Use of Botulinum toxin-A (BoNT-A) for treatment of upper limb spasticity in children with cerebral palsy has become routine clinical practice in many paediatric treatment centres worldwide. There is now high-level evidence that upper limb BoNT-A injection, in combination with occupational therapy, improves outcomes in children with cerebral palsy at both the body function/structure and activity level domains of the International Classification of Functioning, Disability and Health. Investigation is now required to establish what amount and specific type of occupational therapy will further enhance functional outcomes and prolong the beneficial effects of BoNT-A.A randomised, controlled, evaluator blinded, prospective parallel-group trial. Eligible participants were children aged 18 months to 6 years, diagnosed with spastic hemiplegic cerebral palsy and who were able to demonstrate selective motor control of the affected upper limb. Both groups received upper limb injections of BoNT-A. Children were randomised to either the modified constraint-induced movement therapy group  or bimanual occupational therapy group (control). Outcome assessments were undertaken at pre-injection and 1, 3 and 6 months following injection of BoNT-A. The primary outcome measure was the Assisting Hand Assessment. Secondary outcomes included: the Quality of Upper Extremity Skills Test; Pediatric Evaluation of Disability Inventory; Canadian Occupational Performance Measure; Goal Attainment Scaling; Pediatric Motor Activity Log; modified Ashworth Scale and; the modified Tardieu Scale.The aim of this paper is to describe the methodology of a randomised controlled trial comparing the effects of modified constraint-induced movement therapy  versus bimanual occupational therapy  on improving bimanual upper limb performance of children with hemiplegic cerebral palsy following upper limb injection of BoNT-A. The paper outlines the background to the study, the study hypotheses, outcome measures and trial methodology. It also provides a comprehensive description of the interventions provided.ACTRN12605000002684 Cerebral palsy is "a group of permanent disorders of the development of movement and posture causing activity limitation(s) that are attributed to non-progressive disturbances that occurred in the developing fetal or infant brain" . Hemiplegic cerebral palsy, characterised by a clinical pattern of unilateral motor impairment, accounts for 35.1% of all cerebral palsy types in Victoria, Australia  and 31.2The spastic motor type of cerebral palsy is the most common, comprising about 80% of all reported cases . Althougclostridium botulinum [acetylcholine. Injection of BoNT-A into selected muscles produces dose-dependent chemical denervation resulting in reduced muscle activity. The denervation is temporary as sprouting of new nerve terminals from the treated nerves leads to re-innervation. The function of the original terminal is eventually restored leading to the recovery of the affected muscles [BoNT-A is a powerful neuromuscular paralysing agent that is produced by the anaerobic bacterium otulinum . BoNT-A  muscles . The per muscles . The aim muscles .Use of BoNT-A for the treatment of upper limb spasticity in children with cerebral palsy has become routine clinical practice in many paediatric treatment centres worldwide. In Australia, injection of BoNT-A (Botox(r) only) is now an approved and government funded treatment for moderate to severe spasticity of the upper limbs children with cerebral palsy, 2 to 17 years of age inclusive  when comIntramuscular injection of BoNT-A results in relaxation of the affected muscle providing a window of opportunity for maximising therapy outcomes. For children with hemiplegia however, injection of BoNT-A alone does not improve the ability of the child to use the affected limb and clinical experience suggests attempts to use the limb immediately following injection often remain effortful, inefficient and result in clumsy movement. Persistent verbal and physical encouragement by therapists and parents may frequently lead to further frustration and negative or defiant behavior. Therapy must, therefore, create the opportunity, motivation, experience and environment in which a child can learn how to use their affected limb to maximize the effectiveness of the BoNT-A.A recent systematic review of all upper limb interventions in children with cerebral palsy found that occupational therapy in combination with BoNT-A produced the largest treatment effect of all upper limb interventions on activity level outcomes . DespiteFor occupational therapists, bimanual occupational therapy training (BOT) in children with hemiplegia is a commonly applied intervention. Theories of practice have previously been described in the literature -29, althIn 2007, Gordon and colleagues comprehensively described and evaluated a bimanual approach to upper limb training in children with hemiplegic cerebral palsy called Hand Arm Bimanual Intensive Training (HABIT) . DespiteConstraint-induced movement therapy (CIMT) has been adopted as a method of teaching a child to use his/her affected upper limb through use of a restraint on the