrochemistry metabolomics platform total output (digital map), could help classify patients as responders or non-responders to sertraline or to placebo. It suggests that metabolomics and metabolic profiles of patients could be valuable in helping to guide therapeutic choices, thus reducing trial and error in matching the right drug with the right patient. Such findings have the potential to contribute to personalizing therapeutic treatment for patients with a diagnosis of MDD. Confirmation of these results with samples from an unrelated clinical trial of similar design would greatly enhance the impact of these early reported findings. These results provide a powerful stepping stone into a thorough metabolite identification campaign that will highlight the pathways involved in both response mechanisms, and hopefully lead to biomarker discovery usable in clinical practice and clinical trial design. Efforts are already ongoing to collect samples from independent trials to confirm the present findings. Although this study focused on the use of an electrochemistry platform and a specific area of biochemistry, future studies using complementary platforms will enable us to define further the biochemical pathways contributing to response variation. The ultimate goal would be to reduce the complexity of a digital map and identify a subset of known metabolites that are easily measured in a clinical setting. In conclusion, this study supports the feasibility of using metabolomics as a tool for understanding individual variation in response to pharmacotherapy for MDD and hence means to sub-classify patients with depression.

TITLE: An interactive videogame designed to improve respiratory navigator efficiency in children undergoing cardiovascular magnetic resonance ABSTRACT.BACKGROUND: Advanced cardiovascular magnetic resonance (CMR) acquisitions often require long scan durations that necessitate respiratory navigator gating. The tradeoff of navigator gating is reduced scan efficiency, particularly when the patient's breathing patterns are inconsistent, as is commonly seen in children. We hypothesized that engaging pediatric participants with a navigator-controlled videogame to help control breathing patterns would improve navigator efficiency and maintain image quality. ABSTRACT.METHODS: We developed custom software that processed the Siemens respiratory navigator image in real-time during CMR and represented diaphragm position using a cartoon avatar, which was projected to the participant in the scanner as visual feedback. The game incentivized children to breathe such that the avatar was positioned within the navigator acceptance window (±3 mm) throughout image acquisition. Using a 3T Siemens Tim Trio, 50 children (Age: 14 ± 3 years, 48 % female) with no significant past medical history underwent a respiratory navigator-gated 2D spiral cine displacement encoding with stimulated echoes (DENSE) CMR acquisition first with no feedback (NF) and then with the feedback game (FG). Thirty of the 50 children were randomized to undergo extensive off-scanner training with the FG using a MRI simulator, or no off-scanner training. Navigator efficiency, signal-to-noise ratio (SNR), and global left-ventricular strains were determined for each participant and compared. ABSTRACT.RESULTS: Using the FG improved average navigator efficiency from 33 ± 15 to 58 ± 13 % (p < 0.001) and improved SNR by 5 % (p = 0.01) compared to acquisitions with NF. There was no difference in navigator efficiency (p = 0.90) or SNR (p = 0.77) between untrained and trained participants for FG acquisitions. Circumferential and radial strains derived from FG acquisitions were slightly reduced compared to NF acquisitions (−16 ± 2 % vs −17 ± 2 %, p < 0.001; 40 ± 10 % vs 44 ± 11 %, p = 0.005, respectively). There were no differences in longitudinal strain (p = 0.38). ABSTRACT.CONCLUSIONS: Use of a respiratory navigator feedback game during navigator-gated CMR improved navigator efficiency in children from 33 to 58 %. This improved efficiency was associated with a 5 % increase in SNR for spiral cine DENSE. Extensive off-scanner training was not required to achieve the improvement in navigator efficiency. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12968-016-0272-z) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: Cardiovascular magnetic resonance (CMR) can be used to non-invasively assess heart function. In the clinical setting, CMR techniques play an important role in the diagnosis and monitoring of the complex anatomy and physiology of congenital and acquired heart diseases. Moreover, there is a considerable body of pre-clinical research devoted to the development and evaluation of new, advanced imaging techniques, such as 3D displacement encoding with stimulated echoes (DENSE) [1], 3D steady state free precession [2], and 4D flow imaging [3]. These new techniques have demonstrated ability in distinguishing normal and pathological tissue deformation and blood flow and may become beneficial tools in the diagnosis and management of heart disease. Many of these clinical and pre-clinical techniques require scan durations that exceed patients' ability to hold their breath. End-expiratory breath-holds are used by many CMR sequences in order to minimize respiratory-motion artifacts. However, requiring subjects to hold their breath introduces significant limitations on the duration of data acquisition or the quality of the acquired images, particularly for young children or patients with advanced disease. A common alternative is respiratory navigator gating, which works by measuring the diaphragm position during normal breathing and only acquiring data when the diaphragm is within a pre-defined acceptance window (Fig. 1a). The trade-off of navigator gating is significantly increased scan duration because of poor navigator efficiency. For example, previous CMR studies have reported respiratory navigator efficiencies of 20 to 45 % in adults [4–7]. This poor navigator efficiency lengthens the duration of currently used clinical imaging and limits clinical feasibility of emerging advanced imaging techniques.Fig. 1Feedback videogame. a Cropped version of the Siemens Syngo navigator image that was processed in real-time during CMR acquisition to yield the feedback videogame. b Example frame of the navigator