For the majority of patients with osteoarthritis (OA), joint replacement is a successful intervention for relieving chronic joint pain. However, between 10-30% of patients continue to experience chronic pain after joint replacement. Evidence suggests that a risk factor for chronic pain after joint replacement is the severity of acute post-operative pain. The aim of this randomised controlled trial (RCT) is to determine if intra-operative local anaesthethic wound infiltration additional to a standard anaethesia regimen can reduce the severity of joint pain at 12-months after total knee replacement (TKR) and total hip replacement (THR) for OA.300 TKR patients and 300 THR patients are being recruited into this single-centre double-blind RCT. Participants are recruited before surgery and randomised to either the standard care group or the intervention group. Participants and outcome assessors are blind to treatment allocation throughout the study. The intervention consists of an intra-operative local anaesthetic wound infiltration, consisting of 60 mls of 0.25% bupivacaine with 1 in 200,000 adrenaline. Participants are assessed on the first 5 days post-operative, and then at 3-months, 6-months and 12-months. The primary outcome is the WOMAC Pain Scale, a validated measure of joint pain at 12-months. Secondary outcomes include pain severity during the in-patient stay, post-operative nausea and vomiting, satisfaction with pain relief, length of hospital stay, joint pain and disability, pain sensitivity, complications and cost-effectiveness. A nested qualitative study within the RCT will examine the acceptability and feasibility of the intervention for both patients and healthcare professionals.Large-scale RCTs assessing the effectiveness of a surgical intervention are uncommon, particulary in orthopaedics. The results from this trial will inform evidence-based recommendations for both short-term and long-term pain management after lower limb joint replacement. If a local anaesthetic wound infiltration is found to be an effective and cost-effective intervention, implementation into clinical practice could improve long-term pain outcomes for patients undergoing lower limb joint replacement.ISRCTN96095682Current Controlled Trials  Joint replacement is one of the most common elective surgical procedures, with over 150,000 total hip replacements (THR) and total knee replacements (TKR) performed annually in England and Wales in the National Health Service (NHS)-4. ChronIn the context of joint replacement, post-operative pain management needs to provide effective pain relief, while allowing for early mobilisation and rehabilitation. AlthougThe existing literature provides evidence that the use of local anaesthetic wound infiltration as part of a multimodal anaesthetic regimen reduces short-term post-operative pain after joint replacement. However, it is not known if the reduction of this short-term post-operative pain reduces the severity of chronic pain. The aim of this RCT is to determine if using intra-operative local anaesthetic wound infiltration significantly reduces the severity of joint pain at 12-months after primary TKR or THR.The Arthroplasty Pain Experience (APEX) study is a single-centre double-blind RCT that is currently being conducted at the Avon Orthopaedic Centre, one of the largest orthopaedic centres in the UK. The study has been approved by Southampton and South West Hampshire Research Ethics Committee (B) (09/H0504/94) and all participants provide informed, written consent. The trial has been registered with EudraCT (2009-013817-93) and Current Controlled Trials (ISRCTN96095682). The trial is also registered as a Clinical Trial of a Investigational Medicinal Produce with the Medicine Healthcare and Regulary Authority (18524/0215/001-0001).Recruitment into the trial began in November 2009 and 12-month follow-up for all participants is anticipated to be complete by November 2012.Potential participants are screened and recruited by research nurses from pre-operative assessment clinics at the Avon Orthopaedic Centre. Inclusion criteria include being listed for a primary unilateral TKR or THR for osteoarthritis (OA) and being willing and able to provide fully-informed consent. Exclusion criteria include i.) any medical co-morbidity that precludes spinal anaesthesia, regional blocks or the use of strong analgesics post-operatively ii.) severe dementia or psychiatric illness such that they are unable to complete the questionnaires or provide informed consent iii.) listed for simultaneous bilateral joint replacement iv.) having been in the APEX trial for a previous joint replacement v.) being unable to understand English as not all the questionnaires have been translated and validated into other languages. In order to explore generalisability (the patients enrolled in the study being representative of those undergoing joint replacement) anonymised data about age and gender is recorded for all eligible patients.Before participants undergo surgery, they are randomised to either the standard care group or the intervention group. Randomisation is conducted by means of a computer-generated code, administered centrally and communicated via the internet . Randomisation is stratified by operation site (TKR/THR) and minimised by baseline joint pain severity and surgical approach. The operating surgeon and anaesthetist are blind to the results of randomisation until the beginning of the operation, when they are informed of treatment allocation by a member of the research team who is not involved in outcomes assessment. The study participants, research nurses involved in recruitment and assessment, and clinical staff involved in the care of study participants are blind to treatment allocation throughout the study.The standard anaesthetic care for THR participants is a spinal anaesthetic with 3 mls of 0.5% plain bupivacaine placed at the L3,4 or L4,5 interspace. Intra-operatively, the patient is either awake, sedated or under a light general anaesthetic, depending on patient and anaesthetic factors. If there is intra-operative discomfort then rescue analgesia in the form of intravenous fentanyl is titrated to effect. All participants are given 1 g of intravenous paracetamol 30 minutes before the end of the operation. In the recovery area immediately post-operative, participants receive (if no contra-indications are present) 400 mg of ibuprofen administered orally. A patient controlled analgesia (PCA) device is started containing morphine 1 mg/ml, a 1 mg bolus dose and a 5 minute lock-out. If on awakening the patient is in pain with a rating of more than 50 mm on a 100 mm pain visual analogue scale (VAS), a morphine bolus up to 0.2 mg/kg can be administered as rescue analgesia.Each day during their hospital stay, participants receive a visit from a pain specialist nurse. Post-operative analgesia consists of oral or intravenous paracetamol every 6 hours and, if no contraindications are present, oral ibuprofen 400 mg every 8 hours. When the PCA is no longer needed, oral codeine phosphate 30-60 mg every 6 hours, tramadol 50-100 mg every 6 hours and oramorph 10-20 mg are prescribed as rescue analgesia.The intervention group receives the same anaesthetic and analgesic regime as the standard care group, plus an intra-operative local anaesthetic wound infiltration. The local anaesthetic mixture consists of 60 mls of 0.25% bupivacaine with 1 in 200,000 adrenaline. If the patient is below 60 kg or particularly frail, the volume of injectate is reduced to 50 mls or lower if necessary. The surgeon injects the anaesthetic mixture into the joint capsule and short external rotators, fascia, fat and subcutaneous tissue.The standard anaesthetic care for TKR participants is a femoral nerve block and a spinal or general anaesthetic, depending on patient factors. The femoral nerve blockade is achieved by an injection of 20 mls of 0.25% bupivacaine sited with the use of a nerve stimulator and/or ultrasound guidance. Intra-operative analgesia is provided by titration of intravenous fentanyl initially and morphine if deemed necessary in order to achieve haemodynamic stability and a smooth emergence. All participants are given 1 g of intravenous paracetamol 30 minutes before the end of the operation. The post-operative pain management regime for TKR participants is the same as that described for THR participants.The intervention group receive the same anaesthetic and analgesic regime as the standard care group, plus an intra-operative local anaesthetic wound infiltration. The local anaesthetic mixture is the same as described for THR participants. The surgeon injects the anaesthetic mixture into the posterior capsule, medial and lateral capsule, fascia and muscle, and subcutaneous tissues.Participants are followed-up for 12-months after surgery. Assessments are conducted pre-operatively, daily during the hopsital stay, and then at 3-months, 6-months and 12-months post-operative. Outcomes are assessed using self-report questionnaires, joint examinations, analysis of x-rays, pressure algometry and extraction of data from hospital records.The primary outcome is the 12-month Western Ontario and McMaster Universities Index of Osteoarthritis (WOMAC) Pain Scale. The WOMOn the day of surgery, participants are asked to verbally rate their pain severity on a 0-10 scale when they wake up in the recovery room and when they leave recovery, and then on a 4-hourly basis when they are transfeered to a ward. From the day after surgery to discharge (or up to post-operative day 5), participants complete a 100 mm VAS for pain severity at rest and 100 mm VAS for pain severity on movement in the morning, afternoon and evening.At the end of each day, participants complete a 100 mm VAS to indicate their satisfaction with their pain relief.Participants are asked whether they have experienced any vomiting or nausea (yes/no variable) each day. If participants have experienced nausea, they then indicate how distressing it was on a 100 mm VAS .This is calculated from participants admission and discharge dates.The WOMAC and the Pressure pain thresholds, which are a measure of pain sensitivity, are measured on the volar forearm before surgery, on discharge from hospital, and 12-month post-operative using a pressure algometer .This data is recorded from hospital records during the in-patient stay, a telephone call to participants at 3-months post-operative, and a comprehensive joint assessment by a research nurse and an x-ray review at 12-months post-operative. If participants report the occurence of a complication at either 3-months of 12-months post-operative, this is verified by a review of their hospital records.Health service resource data, including staff time and other resources used in the intervention, subsequent inpatient stays, outpatient and community based visits and medication are collected using hospital records and participant self-complete questionnaires. These questionnaires also collect information on social service use, travel, time off work, usual activities, informal care, and include the EQ-5D.A number of possible potential prognostic factors are also being recorded, including socio-demographic factors, medical comorbidities, structural joint damage, health-related quality of life and psychosocial factors. The EQ-5D is beingWithin the RCT, there is a nested qualitative study to explore the acceptability and feasibility of the intervention for both patients and healthcare professionals. Interview participants are purposively sampled to include a range of socio-demographic variables so they adequately reflect those of a range of NHS patients undergoing joint replacement and NHS staff involved in joint replacement surgery. Data collection