non-affected limb and massed practice of movements of the affected limb . It has Developmental disregard is a term used to describe a child with hemiplegia who may disregard, or learn not to use, the affected limb during the development of motor function [function . Childrefunction . Constrafunction . Studiesfunction -39. Sincfunction -42, it ifunction .CIMT is a multi-faceted intervention and studies describing its use in children with cerebral palsy present wide variation in its application in relation to: method of restraint; length of restraint ; type and duration of therapy; intervention environment  and intervention provider . Children included in studies have also varied in age, diagnosis, severity of motor and sensory impairment, cognitive abilities and behavior. Despite the emerging popularity of CIMT in children with hemiplegic cerebral palsy, a Cochrane review  identifiStudies published to date have provided limited evidence of factors that may impact on the effectiveness of CIMT. Response to CIMT does not appear to be age-dependent and children with more impaired bimanual hand function demonstrate greater improvement than less impaired children . AlthougThere is currently no published trial directly comparing a BOT protocol with a CIMT protocol. However, in 2008 Gordon and colleagues  publisheThe aim of this current paper is to describe the methodology of a randomised controlled trial comparing the effects of modified constraint-induced movement therapy  versus conventional bimanual occupational therapy on improving bimanual upper limb performance of children with hemiplegic cerebral palsy following upper limb injection of BoNT-A. Bimanual upper limb performance, assessed using the Assisting Hand Assessment, was selected as the primary measure as the central aim of all upper limb motor-based interventions in children with hemiplegia is to improve a child's actual use of their affected upper limb in a range of daily tasks, including those requiring bimanual performance; rather than what they can do in a clinical setting or how normal their movement appears. This RCT builds on the existing evidence supporting the use of BoNT-A in the upper limb of children with cerebral palsy. It aims to identify the most effective intervention following injection of BoNT-A. A further aim is to explore the individual characteristics of children that impact on response to treatment. Finally, it will also provide additional evidence regarding dose response to treatment, specificity of training effects, and the retention of treatment effects.To compare the effects of modified CIMT versus bimanual occupational therapy (BOT) in improving the bimanual performance of children with hemiplegic cerebral palsy following upper limb BoNT-A injection.Modified CIMT is more effective than conventional BOT in improving the bimanual performance of children with hemiplegic cerebral palsy following upper limb BoNT-A injection.Relative to baseline performance on the Assisting Hand Assessment, modified CIMT results in greater retention of gains in bimanual performance compared with BOT.A. To compare the effect of modified CIMT versus bimanual occupational therapy (BOT) on secondary outcomes relating to: the performance of activities of daily living; the quality of upper limb movement; occupational performance; individual goal achievement; and frequency of use of the affected upper limb of children with hemiplegic cerebral palsy following upper limb BoNT-A injection.Modified CIMT is more effective than conventional BOT in improving performance of activities of daily living, quality of upper limb movement, occupational performance, individual goal achievement and frequency of use of the affected upper limb in children with hemiplegic cerebral palsy following upper limb BoNT-A injection.B. To evaluate the clinical significance of change identified on the primary outcome, the Assisting Hand Assessment.That changes on the Assisting Hand Assessment associated with intervention are of a clinically significant magnitude. Clinical significance was defined as a change score of more than 5 raw score points (2.6 logits) on the AHA .C. To evaluate the impact of age and actual treatment dosage on the primary outcome, the Assisting Hand Assessment.Younger children (irrespective of treatment type) will show greater improvement in bimanual performance following uni-manual or bimanual therapy and upper limb BoNT-A injection.Children who undertake treatment of greatest intensity (irrespective of treatment type) will show greater improvement in bimanual performance.Randomised, controlled, evaluator-blinded, prospective parallel-group trial based on Consolidated Standards of Reporting Trials (CONSORT) Statement for Randomised Trials of Non-Pharmacologic Treatment .Children were eligible to participate if they met the following criteria:• Diagnosis of congenital spastic hemiplegic cerebral palsy as diagnosed and reported in the medical history by a medical specialist .• Aged 18 months to 6 years at time of recruitment.• Active movement of the shoulder, elbow, wrist, digits and thumb of the affected upper limb, such that the:• child is able to reach forward to an elevated position in front with mid range shoulder flexion.