feedback videogame interface, which was shown to the child during CMR (yellow overlay text was not shown to the child) Navigator efficiency is typically poor because breathing patterns can be erratic [8–10] and the patient is generally unaware of the desired acceptance window location. Providing the patient with visual feedback of the diaphragm position during CMR ("navigator feedback") has been shown to improve breathing consistency and scan efficiency in adults [5, 8]. For example, studies have shown efficiency improvements up to 29 % (absolute) compared to traditional acquisitions without feedback [5, 6]. Importantly, these previous studies have demonstrated that image quality from navigator feedback acquisitions is similar to acquisitions without feedback [5, 6]. The potential to achieve similar benefits using navigator feedback with pediatric participants has not been explored. Given the challenge of keeping these participants still and motionless for long periods of time, this improved efficiency could have substantial clinical benefit. Most previous studies involving navigator feedback simply utilized the built-in navigator display. One previous study evaluated a custom videogame interface in a study of adults for increasing navigator efficiency [6]. Such an interface theoretically combines the benefits of visual feedback with an intuitive and engaging design for the user—attributes that are highly desirable for pediatric scanning. Thus, the present study sought to extend and tailor this paradigm specifically for children by providing navigator feedback in the form of an interactive, kid-friendly videogame. Moreover, this study sought to test this design using DENSE, an imaging technique that can be used to quantify advanced measures of function such as cardiac strains. We hypothesized that navigator feedback using an interactive videogame during CMR would improve navigator efficiency and maintain image quality and strains in children. BODY.METHODS.FEEDBACK VIDEOGAME: A navigator feedback videogame (FG), called "Bubble Gulp", was developed using MATLAB (The Mathworks Inc, Natick, MA). Each frame of the navigator image provided within the Siemens Syngo user-interface (Siemens Healthcare, Erlangen, Germany) (Fig. 1a) was captured using an Epiphan DVI2USB 3.0 (Epiphan Systems Inc., Palo Alto, California) frame grabber and processed in real-time during CMR to yield a kid-friendly representation of the diaphragm position (Fig. 1b). Navigator image processing was performed using an externally connected laptop running Windows 7 with an Intel Core i7 processor and 16 GB of RAM. The FG interface was then projected to the participant in the scanner using an angled mirror and a magnetic resonance compatible projector (Fig. 2).Fig. 2Feedback videogame was shown to children during CMR with an angled mirror and MR-compatible projector The diaphragm position relative to the acceptance window (Fig. 1a) was represented by the vertical position of a fish character relative to parallel green lines containing scrolling dots, representing bubbles (Fig. 1b). The objective of the game was to control the fish's vertical position, which was updated with each navigator pulse, so it would "gulp" bubbles and acquire points. To incentivize slow, stable breathing, point values increased as the fish spent more time within the green lines, instead of frequent short-duration breath-holds. However, prior to any use of the FG, children were instructed to not hold their breath for an uncomfortable amount of time and to breathe when needed. Finally, the FG interface displayed text to instruct children how to adjust their breathing in order to place the fish in between the green lines (Fig. 1b). BODY.METHODS.PARTICIPANTS: Fifty children with no significant past medical history were recruited to participate in the study. Participants were recruited from the broader clinical community based out of our university medical center using a wide range of participant recruitment services provided by the University of Kentucky Center for Clinical and Translational Science. All participants were screened with a 12-lead ECG prior to imaging to rule out arrhythmias. The local Institutional Review Board at the University of Kentucky approved the study protocol and all participants and legal guardians provided written informed consent or assent. BODY.METHODS.IMAGING: All imaging was performed using a 3T Siemens Tim Trio (Siemens Healthcare, Erlangen, Germany) with a 6-element chest coil and a 24-element spine coil. For each participant, navigator-gated 2D spiral cine DENSE CMR [1, 11] images from mid-ventricular, 4-chamber, basal, and apical image orientations were separately acquired with no feedback (NF) and then while using the FG. No instructions regarding breathing were given for the NF acquisitions, thus participants were allowed to breathe naturally. Between acquisitions with NF and those with the FG, each participant underwent two 30-heartbeat practice scans to familiarize himself or herself with the FG. DENSE imaging parameters included: number of spiral interleaves = 12, interleaves per beat = 2, FOV = 360 × 360 mm2, pixel spacing = 2.8 × 2.8 mm2, slice thickness = 8 mm, TE = 1.4 ms, TR = 17 ms, variable flip angle = 20°, displacement encoding = 0.06 cyc/mm [12], through-plane dephasing = 0.08 cyc/mm [13], CSPAMM echo suppression [14], view sharing and a dual-navigator strategy [15] with an acceptance window size of ± 3 mm. For each cardiac cycle, the navigator echo occurred immediately after data acquisition. The dual-navigator strategy required the diaphragm position to be within the acceptance window for both the preceding and current cardiac cycles in order for data to be accepted. Prospective ECG gating was performed and 11–25 cardiac phases were acquired depending on participant heart rate. As a result of the imaging parameters, each complete image acquisition required 38 heartbeats that satisfied the navigator gating criteria. Due to erratic respiratory patterns or participant movement, image acquisition can be difficult to complete in children in a reasonable amount of time with NF. As scan session duration increases, the likelihood of patient movement also increases, so we defined criteria for maintaining a target scan protocol duration of 30 min. We defined image acquisition as