comprises of in-depth qualitative interviews with up to 25 patients at 2-3 weeks post-operative and with up to 20 healthcare professionals involved in the trial. Interviews with patients elicit experiences of surgery and post-operative recovery, acceptability of their treatment, identify information needs regarding anaesthesia and the degree to which they would like to be involved in decision-making about pain management. Interviews with healthcare professionals explore the acceptability and feasibility of the intervention and identify any potential barriers to the implementation of the intervention. The interviews are audio-recorded and fully transcribed, and themes are compared within and between one another using constant comparison techniques.The primary outcome for this trial is the WOMAC Pain score at 12-months post-operative. The trial is powered to allow the THR and TKR groups to be analysed separately. Three hundred patients listed for TKR are being recruited into the study, and 150 patients will be randomised to the intervention arm and 150 to the standard care arm. The same number of patients listed for THR are being recruited and randomised. This sample size will provide 90% power to detect a difference of 0.5 standard deviations on the WOMAC Pain Scale with a two-sided 1% significance level, and allows for a 20% dropout rate. Previous research suggests standard deviations of around 17 mm on the WOMAC Pain Scale before surgery. Hence aIn terms of generalisability, the characteristics of those successfully recruited into the trial will be compared with those who were eligible but declined to participate. A CONSORT diagram  will sumAnalysis will consider TKR and THR separately. The primary analysis will be implemented using a multivariable generalised linear model to investigate between-group differences in mean WOMAC pain score at 12 months follow-up, adjusting for minimisation variables and baseline value of the primary outcome. The type of model will depend on the distribution of the WOMAC pain score data. The primary analysis will be conducted on an intention-to-treat (ITT) basis without imputation. However, sensitivity analyses will inform the interpretation of the primary analysis. These will include an estimate of between-group difference for the primary outcome using a per-protocol analysis. In addition, the effect of missing primary outcome data will be investigated using multiple imputation methods and random effects regression models will be used to investigate any clustering of outcomes by surgeon.The same general approach will be followed for analysis of secondary outcome measures. For additional exploratory analyses, data will be considered within each treatment group separately and so treated as two cohort studies. Analyses will examine the impact of potential risk factors, specified a priori , on pain severity at 12-months post-operative independent of any treatment effects.The aim of the economic evaluation is to compare from a societal perspective the costs and effects of the intervention compared to standard care. Resource use in relation to joint replacement is collected from the date of operation until the end of the trial. Health service resource use is valued using the hospital finance department data, as well as routine UK data sources. Patient and informal carer resource use is valued using self-report and routine data sources. The analysis will consider TKR and THR participants separately. The analysis will first estimate the between group differences in mean costs using bootstrapping techniques to obtain bias corrected confidence intervals if the data are skewed. Then, incremental cost effectiveness between the two arms of the trial will be estimated using the WOMAC Pain score and the EQ-5 D to calculate Quality Adjusted Life Years (QALYs) for this population during the trial period. These results will be presented as incremental cost-effectiveness ratios and cost-effectiveness acceptability curves, derived used bootstrapping techniques. These will show the probability of the intervention being cost-effective at a range of 'willingness to pay' threshold levels.The net monetary benefit statistic, using the difference in costs and the difference in QALYs between the two arms of the trial, will also be calculated for the different values of the societal willingness to pay for a year of life. In addition, because of the number of important secondary outcomes, a cost consequence analysis will be conducted in which the difference in costs and the difference in effects between the two arms of the trial are presented in tabular form. Sensitivity analysis will then be conducted to take account of uncertainty and imprecision in measurements, and will include multiple imputation models for missing values. All these analyses will be on an ITT basis and will adjust for minimisation variables.Many people experience chronic pain after a range of different surgical procedures-4,6-8, aRCTs are the highest level of evidence to assess the effectivness of a surgical intervention. However, there is a paucity of well-designed and sufficiently-powered RCTs of surgical interventions, particulary within orthopaedics. Common problems with the design and reporting of orthopaedic-based RCTs include patients and outcome assessors not being blinded to treatment allocation, use of unvalidated or modified validated outcome measures, no sample size calculation being performed prior to the trial, unclear inclusion and exclusion criteria, and lack of information on the method of statistical analysis -51. The The primary aim of the APEX trial is to determine if the addition of an intra-operative local anaesthetic wound infiltration to the standard anaesthetic regime at the Avon Orthopaedic Centre can significantly reduce the severity of joint pain at 12-months after primary THR and TKR. The results from this trial will inform evidence-based recommendations for both short-term and long-term pain management after lower limb joint replacement. If a local wound infiltration is found to be an effective and cost-effective intervention, its implementation into clinical practice could reduce the severity of chronic pain after THR and TKR, and therefore improve long-term pain outcomes for patients undergoing lower limb joint replacement.The authors declare that they have no competing interests.All authors conceived and designed the trialSN designed the health economic evaluationSB and AS designed the statistical analysisRGH and JH designed the nested qualitative studyAll authors drafted the manuscriptThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/53/prepub


Glomus tumors are benign neoplasms that arise from neuromyoarterial glomus bodies, with clinical manifestations that include acute pain, cold intolerance and tenderness. Glomus tumors may occur anywhere in the skin, soft tissue or gastrointestinal tract, but are most frequently encountered in the nail bed of the hands. The present study reports the case of a 30-year-old female with a history of shoulder pain caused by a cystic neoformation. Following surgery, a microscopic examination revealed nests of small cells of a rounded and regular shape. The tumor cells exhibited positive expression for CD34 and smooth muscle actin. This study supports and confirms the fact that a glomus tumor is a benign neoplasm that may occur in multiple locations. Therefore, the significance of a histological and immunohistochemical approach for a correct characterization of this lesion is required. Glomus tumors are neoplasms that arise from modified smooth muscle cells of the glomus body, which is a specialized form of an arteriovenous anastomosis that plays a significant role in the regulation of skin circulation ,2. GlomuGlomus tumors have been reported to account for 1–6% of all soft-tissue tumors and 1–5% of hand tumors . The tumClinical manifestations include paroxysms of pain, cold sensitivity and point tenderness. In certain patients the pain is accompanied by additional signs of hyperesthesia, muscle atrophy or osteoporosis of the affected area .Grossly, the tumor is a small purple nodule that varies between 2 and 20 mm in diameter, though tumors of >3 cm have been reported . HistoloThe present study reports a case of a glomus tumor of the shoulder and discusses the histological and immunohistochemical features.A 30-year-old female was referred to the Department of Thoracic Surgery  with a 1-year history of shoulder pain and paresthesia of the left arm. Upon physical examination, the range of motion of the left shoulder was normal.Ultrasonography of the supraclavicular area revealed a well-defined hypoechoic oval mass measuring ~3.5 cm. MRI of the shoulder confirmed the presence of a cystic neoformation measuring ~4 cm located between the trapezius and levator scapulae muscles .The patient was treated by a wide excision of the mass with a surgical incision in the neck, laterally to the sternocleidomastoid muscle. The patient did not undergo any further treatment following the excision and did not have any recurrence.Excised specimens were fixed in 10% neutral buffered formaldehyde and embedded in paraffin. Routine hematoxylin and eosin staining was performed on the microscopic section for histopathological examination.A paraffin block was chosen for immunohistochemical study. An immunohistochemical evaluation was performed using the avidin-biotin-peroxidase complex method. Antibodies were purchased from Ventana Medical Systems . The antibodies that were used were mouse monoclonal anti-smooth muscle actin, mouse monoclonal anti-CD34, rabbit monoclonal anti-CD31, polyclonal anti-factor VIII, mouse monoclonal anti-CK-Pan, mouse monoclonal anti-CD68, mouse monoclonal anti-S-100, mouse monoclonal anti-CD99, polyclonal anti-calretinin and mouse monoclonal anti-desmin. All the antibodies were pre-diluted.The analysis of the specimens was performed by two pathologists and the differential diagnosis was widely discussed.The specimens were obtained from a 30-year-old female who underwent an excision of a subcutaneous mass of the supraclavicular area.The gross appearance of the specimen was a grayish fragmented cystic lesion, measuring 4 cm at the maximum. The cut sections revealed firm tissue without hemorrhagic or necrotic areas.A microscopic examination showed multiple nests of typical glomus cells surrounded by fibrous tissue with focal myxoid changes. The glomus cells contained epithelioid elements with moderate amounts of clear to eosinophilic cytoplasm and round nuclei with fine chromatin. These cells were closely associated with small vascular channels and nerves. The cells demonstrated a low proliferative activity (Ki-67 <5%) and a low mitotic rate of <1 mitotic figure (MF)/50 high-power fields (HPFs). The lesion was well-circumscribed and there was no evidence of hemorrhage or necrosis.Immunohistochemical staining was positive for SMA and CD34 and negative for CD31, factor VIII, CK-Pan, CD68, S-100, CD99, calretinin and desmin .A glomus tumor is a hamartoma that develops from a neuromyoarterial glomus body and consists of dilated vascular channels surrounded by proliferating glomus and nerve cells. It accounts for 1–6% of all soft tissue tumors and 1–5% of hand tumors . Glomic Glomus tumors are usually located in the deep dermis of the extremities and in the subungual region of the hands. Other sites are the shoulder , thigh , knee 44 and gaset al. The surgical excision of the lesion resulted in a complete disappearance of the symptoms.In conclusion, glomus tumors of the shoulder are not as uncommon as previously believed. On this basis, in cases of unexplained pain in this area, a glomus tumor should be considered in the differential diagnosis.