• child is able to grasp a 2.5 cm cube from a table top and release it in a large container (20 cm × 14 cm).• Able to attend to tasks and follow simple one stage commands.• child is able to actively perform reach and grasp/release activities with verbal prompting.• Moderate levels of muscle tone  and spasticity  and no fixed contracture in target group of muscles to be injected with BoNT-A.• Parents able to commit to an intensive therapy program and agree to cease all other upper limb therapeutic interventions for the 6-month period of the trial.• Assessed as appropriate for upper limb BoNT-A at neuromuscular clinic by rehabilitation specialist (BR).Children who otherwise met the inclusion criteria were excluded if they had:• Congenital quadriplegic or diplegic cerebral palsy.• Previous BoNT-A injections in the upper limb in the past 12 months.• Prior upper limb surgery (i.e. tendon transfer/tendon lengthening).• Existing treatments that are not compatible with those those included in the study treatment package.There were no criteria relating to exclusion of children with mother tongue other than English, presence of co-morbidity or socio-economic status.Potential participants were identified from the Physical Rehabilitation Clinic of a major paediatric metropolitan hospital. In addition, postal and email advertising were sent to local medical practitioners and paediatric therapy networks in metropolitan Melbourne. Potential participants were screened by the chief investigator (BJH) to determine eligibility. Children eligible for inclusion were then assessed by an experienced rehabilitation specialist for suitability for upper limb BoNT-A injection (HBR). Suitable children were then invited to participate in the RCT and informed consent obtained for the participation of the child and the child's parent prior to enrolment in the RCT.The RCT was approved by the Ethics Committee of both Southern Health and La Trobe University. The trial is registered with the Australian New Zealand Clinical Trials Registry ACTRN12605000002684) [00002684 Prior to commencement of the RCT, two highly experienced paediatric therapists  were trained in the administration of all outcome measures. A manual was developed for both therapists to ensure consistency in measurement over the recruitment period. The same occupational therapist collected data for the Assisting Hand Assessment, Quality of Upper Extremity Skills Test, Pediatric Evaluation of Disability Inventory, Pediatric Motor Activity Log, Canadian Occupational Performance Measure and Goal Attainment Scaling for the entire recruitment period. Two physiotherapists collected data for the modified Ashworth and modified Tardieu Scales. Scoring of the Assisting Hand Assessment was undertaken by the assessment developers. The Quality of Upper Extremity Skills Test was scored by a highly experienced paediatric occupational therapist who had significant experience in the assessment and treatment of children following upper limb BoNT-A injections.Children were block randomised into pairs matched by age (± 6 months) using a computer generated set of random numbers.A set of random numbers was used to create an allocation sequence which was contained in individual opaque envelopes for use by the chief investigator (BJH). As children were recruited, the next envelope in the sequence was opened and the child assigned to the stated group.All randomisation, sequence generation, and preparation of group allocation materials were performed by a third party  who had no direct contact with the clinical aspects of the trial. The master list of random numbers was located in locked cabinets at the Institute and only accessible at completion of the RCT for analysis.Due to the overt nature of the interventions, children, their parents and the treating therapist were not blinded to group assignment, however, children and parents were blind to the RCT hypotheses. Outcome measures were administered by a therapist blind to group assignment and scored by different assessors who were blind to group allocation. The Assisting Hand Assessment and Quality of Upper Extremity Skills Test was videotaped, randomised and scored by assessors blind to group allocation and order of assessment.Sample size estimates were based on projected treatment effect on the primary outcome measure, the Assisting Hand Assessment. In 2005, the authors suggested that a change of more than 5 raw score points may represent a clinically meaningful difference on the Assisting Hand Assessment . This was the equivalent to a change in 2.6 logits. Calculation for fractions of logits undertaken by a statistician indicated that in order to detect a change of 2.6 logits a minimum sample size of 17 per group was required. As the RCT design involves a two-group design, a total sample size of 34 was required to provide 80% power to detect clinically meaningful change between groups. Therefore, the study aimed to recruit 40 children to allow for a 10-12% drop-out rate.Outcomes measures were completed on four occasions: baseline (1 to 2 weeks prior to injection), and at 1, 3 and 6 months after