incomplete (data not acquired) following 192 heartbeats without a completed image acquisition. Progressing past 192 heartbeats for a 38-heartbeat scan is equivalent to achieving less than 20 % navigator efficiency, which is worse than previously reported NF values [4–7]. Once any NF image acquisition was marked as incomplete, we proceeded to the FG acquisitions. If a participant moved, the number of acquired image orientations was reduced from four (mid, 4ch, base, apex) to two (mid, 4ch) to ensure at least two images were acquired with NF and FG. BODY.METHODS.CALCULATION OF CARDIAC STRAINS FROM DENSE: DENSE images were analyzed using DENSEanalysis (denseanalysis.com), a custom, open-source MATLAB (the Mathworks Inc, Natick, MA) software. To delineate the myocardium, endocardial and epicardial boundaries were manually drawn on the DENSE magnitude image using an end-systolic and end-diastolic frame. The motion field was reconstructed using a simplified analysis technique [16]. Using manual selection of seed points, which indicated unwrapped phase data, a path following algorithm was used to unwrap the displacement-encoded phase data. Temporal fitting and spatial smoothing was applied to the resulting Lagrangian displacements as previously described [17]. Two-dimensional segmental Lagrangian strains were quantified from the smoothed trajectories over the entire cardiac cycle. Radial and circumferential strain was computed for 6 myocardial segments of the short-axis images and longitudinal strain was computed from the long-axis images. The strain curves of all the cardiac segments were averaged into a single average curve. Global peak strain was quantified by averaging the strain curves from each slice and finding the resulting peak strain of this curve. When computing peak longitudinal strain, pixels within 10 % of left ventricular longitudinal length of the most basal and apical regions were excluded due to increased noise typically observed in the strain curves in those regions. Peak strain was defined as a positive for thickening (radial strain) and negative for shortening (circumferential and longitudinal strain). BODY.METHODS.ANALYSIS: This study measured navigator efficiency and heart rate during image acquisition and used image signal-to-noise ratio (SNR) of the end-systolic DENSE magnitude image as a measure of image quality. Navigator efficiency was defined as the ratio of the number of heartbeats for which image data were accepted to the total number of heart beats required to complete the image acquisition. To compare image quality, signal to noise ratio (SNR) was calculated for each cardiac phase of each DENSE magnitude image. SNR was computed from the average myocardium signal and the standard deviation of the signal (noise) within an area with no signal (free from tissue and imaging artifacts). Due to the Rician distribution of the MR signal, corrections were applied to the measured standard deviation (σM in Eq. 1) and measured myocardial signal (M in Eq. 2) to compute the true SNR [11, 12, 18]. The SNR was defined as the ratio of the true myocardial signal to the true standard deviation.1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \sigma = \sqrt{\frac{2}{4-\pi }}*{\sigma}_M \approx 1.526*{\sigma}_M $$\end{document}σ=24−π*σM≈1.526*σM2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ S = \sqrt{M^2-{\sigma}^2} $$\end{document}S=M2−σ2 For incomplete NF image acquisitions (satisfied stoppage criterion), navigator efficiency and heart rate measurements were computed based on the partial data that were acquired. BODY.METHODS.TRAINING: Off-scanner training has been used by other investigators to ensure participants are comfortable and understand a navigator feedback interface before entering the magnet [5]. We wanted to determine the efficacy of off-scanner training with the FG on navigator efficiency, image quality, and heart rate. Thus, 30 of the 50 enrolled participants were randomized into equal groups to either receive extensive off-scanner training or no off-scanner training prior to scanning; thus, the groups were referred to as 'trained' and 'untrained.' As mentioned above, all subjects (including trained and untrained participants) underwent minimal training in the scanner, which was defined as two 30-heartbeat practice scans prior to FG acquisitions. The remaining 20 participants also received off-scanner training, but they were not included within the trained subgroup for analysis because they were not randomized to this treatment. Each trained participant was introduced to the FG using an MRI simulator prior to the formal study. The MRI simulator utilized a PrimeSense Carmine 1.09 (PrimeSense, Tel Aviv, Israel) 3D camera to precisely measure the chest wall and abdomen excursion as a proxy for diaphragm translation [19, 20]. Each participant had to complete goal-based training before advancing to CMR scanning. Training time was recorded for all trained participants. The training protocol is described in detail in Additional file 1. BODY.METHODS.STATISTICS: Statistical analyses were completed using R version 3.2.2 (R Foundation for Statistical Computing, Vienna, Austria). All continuous measurements were reported as mean ± standard deviation. Navigator efficiency, SNR, heart rate, and global left ventricular strains were tested for normality using a Shapiro-Wilk test. Average navigator efficiency, SNR, heart rate, and strain were compared between NF and FG acquisitions using a paired student's t-test or Wilcoxon Signed-Rank test when appropriate, and compared between untrained and trained groups using a student's t-test or Mann-Whitney U test when appropriate. To determine whether age had an effect on navigator efficiency, age was correlated with navigator efficiency for both NF and FG acquisitions. BODY.RESULTS: Fifty-six children were prospectively enrolled. Six children were excluded from the study due to either being uncomfortable in an MRI scanner, having premature ventricular contractions, having ECG-monitoring equipment fail, or consistently moving during scanning. Thus, this study reported data on 50 children (Age: 14 ± 3 years, 48 % female) with no significant past medical history, which included a subset of 30 children randomized to either the off-scanner trained (n = 15; Age: 15 ± 3 years, 47 % female) or untrained (n = 15; Age: 13 ± 3, 66 % female) groups. All trained participants successfully completed off-scanner training and the mean training duration was 11 ± 2 min. The prescribed stoppage criterion for the NF scans was met in 11 cases, resulting in fewer completed NF images for those participants. Additionally, four participants moved during scanning, which included two during NF scans and two during FG scans, resulting in the completion of the abridged imaging protocol, as described in the methods. BODY.RESULTS.NAVIGATOR EFFICIENCY: Using the FG significantly improved average navigator efficiency compared to NF (58 ± 13 % vs 33 ± 15 %, p < 0.001, Fig. 3a). Average navigator efficiency was not correlated with age for either NF or FG image acquisitions (r = −0.07, p = 0.63; r = 0.14, p = 0.32, Fig. 3b). There was no significant difference in average navigator efficiency between untrained and off-scanner trained groups for FG image acquisitions (57 ± 17 % vs 57 ± 11 %, p = 0.90, Fig. 4).Fig. 3 a Average navigator efficiency for No Feedback and Feedback Game image acquisitions. Use of the feedback game significantly increased navigator efficiency compared to no feedback. The solid red line indicates the mean of each group. b Average navigator efficiency vs age for No Feedback (NF) and Feedback Game (FG) image acquisitions. There was no correlation between navigator efficiency and age for either no feedback (r = -0.07, p = 0.63) or feedback game (r = 0.14, p = 0.32) acquisitions. The solid lines indicate the line of best fit for each groupFig. 4Average navigator efficiency for Off-scanner Trained and Untrained groups. There was no significant difference in navigator efficiency between untrained and off-scanner trained groups for feedback game acquisitions. The solid red line indicates the mean of each group BODY.RESULTS.SNR: Use of the FG significantly improved SNR compared to NF (22 ± 6 vs 21 ± 6, p = 0.01, Fig. 5). There was no significant difference in SNR between untrained and off-scanner trained groups for FG images (22 ± 6 vs 21 ± 6, p = 0.77).Fig. 5SNR for all No Feedback and Feedback Game images. Use of the feedback game resulted in significantly increased SNR compared to no feedbackTable 1Average Heart Rate for Off-scanner Trained and Untrained groupsHeart Rate (bpm)Trained p-valueUntrained p-valueNo FeedbackFeedback GameNo FeedbackFeedback GameMean72 ± 1376 ± 160.0372 ± 978 ± 9<0.001Standard Deviation6.9 ± 5.05.7 ± 2.40.805.3 ± 2.46.0 ± 2.00.17 BODY.RESULTS.HEART RATE: On average, heart rate during FG scans was slightly higher than NF acquisitions (75 ± 13 vs 72 ± 12 bpm, p < 0.001, Fig. 6a), but there were no differences in the standard deviation of heart rate (5.9 ± 2.2 vs 6.1 ± 3.9 bpm, p = 0.30, Fig. 6b). Heart rate was similarly elevated during FG acquisitions in both the untrained and off-scanner trained groups compared to NF acquisitions (p < 0.001 and p = 0.03, respectively, Table 1).Fig. 6Mean and standard deviation of heart rate for No Feedback and Feedback Game acquisitions. a. Use of the feedback game resulted in significantly higher heart rate compared to no feedback. b. There was no significant difference in standard deviation of heart rate between no feedback and feedback game acquisitions. The solid red line indicates the mean of each group BODY.RESULTS.STRAIN: Global circumferential and radial strains derived from FG acquisitions were slightly lower in magnitude compared to NF acquisitions (−16 ± 2 % vs −17 ± 2 %, p < 0.001; 40 ± 10 % vs 44 ± 11 %, p = 0.005, respectively, Table 2). There were no differences in longitudinal strain between NF and FG acquisitions (−13 ± 2 % vs −13 ± 2 %, p = 0.38).Table 2Global peak strain results for NF and FG scansNo FeedbackFeedback Game p-valueCircumferential Strain (%)−17 ± 2−16 ± 2<0.001Radial Strain (%)44 ± 1140 ± 100.005Longitudinal Strain (%)−13 ± 2−13 ± 20.38 BODY.DISCUSSION: Feedback of the diaphragm position during CMR has been shown to improve navigator efficiency in adults [5, 6]. This study explored how the use of a feedback game (FG) affects navigator efficiency compared to traditional no-feedback (NF) acquisitions in children. The results of the study showed that, compared to NF, using the FG resulted in 1) substantially improved navigator efficiency (from 33 to 58 %); 2) slightly improved SNR; 3) slightly higher mean heart rate; and 4) slightly lower global strain magnitudes. Importantly, these results were not affected by the use of an off-scanner training protocol, which suggests that lengthy, robust training (11 min in our protocol) does not need to be a part of the clinical/imaging workflow for this interface. BODY.DISCUSSION.NAVIGATOR EFFICIENCY: Navigator efficiency was improved from 33 to 58 % by using a FG in children (Fig. 3a). This increase in navigator efficiency led to a 43 % reduction in the number of heartbeats required to complete a scan. Studies have shown that feedback of the diaphragm position during CMR results in a more reproducible breath-hold position [5, 8, 21], which can lead to improved navigator efficiency. Previous CMR studies have reported that NF navigator efficiencies can vary from 20 to 45 % in adults [4–7], and we found a comparable NF navigator efficiency of 33 % in children using a conservative dual-navigator acceptance strategy. Visual feedback of the diaphragm position has been shown to improve end-expiratory navigator efficiency from 45 to 56 % [6] and from 42 to 71 % with the addition of supplemental oxygen [5] leading to a 20 % and 41 % reduction in the number of required heartbeats, respectively. With the use of the FG, we found a slightly better improvement of navigator efficiency from 33 to 58 % in children without the use of supplemental oxygen. Average navigator efficiency was not correlated with age (Fig. 3b). Therefore, children ages eight and older should be able to utilize the FG to effectively improve navigator efficiency compared to conventional NF acquisitions. Extensive off-scanner training using an MRI-simulator was not necessary to achieve the observed improvement in navigator efficiency using the FG. Instead, the subjects with minimal training immediately prior to data acquisition had equivalent efficiency as their extensively-trained counterparts. While this finding might suggest that the chest wall excursion-based training method