Port-A catheters are frequently used in pediatric cancer patients. Their dislodgement is potentially seriously risky although the incidence is not high. We analyzed our 11 years of data to address this important problem.From January 2001 to December 2011, 330 port-A catheters of different brands were implanted in pediatric cancer patients. In total, eight children suffered a dislodgement of their catheter. Their ages ranged from four to thirteen years, with a median age of ten. Five patients presented with catheter dysfunction, two presented with a cough and one was identified incidentally during surgery to remove his port.The downstream ends of the dislodged catheters were located in the right atrium (three patients), left pulmonary artery (three) and inferior vena cava (two). Six of the eight catheters were broken at the site of anastomosis to the port and the other two were broken halfway in between. All episodes of dislodgement happened after the chemotherapy regimen was completed. The dislodged catheters were successfully retrieved without complications by transcatheter retrieval using a gooseneck snare.The dislodgment rate of port-A catheter in our series was 2.4%. Chest X-rays can rapidly detect the problem. Most of the catheters were broken at the site of anastomosis. Earlier explantation of port-A catheters after completing chemotherapy may be considered to avoid the dislodgement of catheters, but this needs to be weighed against the possibility of underlying disease recurrence. However, we should re-examine how long port-A catheters need to be retained after chemotherapy considering the improved cure rate of pediatric cancer. Totally implanted central venous access catheters  can provide reliable, long-term vascular access for chemotherapy, total parenteral nutrition and frequent blood sampling . They arComplications, such as infection and thrombosis, with port-A catheters are not uncommon . The disWe reviewed the medical records of patients who received replacement port-A catheters from 1 January 2001 to 31 December 2011, and followed up the clinical course until 30 June 2012. This study was approved by the Institution Review Board of the hospital. There were 330 totally implanted catheters of different brands implanted in 297 pediatric cancer patients. Of these, twenty-six patients had a catheter implanted twice, two patients three times and one patient four times.t test to compare continuous variables. A P value less than 0.05 was considered statistically significant.In total, eight children suffered dislodgement and migration of a catheter. Five patients presented with catheter dysfunction, two presented with a cough and the other one was identified incidentally during surgery for removal of his port-A catheter. All dislodged catheters were successfully retrieved without complication by transcatheter retrieval through the femoral route using a gooseneck snare and one with additional pigtail help. We used chi-squared or Fisher’s exact tests to compare categorical variables. We used Student’s The data for the patients suffering catheter dislodgements and migration are summarized in Table Next, we compared the clinical characteristics of the eight patients who suffered a catheter dislodgement and 322 patients who did not , the left pulmonary artery (three) or the inferior vena cava (two). Tsai et al. reported that the most frequent location of dislodged port-A catheters was between the right atrium and inferior vena cava . The mosThe causes of catheter dislodgment include poor connection to the port, catheter damage at the site of anastomosis, incorrect catheter position, catheter damage by chemotherapeutic drugs and the pinch-off syndrome ,12. The Chang et al. stated that a cephalic vein cut-down performed by a general surgeon had a lower risk of catheter fragmentation than a subclavian vein puncture through the Seldinger technique performed by a vascular surgeon . Wu et aWithout doubt, early explantation of a port-A catheter will definitely prevent related complications. There are still reasons to retain the device during the post-treatment surveillance since cancer patients require further follow-up to rule out recurrence. In our series, there were 144/330 cases of intentional removal of a port-A catheter. Three of these cases needed re-implantation of the port-A catheter. These three patients were all diagnosed with acute lymphoblastic leukemia relapse. They were off chemotherapy for 19, 27 and 24 months, respectively. The intervals from explantation of the old port-A catheter to implantation of the new one were 59, 66 and 61 months, respectively. Furthermore, it may be a challenge to find new vascular access in patients with previous central venous lines or who had a port-A catheter removed. However, with the improved cure rate of pediatric cancer, we should re-examine how long port-A catheters need to be retained after the completion of chemotherapy.The dislodgment rate of port-A catheters in our series was 2.4%. Chest X-rays can rapidly detect the problem. Most catheters were broken at the site of anastomosis. Earlier explantation of a port-A catheter after completion of chemotherapy may avoid dislodgement and migration of the catheter but the possibility of underlying disease recurrence needs to be considered.The authors declare that they have no competing interests.SCW and CHT collected the data and prepared the manuscript; CPH collected data; SYL, SFK, CCH, YCC and JHC managed the patients; JMS supervised and reviewed the manuscript. All authors read and approved the final version of the manuscript.


Retinal pathogenic angiogenesis in the eyes is a causative factor in retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration. This study was designed to examine the pathogenic role of the high-mobility group box-1 (HMGB1) protein and the inhibitory effect of ethyl pyruvate (EP), a well-known antioxidant substance, in retinal pathogenic angiogenesis in mice with oxygen-induced retinopathy (OIR), one of the animal models of proliferative ischemic retinopathy. The OIR mouse model was used for our in vivo studies. The mice were exposed to 75% oxygen from postnatal day 7 (P7) to P11, after which the mice were brought to room air and intraperitoneally injected with EP  for five days. At P17, the mice were perfused with fluorescein isothiocyanate-dextran, and flat-mounted retinas were used to measure nonperfused and neovascular tufts. In OIR mice, an intraperitoneal injection of EP reduced the nonperfused retinal area in the treatment group and significantly reduced the retinal neovascular tufts. In addition, EP inhibited the overexpression of HMGB1 in the retinas of OIR mice. These data suggest that EP could serve as an innovative pharmaceutical agent to prevent retinal neovascularization through inhibiting HMGB1 expression. GL treatment also significantly reduced the area of neovascularization in retinas compared with OIR.Click here for additional data file.


Childhood obesity is becoming more prevalent; however, the influence of obesity or dieting during childhood on outcomes in adulthood is poorly understood. The aim of this study was to examine the effects of short-term calorie restriction (CR) and high-calorie feeding with high-fat or high-sucrose diets during early life on the development of glucose tolerance and diabetic nephropathy in later life of Otsuka Long-Evans Tokushima fatty (OLETF) rats. Neither high-calorie intake nor CR at 7–13 weeks of age affected glucose tolerance of 27-week-old OLETF rats. On the other hand, proteinuria was lower at 27 weeks of age in CR rats than in the other rats. These results suggest that short-term CR at a young age protects against the development of renal injury in later life. In contrast, short-term high-calorie intake or CR at a young age does not appear to affect glucose metabolism in later life. A massive increase in the prevalence of defective glucose metabolism, insulin resistance, and cardiovascular morbidity is anticipated in most developed countries –3. DysfuCalorie restriction is one of the possible treatments for diabetes, and it is also well known to prolong the lifespan. Calorie restriction is often used as initial therapy for diabetic patients; however, lifestyle changes are often only started following the diagnosis of diabetes. It is clear that starting calorie restriction during an earlier phase has greater effects on outcomes. However, for obese children, it would be difficult to limit calorie intake throughout their life, and calorie intake often starts to increase after some time. The effects of temporary calorie restriction at a young age on obesity-related problems, such as insulin resistance and diabetes, in later life are still unclear.Diabetic nephropathy, one of the main complications of diabetes, develops in a time-dependent manner, and the number of patients requiring dialysis or transplantation is steadily increasing. Diabetic nephropathy is considered to be a long-term consequence of diabetes mellitus; therefore, interventions during an early phase are strongly recommended. Both basic and clinical studies have demonstrated that treatment with rennin-angiotensin system (RAS) blockers delays the developments and progression of diabetic nephropathy –10. InteHowever, it remains to be determined whether the effects of either excessive or restricted calorie intake during early life are sustained until later life, or whether such interventions affect the incidence of insulin resistance, diabetes, and complications, such as nephropathy. Therefore, the present study was conducted to examine the effect of short-term calorie restriction, high-fat feeding, and high-sucrose feeding during early life on the development of insulin resistance and renal injury in later life in spontaneously type 2 diabetic Otsuka long-Evans Tokushima fatty (OLETF) rats.n = 16), standard chow with feeding restriction , high-sucrose (60%) chow (n = 16), or high-fat (60%) chow (n = 16) between weeks 7 and 13 of age. We used Long-Evans Tokushima Otsuka (LETO) rats as the lean control of OLETF rats. After week 13, the diet was replaced with an unrestricted normal diet and was given until 27 weeks of age. Twenty-four-hour urine samples were collected from rats at 6 (baseline), 13, and 27 weeks of age. All animals underwent a 24-hour acclimatization period in metabolic cages before urine collection. We chose 27 weeks of age because our preliminary experiments showed the OLETF rats developed significant insulin resistance at 25–27 weeks of age. Animals were fasted for 12 hours at 13 and 27 weeks of age and killed to collect retroperitoneal and epididymal adipose tissues and the kidneys. The other tissues were snap frozen in liquid nitrogen and stored at −80°C until use.All experimental procedures were performed according to the guidelines for the care and use of animals established by the Kagawa University. Six-week-old male OLETF rats  were divided into four groups and fed either a standard chow . The oligonucleotide primer sequences for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and SIRTs were as previously described . All datOGTT was performed 1 week before the end of the experimental period, as previously described . The ratPlasma levels of triglycerides, nonesterified fatty acid (NEFA), total cholesterol, glucose , and insulin  were measured using commercially available kits. Urinary protein excretion was determined using a protein assay kit .μm-thick slices and stained with periodic acid Schiff (PAS). Kidney sections were evaluated by light microscopy, as previously described [Kidneys were fixed with 15% formalin and embedded in paraffin. Tissue was sectioned into 20 escribed .post hoc test. Values of P < 0.05 were considered statistically significant.Values are presented as means ± standard error of the mean. Differences between groups were compared using one-way analysis of variance followed by the Newman-Keuls The body weight of normal-diet-fed OLETF rats was significantly greater at 13 and 27 weeks of age than that of LETO rats . Short-tNormal diet-fed OLETF rats developed significant proteinuria at 27 weeks of age . Short-tTo determine the mechanism by which short-term calorie restriction at a young age delayed or prevented the development of proteinuria in obese animals, we measured sirtuin mRNA expression in the kidney of animals at 13 and 27 weeks of age. However, we found no significant differences in SIRT1 and SIRT3 expression at either 13 or 27 weeks of age between the groups .The prevalence of obesity in young people is increasing, and many children are now at risk of disorders associated with obesity, particularly endocrine abnormalities such as type 2 diabetes . It appeDiabetic nephropathy is a major complication in diabetic patients. It has been shown that the probability of developing albuminuria in patients with childhood-onset type 1 diabetes is similar to that in patients with adult-onset type 1 diabetes . Thus, wTransient high-fat or high-sucrose feeding in early life does not appear to cause irreversible or sustained changes in either metabolic or kidney function in animals that are genetically obese and type 2 diabetic because of overfeeding. On the other hand, calorie restriction, even if it is temporary, confers protective effects against diabetes-related renal injury. These findings provide further support to the effectiveness of calorie restriction, although it should be noted that it failed to induce sustained benefits on metabolic parameters.


Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies, and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is present in the blood.Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC), contains the integrin-binding RGD motif. RGD mediates tumor-homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR) motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular “zip code” of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors  A major problem in systemic therapy is that only a small proportion of administered drug reaches its intended target site(s). Selective delivery of the drug to the target tissue can alleviate this problem. Affinity-based physical targeting  makes use of molecular markers that are specifically expressed at the target, and not elsewhere in the body, to accomplish selective targeting of systemically administered drugs . The desSynaphic targeting efforts have led to improved cancer drug delivery, but this approach only partially solves the selective delivery problem. Delivering a payload to a molecule specifically expressed on the surface of vascular cells in the target tissue can be effective because the vasculature is readily available for blood-borne probes. Thus, anti-angiogenic and vascular disrupting compounds can benefit from this approach. In fact, many of these compounds inherently target the vascular endothelium. An obvious example is antibodies that block the vascular endothelial growth factor receptors . These The endothelia of vessels in different organs, even when morphologically indistinguishable, are molecularly  different . MoreovIn vivo phage display has been instrumental in establishing the extent of the molecular specialization in the vasculature and has contributed a number of new markers of tumor vasculature  with positive selection at the target tissue , with the ability to take the phage expressing it to the lymphatic vessels and hypoxic areas in tumors . SurprisTumor-penetrating peptides like iRGD and LyP-1 contain three independent modules: a vascular homing motif, an R/KXXR/K tissue penetration motif, and a protease recognition site. These modules cooperate to ensure a multistep, highly specific process of tumor-homing and penetration. The sequence of the prototypic tumor-penetrating peptide, iRGD, is CRGDR/KGPDC. We mostly use the K-variant, CRGDKGPDC, because it appears to provide stronger tumor-homing than the R-variant. Following systemic administration, the iRGD peptide is first recruited through its RGD motif to αv integrins, which are overexpressed on tumor endothelial cells. After the initial binding, proteolytic processing exposes the internal R/KXXR/K motif at the C-terminus of the truncated peptide. We have termed the R/KXXR/K motif the C-end Rule or CendR motif (pronounced sender) because of the requirement of C-terminal exposure for activity. The C-terminal CendR motif interacts with neuropilin-1 (NRP-1), and the NRP-1 interaction triggers the activation of a transport pathway (CendR pathway) through the vascular wall and through extravascular tumor tissue , 13. TheWe came across the CendR phenomenon while screening phage libraries for peptides that would bind to and internalize into cells isolated from tumors grown in mice. We were initially disappointed to find that, independent of the starting library configuration (we used cysteine-flanked cyclic and linear random heptapeptide libraries), the selected peptides all looked similar; they all had a C-terminal arginine or lysine residues with another basic amino acid at the −3 position. However, we soon realized that the consensus motif, R/KXXR/K, had to be some kind of a master cell internalization signal and set out to study it. It is worth noting that, while our laboratory used the filamentous phage display system introduced by Smith , 15 in oL-amino acids and that the binding can be inhibited by excess of free peptide, suggesting the existence of a saturable receptor with a chiral recognition specificity. In contrast, cell-penetrating peptides, widely used for intracellular delivery of payloads in vitro are independent of position and chirality, and no specific receptors for them have been identified.The binding and internalization of R/KXXR/K-displaying phage or synthetic nanoparticles required the presence of free C-terminal arginine or lysine residues as addition of additional amino acid residues to the motif or amidation of the carboxyl terminus resulted in loss of activity . In addiAffinity chromatography with RPARPAR identified NRP-1 as the main binding molecule for RPARPAR. NRP-1 is a trans-membrane receptor with major roles in cell migration and endothelial cell sprouting in blood vessels, while NRP-2 with a similar, but not identical binding specificity is abundant and plays an important role in lymphatic vessels , 18. NRPThe b1b2 domain of NRP-1 that contains the binding pocket for the CendR motif has been crystallized together with tuftsin . MoleculBased on molecular simulations and phage binding to purified NRP-1 protein it appears that formation of a stable complex between a CendR peptide and NRP-1 requires interaction of the -2 and -3 residues with loop III of the b1 domain of the NRP-1, as in the case of RPAR, RRAR, RDAR, RPDR, RPRR, and RPPR . For a sin silico screening of CendR analogs and evaluation of low molecular weight compounds resulting from high throughput screening. The molecules that bind to the CendR binding pocket on b1b2 domain of NRP-1 will be either acting as agonists or antagonists with potential applications in cancer drug delivery, and in diseases associated with elevated vascular permeability and pathogen spreading in tissues (see below).Interestingly, the D-conformer of RPARPAR is a poor fit with the binding pocket, suggesting that the D-Tat, even with a C-terminal arginine would not bind to NRP-1. The modeling studies also indicate that under some circumstances a cyclic peptide could fit into the binding pocket . Indeed,A wide range of other receptors have been reported to use NRP-1 as a co-receptor, earning NRP-1 designation as a “hub” receptor , but it The ability of VEGF and semaphorins to increase vascular permeability has been recognized for some time. Dvorak and Feng  showed tin vivo and ex vivo, and its energy dependence  at sites that create an active CendR motif is a recurrent theme seen in many pathogens. Examples include the Human T-lymphotropic virus-2, Crimean–Congo hemorrhagic fever virus, tick-born encephalitis virus, and Ebola viruses, as well as anthrax toxin –43. CendVascular edema is associated with many diseases . Several proinflammatory vasoactive (poly)peptides capable of increasing vascular permeability display a functionally important arginine residue at their C-terminus. Examples include complement C3a and C5a anaphylatoxins  as well as kinins . Intriguingly, we have observed that phage that display peptides corresponding to the C-terminal amino acids of C5/3a and bradykinin bind to the recombinant b1b2 domain of NRP (in preparation) and that the binding is reversed by an excess of the free peptide. It remains to be seen whether the NRP/CendR axis plays a role in the activity of C3/5a, bradykinin, and/or other vasoactive peptides.Having worked out the two-motif requirement for a tumor-homing peptide to have CendR activity, we tested the universality of the concept by designing a new peptide with such activities. We used as the starting point the NGR tumor-homing motif previously identified by our laboratory , 45, whiTo reach tumor cells and tumor-associated parenchymal cells , drugs must cross the vascular barrier and penetrate into the extravascular stroma. Cancerous tissue is heterogeneous, with striking regional differences in tumor structure , and physiology . These features translate into steep drug gradients and variability in the uptake and distribution of anti-cancer drugs within tumor parenchyma . For exaThe tumor-homing CendR peptides provide a solution to the drug penetration problem. A probe or drug attached to iRGD or LyP-1 is delivered to extracellular tumor tissue more effectively than the drug alone. We have extensively demonstrated the tumor penetration with fluorescein (FAM)-labeled peptides. Intravenously injected FAM-iRGD, LyP-1, and iNGR are found dispersed in tumor parenchyma minutes after administered, whereas FAM-labeled inactive control peptides do not appear in the tumors at all. FAM-labeled homing peptides that lack a CendR motif bind to the blood vessels, but do not penetrate into the rest of the tumor , 13, 48.The observations with the fluorescent probe described above prompted us to study the ability of iRGD and the other CendR peptides to enhance the delivery of actual anti-cancer drugs to tumors. We have shown that therapeutics as diverse as a small molecular weight drug (doxorubicin), trastuzumab anti-Her2 antibody), and the nanoparticle drugs Abraxane and Doxil can benefit from iRGD-enhanced delivery , 38. In  antibodyTiming measurements have shown that the CendR pathway is active for about 1 h, with peak activity about 30 min after the administration of the peptide . The timLyP-1 coupled to Abraxane nanoparticles also increased the efficacy of the drug  and iNGRThe tumor-penetrating peptides can also enhance tumor imaging, as demonstrated by coating iron oxide nanoparticles with iRGD for MRI imaging. iRGD gave stronger images than a conventional RGD peptide, CRGDC; the main difference was that iRGD spread into the whole tumor, whereas only highlighted the tumor vessels . LyP-1 hThe discovery of tumor-penetrating peptides has led to the identification of a new trans-tissue transport pathway, the C-end Rule or CendR pathway. The physiological function of the CendR pathway and its molecular workings are obviously important questions to be answered in future studies. Activating the pathway in a tumor-specific manner, which is accomplished with peptides the CendR motif of which is activated in tumors, provides a way of increasing the activity of anti-cancer drugs and enhancing tumor imaging. Thus, the tumor-penetrating CendR peptides represent a potentially significant advance in cancer treatment.Tambet Teesalu, Kazuki N. Sugahara, and Erkki Ruoslahti are shareholders in CendR Therapeutics Inc., and Erkki Ruoslahti is a shareholder in EnduRx Pharmaceuticals. The companies have rights to some of the technology described in the paper.