was ineffective, it is more likely that the intuitive interface design was easy to learn and therefore the children did not require much training. Importantly, the two 30-beat practice scans provided some degree of training in both cases, which is intuitively necessary. Future efforts can optimize that practice time to provide the needed minimal training in the most efficient manner. BODY.DISCUSSION.SNR: We found that using the FG slightly improved the SNR of the end-systolic magnitude images of our spiral DENSE sequence by 5 % compared to NF for all images combined (p = 0.01, Fig. 5). This finding contrasts with previous studies, which reported image quality score using 2 expert reviewers and found that the use of diaphragmatic feedback maintained image quality compared to NF acquisitions [5, 6]. The difference in image quality is likely sequence dependent. The previous studies were performed using steady-state free precession. Additionally, it is likely that quantitative measurement of SNR is more sensitive at detecting differences in image quality compared to subjective image scoring by expert reviewers. BODY.DISCUSSION.HEART RATE: A potential negative finding of this study was the slight increase in heart rate observed with the use of the feedback game. To be clear, this difference did not represent an increase in heart rate variability—as evidenced by the comparable standard deviation values—but simply a higher baseline value. Such findings are not unprecedented, as a previous CMR study found a mean heart rate increase of 5 beats/min with use of navigator feedback in adults (compared to our 3 beats/min), and similarly no differences in heart rate variability between NF and navigator feedback [5]. A likely reason for this difference is the longer breath-holds performed during the FG, which could have increased the heart rate, compared to relaxed breathing during NF. Another mechanism could be related to stimulation and adrenaline associated with playing the game, compared to the relaxed, passive state associated with NF. The importance and implications of this potential heart rate difference likely depends on the imaging application. While it may mean very little for purely anatomic evaluations, functional measures, such as strains, may be affected by changing loading conditions and contractility [22]. To counteract such effects, if undesirable, patients could be coached to relax when playing the game and to not be too competitive. The design of the game could be modified to enforce such behavior; for example, by programmatically requiring the participant to inhale/exhale after a fixed period of time, or instructing him/her to periodically take a series of relaxed breaths between cycles of breath-holding. BODY.DISCUSSION.STRAINS: We observed small, but statistically significant decreases in global circumferential and radial strains with use of the FG, compared with NF. There was, however, no difference in longitudinal strain. While these findings warrant further study and consideration, the clinical relevance of such small differences (1 % for circumferential strain, 4 % for radial strain) is likely minimal as they are smaller than previously observed inter-test (±2.0 % for circumferential, ±13 % for radial) and inter-observer (±1.4 % for circumferential, ±14 % for radial) 95 % limits of agreement for DENSE [11, 12]. BODY.DISCUSSION.CLINICAL IMPLICATIONS: Importantly, the equipment needed to utilize the FG is minimal and does not directly integrate into an imaging sequence; it connects externally to the scanner user interface. Due to the minimal equipment needed and non-invasive connection to the MRI scanner, we anticipate that the FG system can be easily adopted at research and clinical sites that perform CMR navigator gating, especially in children. Since navigator efficiency can be increased from 33 to 58 %, leading to reduced acquisition times, use of the FG can help improve the clinical feasibility of advanced imaging techniques. While reducing the acquisition time would likely be the most common use of increased navigator efficiency from the FG, the saved time could be allocated to improve image spatial or temporal resolution [5]. Importantly, pre-scan training was not necessary for navigator efficiency improvement with our system, so clinical and research sites would not have to invest in an MRI simulator environment or spend significant time training children. Navigator feedback has been shown to reduce acquisition time in adults [5], thus, the use of the FG will likely work well in adults also. Since we only acquired DENSE images for this study, the specific findings are only definitively relevant for DENSE. However, it is reasonable to expect that these findings are generalizable to many other CMR acquisitions that utilize a respiratory navigator. Possible exceptions include higher resolution applications, such as coronary MR angiography, which may be more sensitive to registration issues. Further study is needed to test this technique for these applications. BODY.DISCUSSION.COMPARISON WITH PREVIOUS WORK: A previous study presented a respiratory biofeedback game and continuously adaptive windowing strategy (CLAWS) to increase navigator efficiency of imaging the thoracic aorta. The authors reported an increase in efficiency in that study from 45 to 56 % in adults [6], which represents a smaller magnitude of improvement (25 % vs. 11 %) but a similar end result (58 % vs. 56 %) compared to our study. Although the two studies are similar, there are distinct differences in design. Most notably, the previous study was in adults; whereas we exclusively focused on children, based on their limited ability to breath-hold and thus potentially greater need for respiratory navigated sequences. Additionally, the previous study modified their pulse sequence to allow acquisition of multiple navigator echoes, likely providing a smoother game experience. We did not modify our cine pulse sequence in our evaluation—we had a single navigator echo per cardiac cycle—in order to ensure more general clinical applicability. Collectively, these studies demonstrate the potential utility of user-friendly interfaces for improving efficiency and image quality of cardiovascular imaging sequences using a respiratory navigator in a broad array of patients. BODY.DISCUSSION.LIMITATIONS: This study used a dual-navigator strategy when performing image acquisition. Dual-navigator strategies have stricter data acceptance criteria compared to previously used single-navigator strategies [1], and, given the same imaging parameters, will likely result in lower navigator efficiencies. However, a previous study using a single-navigator strategy with navigator feedback reported similar navigator efficiency results compared to our study. Therefore, the use of the FG with a single-navigator strategy will likely have similar results to this study except that both NF and FG acquisitions may have improved navigator efficiency compared to a dual-navigator strategy. The respiratory navigator gating sequence used in this study only measured the diaphragm position once per cardiac cycle. This low refresh rate can make fine control of the diaphragm position more challenging, especially for participants who may have lower heart rates. Increasing the number of navigator echoes per cardiac cycle could therefore improve performance, but such modifications may not be possible for all sequences, as is the case for DENSE. Furthermore, even with this limitation, we still found substantial improvement in navigator efficiency when using the FG compared to NF acquisitions. Due to the randomization of the participants into the trained and untrained groups, there was no attempt to balance age between groups. Therefore, the average trained participant was about 2 years older than the average untrained participant. We found that there was no difference in FG navigator efficiency between trained and untrained participants. Even though there was an age difference between trained and untrained groups, there was no correlation between age and navigator efficiency with the FG (Fig. 3b); thus, the results of the study apply to all children aged eight to eighteen. In order to accurately assess the NF navigator efficiency as it would be in the clinical setting, we did not want to influence the children's natural breathing pattern. In particular, we did not want the breathing pattern performed during the FG acquisitions to influence the NF breathing pattern. Therefore, NF acquisitions were always performed before FG acquisitions. Since the order of NF and FG acquisitions was not randomized, this may have affected the results as participants may have become more comfortable as they spent more time in the MRI scanner. However, performing this randomization likely would have resulted in similar conclusions and we feel that it was important to accurately measure the navigator efficiency of the NF acquisitions. Due to the potential for patient movement or erratic breathing patterns, we utilized a stoppage criterion to attempt to maintain a 30 min protocol length. We observed eleven cases which satisfied stoppage criterion and four cases of patient movement (one which also satisfied stoppage criterion). In these participants, we estimated navigator efficiency, SNR, and heart rate from fewer acquisitions than the remaining participants. However, since we used all of the data that we did acquire for each participant, the computed values are appropriate. The two 30-heartbeat practice scans were not included in the computation and analysis of navigator efficiency for the FG technique. Their inclusion would only slightly decrease the reported gains in efficiency (for example, if we used the FG to acquire 300 heart beats of actual data, the reduction in scan time would change minimally from 43 to 37 % after accounting for the two practice scans); however, it must be noted that the selection of those practice parameters was arbitrary and not optimized. In reality, less training is likely required to familiarize the subject with the interface, so factoring this specific training design into the analysis is not critical. We performed this study in children with no significant past medical history. While we did attempt to recruit from a broad clinical population using recruitment services at our Center for Clinical and Translational Science, the population we ultimately studied may not be entirely representative of a standard pediatric clinical population that would routinely undergo cardiac MRI. For example, approximately 25 % of patients with tetralogy of Fallot may have learning and behavioral difficulties [23], which may impair their ability to benefit from the feedback game. It is therefore reasonable to expect that the true benefit of the feedback game in a standard clinical population will be smaller than what was measured in the current study, but still better than what can be expected without the use of feedback. Even if only half of the patients benefit to the extent shown in the current study, the overall navigator efficiency for the clinical population as a whole would still increase from 33 % efficiency to 46 % efficiency (a 38 % relative benefit). Future research will seek to evaluate this in further detail as we implement the feedback game during routine clinical workflows. BODY.CONCLUSION: Use of a respiratory navigator feedback game designed to engage children during navigator-gated CMR improved navigator efficiency in children from 33 to 58 %. This improved efficiency reduces the number of heartbeats and corresponding scan durations by 43 %, and is also associated with a 5 % increase in SNR for spiral cine DENSE. Pre-scan training on how to use the feedback game is not necessary to achieve the improvement in navigator efficiency. These findings should generalize to all CMR acquisition sequences that utilize a respiratory navigator.

TITLE: Comparison of treatment response to intravitreal injection of triamcinolone, bevacizumab and combined form in patients with central retinal vein occlusion: A randomized clinical trial ABSTRACT.BACKGROUND: Central retinal vein occlusion (CRVO), is the second-most common disorder after diabetic retinopathy that threatens the vision related to retinal vessels. One of the main reasons of vision loss is CRVO in acute and chronic forms. ABSTRACT.OBJECTIVE: The aim of this study was to investigate the response to intravitreal injection of triamcinolone, bevacizumab, and a combined form in patients with CRVO. ABSTRACT.METHODS: This study was a double-blinded randomized clinical trial conducted on patients with CRVO who were referred to the ophthalmology clinic of Urmia Imam Khomeini Hospital from May 2015 to May 2016. In total, ninety patients were divided into three groups using random numbers table. The first group received intravitreal triamcinolone acetonide (IVT) for treatment of macular edema due to CRVO, the second group received intravitreal bevacizumab (IVB) and the third group received a combination of IVT and IVB. The best corrected visual acuity (BCVA) and central macular thickness (CMT) were recorded and analyzed using optical coherence tomography system. ABSTRACT.RESULTS: In this study a total of 90 eyes were studied, 41 case (45.55%) were male and 49 cases (54.44%) were female. The mean age of patients was 68.41±8.32 years. The mean score of final visual acuity was 0.293±0.11 in the IVT group, 0.25±0.10 in the IVB group and 0.48±0.15 in the IVB+IVT group. The differences between groups considering final visual acuity was significant (p<0.001). The mean thickness of final macular was 383.33±97.70 micrometer in IVT group, 386.33±136.79 micrometers in IVB group and 307.33±110.79 micrometers in IVT+IVB group which were significant (p=0.014). ABSTRACT.CONCLUSION: Using a combination of bevacizumab and triamcinolone in the treatment of central retinal vein occlusion compared with using each of them separately, had a better result and can be used as a solution in this disease. ABSTRACT.TRIAL REGISTRATION: The trial was registered at the Thai Registry of Clinical Trials (http://www.clinicaltrials.in.th) with the TCTR ID: TCTR20170612005. ABSTRACT.FUNDING: The authors received no financial support for the research, authorship, and/or publication of this article. BODY.1. INTRODUCTION: Central retinal vein occlusion (CRVO), is the second-most common disorder after diabetic retinopathy (1). One of the main reasons of vision loss is CRVO in acute and chronic forms. There are many risk factors associated with CRVO including age, hypertension, diabetes mellitus, retinal artery atherosclerotic changes, open-angle glaucoma and hyperopia (2). Its prevalence increases with age and varies from 0.1% to 5%. Visual acuity is a reflection of the severity of the disease, retinal macular hemorrhage, cystoid macular edema and ischemia (3). Macular edema is one of the fundamental causes of vision loss in chronic and acute CRVO as well as ischemic and non-ischemic forms (4). CRVO study showed that although the macular network photocoagulation decreased angiographic edema, the vision did not improve (3). Recently, the standard treatment for central retinal vein occlusion was limited to photocoagulation for neovascular adverse effects and there was no solution to macular edema (3). The main trigger for the formation of edema and macular neovascularization in patients with CRVO is the production of vascular endothelial growth factor (VEGF (caused by hypoxia, which is an angiogenic factor, causing angiogenesis and increase of vascular permeability (5). It has been shown that vascular endothelial growth factor increases in eyes with CRVO (6). Some studies, as noted, have shown that the injection IVB and injection of IVT have beneficial effects for these cases. However, due to their half-life, repeated injections are required (22). Studies did not have the same results on the effectiveness and safety of these therapies in CRVO, and so, this shows the importance of this study in the world. Thus, the aim of the study was to compare the effectiveness of IVT and IVB separately and combined, for the treatment of patients with CRVO, to obtain the best choice. BODY.2. MATERIAL AND METHODS: This study was a double-blinded randomized clinical trial conducted on patients with CRVO who were referred to the ophthalmology clinic of Urmia Imam Khomeini Hospital from May 2015 through May 2016. In total, ninety patients were divided into three groups using a random numbers table. The first group received IVT 2 mg for treatment of macular edema due to CRVO, the second group received IVB 1.25 mg and the third group received a combination of IVB 1mg and IVT 1.5 mg. All injections were carried out by one ophthalmologist after prep and drape of eye if interest in the special room of eye ward. Inclusion criteria were patients with CRVO and satisfaction to participate in the study. Exclusion criteria were the need to use macular photocoagulation in the treatment process, and intravitreal injection of any drug affecting IVB and IVT. This study had some confounding factors including systemic inflammatory disorders such as autoimmune disease, systemic risk factors, such as hypertension, diabetes, systemic vascular disease, glaucoma, hypercoagulable states, dyslipidemia, and elevated plasma levels of homocysteine, concomitant ocular pathology, e.g., diabetic retinopathy or glaucoma, vitrectomy, history of cataract surgery, contraindications for bevacizumab or triamcinolone and pregnancy. This study was approved by the Ethics Committee of Urmia University of Medical Science (ID code: IR.UMSU.REC.73950114), and the objectives of the study were explained to all participants, and all of them agreed to participate and were assured of the confidentiality of their individual information as well as the voluntary nature of participating in the study. All patients were informed about the protocol of our study and written informed consent was taken. The goals of the study were explained to all participants and all of them agreed to participate and were assured considering the confidentiality of their individual information as well as the voluntary nature of participating. The best corrected visual acuity (BCVA) and central macular thickness (CMT) were recorded and analyzed using optical coherence tomography (OCT). The outcomes were checked at 10 a.m., and considering the blinded nature of this study, neither participants nor analyzer, had any information about the group assigned to the patients. Of a total of 90 patients, 41 patients (45.55%) were male and 49 patients (54.44%) were female. The mean age of the patients was 68.41±8.32 years. The Kolmogorov Smirov was done indicating the normal distribution of the data. The data were analyzed using software SPSS version 21. All data entered into SPSS version 21 software and data as frequency (percentage) and mean ± SD were reported. ANCOVA (Analysis of Covariance) test was used to compare three