Recently it was shown that following infection of HeLa cells with Newcastle disease virus (NDV), the matrix (M) protein binds to Bax and subsequently the intrinsic pathway of apoptosis is activated. Moreover, there was very little alteration on mRNA and protein levels of Bax and Bcl-2 after infection with NDV.In order to further investigate the role of members of the Bcl-2 family, Bax-knockout and wild-type HCT116 cells were infected with NDV strain AF2240. Although both cells underwent apoptosis through the activation of the intrinsic pathway and the release of cytochrome c from mitochondria, the percentage of dead Bax-knockout cells was significantly lower than wt cells (more than 10% at 48 h post-infection). In a parallel experiment, the effect of NDV on HT29 cells, that are originally Bcl-2-free, was studied. Apoptosis in HT29 cells was associated with Bax redistribution from cytoplasm to mitochondria, similar to that of HeLa and wt HCT116 cells.Although the presence of Bax during NDV-induced apoptosis contributes to a faster cell death, it was concluded that other apoptotic protein(s) upstream of mitochondria are also involved since cancer cells die whether in the presence or absence of Bax. Therefore, the classic Bax/Bcl-2 ratio may not be a major determinant in NDV-induced apoptosis. Avulavirus from the family of Paramyxoviridae[Newcastle disease virus (NDV) is a highly contagious avian virus that belongs to the genus xoviridae,2. NDV ixoviridae,4. NDV sxoviridae,6, wherexoviridae. On the xoviridae. It has xoviridae,9. The pxoviridae. Bcl-2 pxoviridae. All memxoviridae. Bcl-2, xoviridae-16 to acxoviridae. This inxoviridae,18-20.6 cultured cells)[In the current study, the effect of NDV on cancer cells in the absence of Bcl-2 and Bax was investigated; HT29 human colon adenocarcinoma cell line , HCT116 Bax−/−  and wt colorectal carcinoma cell lines  were cultured in 6-well plates and infected with NDV strain AF2240 according to a standard protocol (30 HA units of NDV for 1.0 × 10d cells). Bax-defMorphological changes and membrane blebbing was completely evident in these cell lines following infection. At different time points post-infection , the cells were checked by flowcytometry and PI staining for the number of dead cells, and graphs were drawn accordingly  Figure. SimilarIn addition, the amount of cytochrome c in the cytosolic fraction of the NDV-infected HCT116 Bax−/− cells also increased by time, suggesting that the mitochondrial pathway of apoptosis in this cell line is activated even in the absence of Bax.In this study, after several trials, it was found that the percentage of cell death in NDV-infected Bax-knockout cells was lower compared to wt cells. This showed that the presence of Bax  speeds up the apoptosis event. Since the Bax-knockout cells die following infection with the virus, it can be concluded that NDV is able to interact with apoptotic proteins other than Bax as well. The release of cytochrome c into cytosol proves that the mitochondrial pathway is activated through some other interaction(s) that directly or indirectly affected the proteins of the surface of mitochondria, allowing formation of pores that release mitochondrial factors.Many Bcl-2 family members interact with each other via the BH domains. For insBcl-2 normally blocks Bax oligomerization to inhibit its insertion into the mitochondria. Since NBak and Bax have almost the same protein structure, share similar BH domains and are the only two pro-apoptotic Bcl-2 family proteins discovered so far that form oligomeric pores on the mitochondria surface. Bak can independently release cytochrome c from mitochondria in absence of Bax, and theBH3-only members such as Bid and Bad are located upstream of mitochondria that upon activation move towards mitochondria to, respectively, activate Bak and Bax and inhiOverall, it was concluded that Bax is not the only cellular protein that NDV proteins interact with to trigger apoptosis. However, Bax plays a role in the apoptosis of HCT116 cells, since the lack of Bax contributes to slower cell death. This further suggested that the interaction between Bax and NDV proteins, such as that with the M protein, is crucATCC: American type culture collection; BH domain: Bcl-2 homology domain; wt: Wild-type.The authors declare that they have no competing interests.AM designed and performed the experiments and drafted the manuscript. KY participated in the design of the study, financial support and contributed to writing the manuscript. Both authors have read and approved the final manuscript.


Although effective smoking cessation interventions exist, information is limited about their cost-effectiveness and cost-utility.To assess the cost-effectiveness and cost-utility of an Internet-based multiple computer-tailored smoking cessation program and tailored counseling by practice nurses working in Dutch general practices compared with an Internet-based multiple computer-tailored program only and care as usual.The economic evaluation was embedded in a randomized controlled trial, for which 91 practice nurses recruited 414 eligible smokers. Smokers were randomized to receive multiple tailoring and counseling (n=163), multiple tailoring only (n=132), or usual care (n=119). Self-reported cost and quality of life were assessed during a 12-month follow-up period. Prolonged abstinence and 24-hour and 7-day point prevalence abstinence were assessed at 12-month follow-up. The trial-based economic evaluation was conducted from a societal perspective. Uncertainty was accounted for by bootstrapping (1000 times) and sensitivity analyses.No significant differences were found between the intervention arms with regard to baseline characteristics or effects on abstinence, quality of life, and addiction level. However, participants in the multiple tailoring and counseling group reported significantly more annual health care–related costs than participants in the usual care group. Cost-effectiveness analysis, using prolonged abstinence as the outcome measure, showed that the mere multiple computer-tailored program had the highest probability of being cost-effective. Compared with usual care, in this group €5100 had to be paid for each additional abstinent participant. With regard to cost-utility analyses, using quality of life as the outcome measure, usual care was probably most efficient.To our knowledge, this was the first study to determine the cost-effectiveness and cost-utility of an Internet-based smoking cessation program with and without counseling by a practice nurse. Although the Internet-based multiple computer-tailored program seemed to be the most cost-effective treatment, the cost-utility was probably highest for care as usual. However, to ease the interpretation of cost-effectiveness results, future research should aim at identifying an acceptable cutoff point for the willingness to pay per abstinent participant. Smoking is the single most preventable cause of illness and premature death in the world and is an important risk factor for 6 of the 8 leading causes of death, including several types of cancer, cardiovascular diseases, and respiratory diseases . ConsequExtensive evidence exists on the clinical effectiveness of behavioral interventions for smoking cessation -7. BriefJournal of Medical Internet Research for research to economically evaluate eHealth interventions [Despite the proven clinical effectiveness of behavioral smoking cessation interventions, information about their relative cost-effectiveness is limited. Previously, several cost-effective smoking cessation interventions have been developed -28. Howeventions , cost-efventions  and deprventions ,33 have Therefore, the objective of the present study was to compare the cost-effectiveness and cost-utility of (1) an Internet-based multiple computer-tailored smoking cessation program combined with a single tailored counseling session by a practice nurses, (2) only an Internet-based multiple computer-tailored smoking cessation program, and (3) care as usual, defined as practice nurses’ standard care regarding smoking cessation.Economic evaluation studies aim to determine the costs and effects associated with an intervention and to compare these with the costs and effects of other interventions and/or current practice . They usStep 1. Identification of relevant costs and effectsBased on a chosen perspective , relevant costs and effects are identified.Step 2. Measurement of costs and effectsCosts can be assessed prospectively by means of cost diaries, or retrospectively using cost questionnaires. Effects are usually assessed in terms of quality of life.Step 3. Valuation of measured costs and effectsHealth care and patient costs are usually valued in a monetary currency using manuals for cost analysis in health care research. Effects on quality of life are usually valued in QALYs gained or lost.Step 4. Calculation of a cost-effectiveness ratioi–Cc)/(Ei–Ec). When comparing more than 2 interventions, a net monetary benefit (NMB) should be calculated using the willingness to pay (WTP): NMB=(Ei–Ec)×WTP–(Ci–Cc). A description of both formulas can be found in When comparing two interventions, an incremental cost-effectiveness ratio (ICER) can be calculated: ICER= that tested the effectiveness of an Internet-based multiple computer-tailored smoking cessation program and tailoring counseling by practice nurses. This 3-armed RCT was conducted among Dutch adult smokers and had a follow-up period of 12 months. From May 2009 to June 2010, 91 practice nurses working in different Dutch general practices throughout the Netherlands recruited smoking patients for participation in the RCT. To aid recruitment, several recruitment materials were provided . Smokers interested in participation could sign up for the study on the study website. There, information was provided about the objectives of the study, the randomization procedure, and the incentive provided when respondents completed all questionnaires . When signing up, participants were able to choose their own username and password and were informed that no one but the research team would be able to retrieve these passwords. After providing informed consent, participants were randomized into 1 of the 2 intervention groups (multiple tailoring and counseling or multiple tailoring only) or into the usual care control group. Randomization took place at the participant level by means of a computer software randomization device.The trial design was approved by the Medical Ethics Committee of Maastricht University and the University Hospital Maastricht , and is registered with the Dutch Trial Register (NTR1351). A more detailed description of the study design has been published elsewhere .Participants were eligible for participation if they smoked, were motivated to quit within 6 months, were 18 years or older, and were able to read and understand Dutch sufficiently to read study materials and participate in the trial. Moreover, they had to have access to the Internet. This resulted in a total of 414 eligible smokers.The Internet-based multiple computer-tailored smoking cessation program was based on a previously developed single computer-tailored intervention ,15 for wAfter receiving the first tailored feedback, participants in the multiple tailoring and counseling group were prompted to schedule a counseling meeting with their practice nurse within 6 to 8 weeks. They received this counseling session instead of the third tailored feedback letter at the 6-week follow-up. A counseling protocol was provided to assist practice nurses in guiding these counseling sessions. This protocol consisted of 3 chapters guiding on 3 different types of participants: smokers who had quit successfully, smokers who had quit but relapsed, and smokers who had not yet quit. The content of the counseling session was developed to be as similar as possible to the content of the computer-tailored feedback and was also tailored to the participant characteristics mentioned previously. After 6 months, practice nurses were instructed to call their patients to ask them about their progress toward permanent cessation and, if needed, to provide them with additional cessation support.Participants randomized in the usual care group received smoking cessation guidance according to participating practice nurses’ standard practice, which can vary from a brief intervention consisting of a single recommendation to stop smoking to more intensive interventions ,44.Self-reported online questionnaires were used to assess both costs and effects. Questionnaires were administered at baseline and at 6-week, 6-month, and 12-month follow-ups. When follow-up questionnaires were not completed by 1 week after the invitation, an email reminder was sent. At 12-month follow-up, this email reminder was followed by a phone call to collect data.The present economic evaluation study was conducted from a societal perspective. This implies that intervention costs, health care costs, and patient costs were identified as relevant. Intervention costs consisted of all costs that could be attributed to the delivery of the intervention, such as hosting costs for the Internet-based program and costs associated with counseling sessions. Costs for the development of the intervention and research-specific costs were excluded because these costs are sunk costs, costs that would already be spent before the intervention is implemented. In total, intervention costs were €57.70 per participant in the multiple tailoring and counseling group and €7.70 per participant in the multiple tailoring group. Interventions costs in the usual care group were considered zero because no intervention materials needed to be developed for this group. Health care costs related to general practitioners’ or practice nurses’ (telephone) consultations or home visits (other than the counseling session which was part of the multiple tailoring and counseling intervention), inpatient and outpatient specialist care, alternative medicine, mental health care, prescribed and over-the-counter (OTC) smoking cessation medication, hospital admissions, smoking cessation aids, and other care . Patient costs consisted of traveling and time lost due to participation in the intervention. However, for primary and secondary analyses, patient costs were not valued in monetary costs, but considered as reflected in participants’ reported quality of life .Self-reported health care use was assessed during a 12-month follow-up period using a 3-month retrospective costing questionnaire that consisted of open-ended questions. Participants indicated whether they had received each type of care during the past 3 months, and if so, how often. The time participants spent using the online tailoring program was tracked by computer-registered log-in and log-out data. To assess time spent on counseling, we used a mean time of 20 minutes for face-to-face counseling sessions and an average of 10 minutes for telephone consultations. Traveling time was measured based on average travel distances to a general practitioner in the Netherlands .To valuate health care usage and patient costs, the updated Dutch manual for cost analysis in health care research was used . In geneThe primary outcome measure used in the cost-effectiveness analysis (CEA) was prolonged abstinence measured at 12-month follow-up. This was assessed by 1 item asking whether the participant had refrained from smoking since the previous measurement at 6-month follow-up . Secondary outcome measures were 7-day point prevalence abstinence assessed by 1 item asking whether the participant had refrained from smoking during the past 7 days  and addiction level measured by the abbreviated Fagerström Test for Nicotine Dependence  . Self-reThe primary outcomes measure for the cost-utility analysis (CUA) was quality of life, measured in terms of QALYs. The valuation of effects on quality of life implies that utility scores need to be computed. In the present study, utilities were measured by the EuroQol EQ-5D ,50, whicAll analyses were conducted according to the intention-to-treat principle. Missing data for costs, EQ-5D items, overall tobacco consumption, and addiction level were replaced by mean imputation by using participants’ scores on the previous and next measurement. When mean imputation was not possible because of missing data on multiple measurement points, missing data were replaced using the last observation carried forward (preferred choice) or next observation carried backward method. Missing data for smoking abstinence were replaced using a negative scenario; participants lost to follow-up were considered still smoking.t tests and chi-square tests.To investigate the comparability of the 3 groups with regard to demographics, baseline values of outcomes, and health care–related costs over the past 3 months, 1-way analyses of variance (ANOVA) with Tukey post hoc tests and chi-square tests were conducted. To determine whether selective dropout had occurred, a comparison was made between those lost to follow-up and those who remained in the study after 12 months using 2-sided The 3 groups were compared with regard to their mean annual costs using nonparametric bootstrapping (5000 times) with 95% confidence intervals in percentiles . To compi–Cc)/(Ei–Ec). In this formula, Ci is the adjusted annual costs of the intervention group, Cc is the adjusted annual costs of the control group, Ei is the adjusted effects for the intervention group, and Ec is the adjusted effects of the control group. With regard to quality of life, ICERs are often called incremental cost-utility ratios (ICURs). However, because ratios such as the ICER or ICUR can compare only 2 groups, to compare the 3 groups in our study a net monetary benefit (NMB) was calculated for each of 3 treatments. The NMB can be calculated by valuing the effectiveness and utility outcomes in monetary values using a threshold for society’s willingness to pay (WTP) per abstinent participant and per QALY gained [i–Ec)×WTP–(Ci–Cc). In the present study, we used a WTP of €18,000 because this is an accepted Dutch cutoff point for the WTP per QALY [First, incremental costs and effects were calculated for each of the 3 treatments studied. Subsequently, an incremental cost-effectiveness ratio (ICER) was calculated to compare costs and effects between pairs of study groups according to the following formula: ICER= were not valued in monetary costs but considered as reflected in participants’ reported quality of life . HoweverBootstrap analyses were conducted using Microsoft Office Excel 2003. All other analyses were conducted using SPSS 17.0 .P=.01). Of the 46 participants who reported prolonged abstinence at the 12-month follow-up, 30 successfully completed a cotinine test and all cases of self-reported abstinence were confirmed. After imputation of missing values, total cost data were available for 370 participants (89.2%) whereas effect data were available for 414 (100%) participants for abstinence measures, 409 (98.8%) for addiction level, and 384 (92.8%) for QALYs.Of the 414 participants who were eligible for participation, 163 were randomized into the multiple tailoring and counseling group, 132 into the multiple tailoring group, and 119 into the usual care group. No baseline differences were found between the 3 groups . After 1A significant difference was found between the multiple tailoring and counseling and the usual care groups with regard to annual health care–related costs per patient, with significantly higher costs in the multiple tailoring and counseling group . No diffThe CEA showed that until a threshold value for the WTP of €5100 per abstinent participant, usual care was probably the most efficient treatment. However, from a WTP of €5100 or higher, multiple tailoring was probably most cost-effective . With thResults from secondary analyses showed that with 7-day point prevalence abstinence, a high probability was found  that multiple tailoring was the most cost-effective treatment. Regarding the level of addiction, however, it was most probable that multiple tailoring would be least efficient .With regard to QALYs gained, With a WTP of €18,000 per abstinent participant, the CUA showed that usual care would probably  be the most efficient treatment . AlthougTo our current knowledge, this was the first study to determine the cost-effectiveness and cost-utility of a behavioral smoking cessation intervention consisting of Internet-based computer tailoring with and without counseling by a practice nurse. The results presented suggest that participants who received the Internet-based multiple computer-tailored program and tailored counseling by their practice nurse reported significantly more annual health care-related costs than participants who received care as usual. A potential explanation for this finding might be that smokers were prompted by the tailored feedback they received to ask for more smoking cessation guidance, eg, additional counseling sessions with the practice nurse or a prescription for smoking cessation medication. Although participants who received the Internet-based program only might have had the same tendency, the practice nurses for participants in the multiple tailoring and counseling group might have been prompted by their patients’ visit to offer them more smoking cessation help. Because the current smoking cessation guidelines in the Netherlands recommend more than 1 counseling session ,58, thisFurthermore, the present study showed that the Internet-based multiple computer-tailored smoking cessation program would probably be the most cost-effective of the 3 treatments under study. Although no similar studies yet exist within the field of smoking cessation, this finding is in-line with findings from recent studies toward the cost-effectiveness of Internet-based interventions aimed at other health-related behaviors or health problems ,32. CompRegarding cost-utilities, the results suggest that care as usual would probably be the most preferable of the treatments studied. A potential explanation for this finding might be that the follow-up period of 12 months was not sufficiently long for the beneficial effects of the intervention on smoking abstinence to be translated into detectable changes in quality of life, as recent ex-smokers are known to suffer from withdrawal symptoms . A potenThe present study aimed to contribute to the literature by examining the cost-effectiveness and cost-utility of an Internet-based smoking cessation intervention, something that has not been done before to our knowledge. In the present study, in addition to generic quality of life, disease-specific effects of the intervention  were taken into account. To facilitate the comparison of the cost-effectiveness of interventions targeting different diseases, effects are usually assessed in terms of quality of life. However, to compare smoking cessation interventions more specifically, disease-specific effect measures might be more informative.Nevertheless, the present study also had its limitations. First, it suffered from relatively high dropout rates. High rates of attrition seem to be inherent to many Internet-based interventions and dropout rates of 44% are not uncommon ,65-67. AThe Internet-based multiple computer-tailored program seemed to be the most cost-effective treatment when smoking abstinence was the outcome measure. However, the cost-utility, using quality of life as the outcome measure, was probably the highest with care as usual. To enable the interpretation of the incremental costs per abstinent participant found in the cost-effectiveness analyses, future research should aim at identifying an acceptable cutoff point for the WTP per abstinent participant.