groups. Less than 0.05 was considered as the level of significance (p<0.05). BODY.3. RESULTS: In this study, a total of 90 eyes were studied including 30 patients in an IVT injection group, 30 patients in an IVB injection group and 30 patients in a combination of IVT and IVB injection group, 41 cases (45.55%) were male and 49 cases (54.44%) were female. The mean age of the patients was 68.41±8.32 years. The mean score of final visual acuity was 0.293±0.11 in the IVT group, 0.25±0.10 in the IVB group and 0.48±0.15 in the IVB+IVT group. The differences between groups, considering final visual acuity, was significant (p<0.001). The patients were followed up two times and the mean of BVA was 0.1±0.05 micrometer in the IVT group, 482.11±120.08 micrometers in the IVB group and 0.34±0.16 micrometers in the IVT+IVB group (Table 1). The average of baseline visual acuity was obtained as following, in the IVT group, 301±0.04, in the IVB group, 101±0.04 and in the IVT + IVB group, 501±0.05. The difference between the groups was not statistically significant in terms of baseline visual acuity (p=0.968) (Table 2). The mean thickness of baseline macular was 480±107.99 micrometer in the IVT group, 476±133.17 micrometers in the IVB group and 490.33±121.44 micrometers in the IVT+IVB group which were not significant (p=0.895) (Table 2). The average of final visual acuity was obtained as following, in the IVT group, 0.293±0.11, in the IVB group, 0.25±0.1 and in the IVT + IVB group, 0.48±0.15. The difference between the groups was statistically significant in terms of final visual acuity (p<0.001) (Table 2). The average of final macular thickness was 383.33±97.70 micrometers in the IVT group, 386.33±136.79 micrometers in the IVB group and 307.33±110.79 micrometers in the IVT+IVB group, which were significant (p<0.014) (Table 2). The improvement of final visual acuity and macular thickness compared to baseline in all three groups were shown in Figures 1 and 2. BODY.4. DISCUSSION: Results of analysis of data obtained in this study showed that intravitreal administration of a combination of IVT and IVB drugs, had a greater impact on visual acuity and CMT in the CRVO compared to each of the drugs. During the last ten years, IVT is widely used for the treatment of proliferative diseases, ocular neovascular edematous and central retinal vein occlusion (29). A disproportionate distribution of inflammatory cytokines and antigenic-related retinal vein occlusion has been reported (30) and experimental and clinical studies have showed the temporary impact of the anti-inflammatory effect of triamcinolone in the CRVO (29). The two main complications of IVT are the increase of intraocular pressure and cataracts caused by steroid injections (31–34). Instead, studies of bevacizumab by Rosenfeld et al, and another researcher have showed the improved visual acuity and decrease in macular thickness, and only minor complications in patients with CRVO have demonstrated (35–38). The rise of anti-VEGF compounds caused significant progress in the treatment of various diseases of eyes. VEGF-A has various effects on physiological and pathophysiological processes such as the vascular permeability, chemotaxis, inflammation and mythogenesis (39). VEGF stimulates neovascularization, and is a crucial factor for the creation of blood vessels and neuron cells (40). On the one hand, hypoxia and oxygen free radicals are known as agents for the stimulation of VEGF and on the other hand, there are low levels of the VEGF in the context of epithelium pigment of the retina (39, 41). All VEGF isoforms are possible using bevacizumab and ranibizumab. There is evidence supporting the theory that the use of selective inhibitor of VEGF-165 isoforms can be the means for reducing the pathological effects while maintaining its normal physiological function (42). Thus, according to available evidence, the use of each of these treatments alone, the more probable the harms and risks can be expected for the patients. So, in this study, effects of a combination of the drugs were compared with each of these drugs alone. In the study by Ekdawi et al., the authors reported a CRVO resistant to monotherapy bevacizumab or triamcinolone which the combination therapy of triamcinolone and bevacizumab resulted in an improvement in visual acuity and central macular thickness (43), and this study confirmed that the two-drug treatment was better. In another study of Ehrlich et al, the authors suggested that the combination of bevacizumab and triamcinolone improves the structure outcome in patients with retinal vein occlusion, but the authors have stated that they have no priority in using a combination of the two drugs six months after treatment compared with other studies in which either of these drugs alone had been used to achieve their objective. The last part of this study was not consistent with our study, which can be due to the longer duration of follow-up, as well as the lack of monotherapy. Also, the structure of studied populations differed in the two groups that these differences can be effective (44). In the study of Ramazani et al., the results showed the better response to treatment in the group of IVB, so that our study showed the better response to treatment in the group of combined IVB and IVT, which was inconsistent with our results (22). But the results of the study of Jin et al., were consistent with our results and had the better outcome in the group of combined IVT and IVB (45). Wang et al., reported that there was no significant difference in terms of CRVO treatment using IVT and IVB, which was inconsistent with our results (46). There is still need for further studies in order to access additional information about the choice treatment for CRVO. Other limitations of the current study were the small sample of participants and performing in the single center. BODY.5. CONCLUSIONS: In general it can be concluded that the combination of bevacizumab and triamcinolone had better effect in the treatment of CRVO compared to the use of them alone, and can be used as a solution in the treatment of this disease. We suggest that the solution of the bevacizumab and triamcinolone be made by pharmacology factories, but before that, future studies in a larger sample and in different populations may approve the results of our study.