This study is aimed at gaining insights into the brain site-specific proteomic senescence signature while comparing physiologically aged brains with aging-related dementia brains ). Our study of proteomic differences within the hippocampus (Hp), parietal cortex (pCx) and cerebellum (Cb) could provide conceptual insights into the molecular mechanisms involved in aging-related neurodegeneration. Using an isobaric tag for relative and absolute quantitation (iTRAQ)-based two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D-LC-MS/MS) brain site-specific proteomic strategy, we identified 950 proteins in the Hp, pCx and Cb of AD brains. Of these proteins, 31 were significantly altered. Most of the differentially regulated proteins are involved in molecular transport, nervous system development, synaptic plasticity and apoptosis. Particularly, proteins such as Gelsolin (GSN), Tenascin-R (TNR) and AHNAK could potentially act as novel biomarkers of aging-related neurodegeneration. Importantly, our Ingenuity Pathway Analysis (IPA)-based network analysis further revealed ubiquitin C (UBC) as a pivotal protein to interact with diverse AD-associated pathophysiological molecular factors and suggests the reduced ubiquitin proteasome degradation system (UPS) as one of the causative factors of AD. Although the occipital lobe cortex retains nearly normal function, frequently even in terminal-stage patients, the temporal lobe cortex, in contrast, as one of the most fragile parts of the brain, is extremely vulnerable to neuronal death.13 Thus, it might be possible to identify those biomarkers that cause aging-related AD in the temporal lobe. Biomarkers that are capable of preventing neurodegenerative processes within the occipital lobe at early stages of AD may also be identified.15 Because the prevailing assumption is that the cerebellar proteome of the central nervous system area is relatively unaffected by AD, this is a less investigated region and often neglected in AD studies. However, it must be noted that several studies indicated that in AD the cerebellum (Cb) also shows some signs of morphological and metabolic dysfunctions.16 A number of pathological changes, such as widespread deposits of diffuse amyloid, ubiquitin-immunoreactive dystrophic neurites and increased microglia, have been revealed in the AD-affected Cb by immunocytochemical studies. However, tau-immunoreactive NFTs have not been seen. Although the observed changes may be merely epiphenomenal to the pathological processes occurring in the AD neocortex and Hp, the morphological and immunocytochemical differences between cerebral and cerebellar cortices of AD patients may nonetheless give insights into the molecular factors involved in the development of the neuropathological lesions of the AD brain.16 Consequently, more attention has also been given to this brain area in recent years, though with little focus at the molecular level.24Understanding the unique alterations in the brain proteomic signature induced by cellular senescence as a result of normal and pathological aging is essential for identification of a specific cure for various neurodegenerative disorders. Alzheimer's disease (AD) is a progressive, aging-related neurodegenerative disorder of the central nervous system and is the most common cause of dementia in the elderly worldwide. It is characterized by impaired memory and the deterioration of higher cognitive functions.25 Accordingly, in this study, we applied the two-dimensional (2D) liquid chromatography coupled with tandem mass spectrometry-based iTRAQ (2D-LC-MS/MS-iTRAQ) technique for quantitative profiling of aging-related brain site-specific proteome changes. We sought to investigate the mechanisms of senescence and pathological aging-related neurodegeneration at the proteomic level in different areas of the brain, including the Hp, the parietal cortex (pCx) and the Cb, by comprehensively identifying the proteins with altered expression levels in response to aging and aging-related dementia . Subsequently, we exploited publically accessible bioinformatic databases to infer the functional role and networks linked to the proteins that we found differentially expressed.Thus far, proteomic analyses have been considered worldwide with greater emphasis on improving the diagnosis of early stages of AD by identifying novel specific biomarkers from the cerebrospinal fluid or plasma to complement the existing diagnostic methods that are based on autopsy or neuroimaging techniques. Isobaric tags for relative and absolute quantitation (iTRAQ) is a widely accepted approach for quantitative proteomics.Our data also reveal protein pathway modulations acquired by the Cb to counteract neurodegeneration and thus could eventually become a lucrative target for the identification of plausible therapeutic drugs.Unless indicated, all reagents used for biochemical methods were purchased from Sigma-Aldrich . Materials and reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis were from Bio-Rad . The iTRAQ reagent eight-plex kit was bought commercially .26 as described previously.28 Controls were elderly patients who were age matched but without significant neurological disorders. Patients were also cognitively evaluated by neuropsychological tests using the mini-mental state examination (MMSE) and Hasegawa's dementia scale ), which is commonly utilized in Japan.29Brain tissues were obtained from the brain bank of the Choju Medical Institute of the Fukushimura hospital , and the protocols utilized were approved by the local ethics committee of the Fukushimura hospital. The scientific use of human material was conducted in accordance with the Declaration of Helsinki, and informed consent was obtained from the guardians of the patients. Brain tissues were weighed at the time of autopsy, snap frozen with liquid nitrogen and stored at –80 °C. Patients with sporadic AD  as a hemisphere block. Samples used for diagnostic purposes were taken from the frontal, temporal, parietal and occipital lobes; hippocampal formation; amygdala; basal ganglia; thalamus; and midbrain, including the substantia nigra, pons, medulla and the Cb. The specimens were embedded in paraffin and processed into 5-mm sections for conventional histological and immunohistochemical examination. For standard tissue characterization, specimens were stained using hematoxylin–eosin (to display morphology) and Klüver–Barerra staining methods. Methenamine silver and modified Gallyas–Braak staining were used to detect senile plaques, cerebral amyloid angiopathy and NFTs. Further characterization by immunohistochemistry was performed with specific antibodies against ubiquitin, α-synuclein, Aβ (1–40/42), and MAPT . Immunostaining methods were applied using the antibodies in a dilution of 1:1000 and a standard Avidin–Biotin Complex (ABC) method using a VectaStain Elite ABC kit  and the protocols provided by the supplier. After being extensively washed, immunoreactive products were detected with DAKO Envision+  and visualized after the addition of diaminobenzidine as the chromogen.g/4 °C/30 min), supernatant was collected and stored at −80 °C until further use. The protein was quantified by a ‘2-D Quant' kit  according to the manufacturer's protocol.29Briefly, brain tissues from Hp, pCx and Cb  were exc31 including postproteomic data verification by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses  using the above-mentioned protocol. Detailed diagnostic features used in this study, such as stages of plaques and NFTs of AD patients and control patients, are described in http://www.jvirgel.de/), a database that creates a virtual 2D-gel picture  to classify the functions of the iTRAQ-identified proteins modulated in the AD brains. Further biocomputational network analysis of the iTRAQ-identified proteins using the Ingenuity Pathways Analysis  offered us additional valuable clues about the complex interactions between these proteins in the AD brain.In addition, we used online databases  at Following the database search and classification of proteins, western blots were performed on randomly selected proteins to verify the iTRAQ values. The western blot images correlated well and thus validated the obtained iTRAQ values .t-tests were used to determine statistical significance. SPSS 19.0  for Windows was used to perform an analysis of variance followed by Fisher's protected least significant difference post hoc tests, when warranted. For the iTRAQ analysis, ProteinPilot Software 3.0 (Applied Biosystems) was used. To be considered statistically significant, we required a probability value to be <0.05 .33The data obtained by the western blot analyses are illustrated as the mean±s.d. Student's P-values are the average P-values of batch-I and batch-II; s.d. values are s.d. of batch-I and batch-II)).We identified a total of 950 proteins in the Hp, pCx and Cb through the use of iTRAQ. Of those identified proteins, 825 were quantified with a strict cutoff of an unused ProtScore ⩾2 as the qualification criteria. These results correspond to a peptide confidence level of 99% and an applied false discovery rate of 0.33% (<1.0%). A total of 31 proteins showed an altered expression level in the investigated brain sites  in the Hp , 2 proteAs expected, APP was found to be upregulated in the AD Hp, whereas MAPT and MAP1A were upregulated in both AD Hp and Cb. It is noteworthy that Clusterin (CLU) was downregulated in the Hp, AHNAK was upregulated in the Hp and downregulated in the Cb, the extracellular matrix protein Tenascin-R (TNR) was downregulated in the Hp, HIST1H1D/E were upregulated in the pCx and the Hp and downregulated in the Cb and MECP2 was upregulated in the Hp of AD brains. In addition, the antiapoptotic protein SOD2 was upregulated in all three areas  of AD subjects.Next, we classified all of the 31 differentially regulated proteins from the Hp, pCx and Cb of AD brains based on their classes as well as cellular and molecular functions using the Panther database , 6, 7, 8http://www.ingenuity.com). IPA identifies protein networks based on the known interactions (either direct or indirect) between proteins. In addition, IPA defines common functional and canonical pathways, thereby offering additional information about the complex interactive links between these proteins in the brain  MAPT in the Cb could also be involved in aging-related neuropathologies such as AD. However, this suggests that the Cb exploits cellular tools to inhibit hyperphosphorylated MAPT-based NFT formation. Furthermore, we identified several other proteins critically regulated in AD brains that could eventually become potential therapeutic targets for various neurodegenerative disorders. In the following sections, we describe some of these proteins with respect to AD.Hyperphosphorylation of MAPT (tau) is closely related to various neurodegenerative diseases, including aging-related dementia such as AD.50 Association of CLU gene polymorphisms with late-onset AD has been reported recently,55 and additional data provide a possible link between the CLU and APOE genotypes in the etiology of AD.57 Some in vitro studies have demonstrated that at certain concentrations, purified CLU can interact with Aβ and result in an inhibition of fibril formation and thus functions as an extracellular chaperone that prevents the aggregation of nonnative proteins.60 In contrast, in a mouse model of AD, CLU was found to cause neuritic dystrophy by promoting Aβ plaque formation.61 This effect of CLU varied according to the amount of the prefibrillar substrate.62 Regardless, CLU could play an antiapoptotic function by interfering with the activity of the proapoptotic protein Bax.63 Overexpression of CLU has also been found in several human gland cancers.64 Thus, our data support the hypothesis that the strict downregulation of CLU in the AD Hp could contribute to neurodegenerative processes because of a reduced chaperone function and decreased antiapoptotic activity.CLU is another important protein involved in neurodegenerative diseases.66 Our data are consistent with these reports, indicating that TNR was significantly downregulated. However, this TNR downregulation only occurred in the Hp, and not in the pCx or Cb. Thus, we postulate that the downregulation of TNR expression might be one specific direct cause of impaired cognitive abilities in AD. Interfering with the TNR protein expression level could potentially be useful for novel AD treatment strategies especially through an improvement of Hp function.TNR is an extracellular glycoprotein known to be primarily involved in signal transduction and cell–matrix adhesion. Loss of TNR impairs cognition, synaptic plasticity and motor abilities in mice. Homozygous deletion of TNR is associated with intellectual disability and cognitive deficits.68 because the development of tau lesions in AD is traceable to differences between early- versus late-maturing oligodendrocytes and to the exceptionally protracted myelination of late-developing portions of the human brain,71 AHNAK becomes of pivotal interest for future investigations.Recent findings have disclosed the crucial role of AHNAK in myelination processes during development, neuronal plasticity and neuro-re-/de-generation events;73 However, the expression level of GSN in the brain of AD patients has been discussed controversially.75 Our data indicate that GSN is significantly downregulated in the AD Cb, which is consistent with a recent report that a reduced plasma GSN level in AD patients was found. This suggests that GSN may function as an additional plasma biomarker candidate that could contribute to the diagnosis of early-stage aging-related AD.75Previous studies have identified the antiamyloidogenic role of gelsolin in AD. Gelsolin can reduce the amyloid burden by acting as an inhibitor of Aβ fibrillization and as an antioxidant and antiapoptotic protein.76 MECP2 was found to be involved in various neurodevelopmental disorders77 and to promote neuronal death.78 Thus, the extremely high expression of MECP2 in the AD Hp could contribute to neurodegenerative pathways.Environmental factors, including metals and dietary factors, operate by interfering with the interaction of methylated CpG clusters and binding proteins, such as MeCP2 and SP1. The impact of these factors on AD has been discussed previously.83 Aside from its post-translational modifications, the expression levels of histones are also important for cell cycle control.84 Here, we show that the expression of HIST1H1E was increased in the AD Hp, whereas the expression of HIST1H1D was increased in the AD pCx but decreased in the AD Cb. In general, increased levels of histone variants were found in affected areas (pCx and Hp) in AD, whereas the Cb reflected reduced levels of another cell cycle protein, CEND1. The different changes in expression levels of histones in different brain regions indicate their brain site-specific roles in the pathology of AD. However, further studies are required to unravel the link between the expression levels and cellular functions of these proteins to accurately evaluate their specific pathological roles in AD and other neurodegenerative disorders.The post-translational modifications of histones, such as acetylation and deacetylation, have been increasingly recognized as critical factors affecting gene activation and silencing in the brain of individuals with neurodegenerative disorders.85 The correlation of neurodegeneration and deregulation of the cell cycle in AD have been frequently discussed over the past few years.92 Brain site-specific CEND1 expression changes (increased in the Hp and decreased in the Cb) may cause neurodegeneration by promoting abnormal cell cycle re-entry.93 Thus, increased CEND1 expression in the Hp could be responsible for a reduced regeneration capacity, whereas its downregulation in the Cb might be a survival strategy acquired by this area to induce dynamic regeneration. Thus, our study suggests a potential therapeutic usage of CEND1 in neurodegenerative disorders.CEND1 is another neural stem cell-specific protein involved in cell cycle exit and neuronal differentiation.97 and thus emphasize the importance of the unfolded protein response and proteostasis in AD .114 However, it is not fully understood why ubiquitin is accumulated in intra- and extra-cellular deposits or how it is involved in AD pathogenesis. Our iTRAQ analysis provides compelling evidence that many of the altered molecular factors in AD brains interact with UBC. Thus, the interconnection of AD-related proteins with UBC implicates a pivotal role of ubiquitin UPS in aging-related neurodegeneration such as AD.Recently, it has also become clear that the UPS is perturbed in aging-related dementia such as AD.115 quantitative proteomics analyzing aging brain tissue has yet to be evaluated and reviewed with critical considerations. There are currently a combination of different methods used, such as (1) 2D difference gel electrophoresis (2D-DIGE) combined with MALDI-TOF-MS/MS (matrix-assisted laser desorption/ionization time-of-flight MS/MS) proteomics116 or (2) 2D-LC-MS/MS-iTRAQ/ICAT (isotope-coded affinity tag); some proteins appear more frequently , whereas others are hardly detected .118 Despite improved sensitivity of our 2D-LC-MS/MS analyses, limitations of ‘undersampling' in LC-MS/MS still restrict the number of peptides that can be identified in a specific sample. Though this technical limitation can be assessed by the inclusion of technical replicates or identical samples that are processed in parallel,119 further technical improvements of quantitative 2D-DIGE/LC-MS/MS (iTRAQ/ICAT) proteomics are required to make more significant translational research-based contributions that may provide more clinically relevant data.Although our proteomic data are in agreement with others,Further studies using higher numbers of control and AD patients, shorter post-mortem intervals and more individual patient-to-patient comparisons may certainly improve our knowledge regarding the neurodegenerative processes in the aging brain.In conclusion, our current study demonstrates a brain site-specific aging-related proteome pattern and emphasizes the importance of UPS in the AD brain. We report higher MAPT expression levels in the AD Cb suggesting transmission of neurodegeneration signals to this area. Our study also indicates a crucial role of AHNAK in processes of aging-related dementia, and we hypothesize that the Cb exploits proteins such as CEND1 or GSN to fight against neurodegeneration. An even more comprehensive study of the cerebellar proteome would most likely shed further light on how this area counteracts NFT formation and AD-induced neurodegeneration.In conclusion, our findings have unraveled the complex proteome changes that occur in aging brains and open a new avenue for drug discovery in aging-related brain disorders such as AD.