injection. Outcomes included measures from across the International Classification of Functioning, Disability and Health (ICF)  spectrumThe effects of upper limb BoNT-A injection in both groups were monitored using measures of muscle spasticity and muscle tone including the Modified Ashworth Scale (MAS) and Modified Tardieu Scale (MTS).The primary outcome measure was the Assisting Hand Assessment (AHA) . The admThe AHA is conducted by video observation of the child involved in a 10-15 minute play session using the AHA test kit with specific toys. The twenty-two items defining different actions are then scored on a 4-point scale rating the quality of the performance. Four being effective, 3 somewhat effective, 2 ineffective and 1 indicating the child does not perform the action. The sum of raw scores (sum score) varies between 22 (low ability) to 88 points (high ability). Raw scores are converted to scaled scores ranging from 0 to 100. Using a computer generated logarithmic transformation, ordinal data are then converted to equal intervals in the unit logits . The ranThe psychometric properties of the AHA have been described in several studies -54. UsinThe Modified Ashworth Scale (MAS) is a 6-point, criterion referenced ordinal scale deigned to measure the resistance that is encountered when a limb is moved passively . Scores The MAS has often been described in the literature as a measure of spasticity ,56, howeThe modified Tardieu scale (MTS) was adapted from the original scale developed by Tardieu and colleagues . The MTSIn this RCT, administration of the MTS was jointly performed by an occupational therapist and physiotherapist both blinded to group assignment. Movements measured included: shoulder abduction and flexion; elbow extension and flexion; forearm supination and pronation; wrist extension and flexion; ulnar and radial deviation and thumb abduction. The velocity used to determine the angle of catch was V3 (as fast as possible), as recommended by Morris . The MTSThe secondary outcomes included measures of: quality of upper limb movement ); caregiver questionnaires relating to functional status of the child ) and perceived use of the affected upper limb (Pediatric Motor Activity Log (PMAL)); individual occupational performance ); individual goal attainment ); muscle tone ); and spasticity ).The Quality of Upper Extremity Skills Test (QUEST) is a descriptive, impairment-based measure designed to evaluate movement patterns and hand function in children with cerebral palsy . The QUEThe QUEST is a reliable and valid measure for evaluating the quality of movement in children with cerebral palsy. Inter-observer ICC's range from 0.90 to 0.96 with test-retest correlations of 0.95 . CurrentThe Pediatric Evaluation of Disability Inventory (PEDI)  is a staDue to the nature of interventions provided in this trial being targeted at upper limb function rather than mobility and social function, only the self-care domain of the PEDI was administered. This is consistent with the use of the PEDI in many other trials evaluating BoNT-A and occupational therapy ,26,72,73PEDI scale construction was developed using Rasch measurement model and analytic techniques to evaluate construct validity and develop scaled scores. The PEDI has established validity ,74-76. HThe Canadian Occupational Performance Measure (COPM)  is a cliDue to the age of the children in this RCT, parental responses to the COPM were obtained rather than the child's. This adaptation has been supported in findings by Cusick et al.  who demoThe Goal Attainment Scale (GAS) is an individualized, criterion-referenced measure of treatment-induced change . The GASUsing mathematical formulae, Kiresuk et al.  provide The GAS has been used in five out of ten RCT's evaluating the use of BoNT-A and occupational therapy in children with cerebral palsy making it the most commonly used outcome measure across these trials. Despite its popularity and reported sensitivity to detect change, the validity and reliability of GAS is largely unknown . ValiditThe Pediatric Motor Activity Log  Version Despite its use in studies evaluating CIMT ,94, and biceps brachii, brachialis, brachioradialis, pronator teres, pronator quadratus, flexor carpi ulnaris, flexor carpi radialis, flexor digitorum profundus, flexor digitorum superficialis, flexor pollicis longus, adductor pollicis, and opponens pollicis. If indicated, children also received injections of BoNT-A into lower limb muscles during the same injection session. This was considered ethically appropriate for the child's overall management and was considered unlikely to interfere with upper limb outcomes. Injections were performed in theatre under a light general anaesthetic and all children were discharged on the same day. Muscle localisation was undertaken by the use of Teflon coated Botox injection needles  allowing electrical stimulation. Doses of BoNT-A were administered at a maximum dose of 15 U/kg (or 400 U). Dilution was 100 U/1 ml. During the first month following injection, the chief investigator (BJH) reviewed all children on two occasions to monitor the effect of the BoNT-A, identify potential adverse events and provide splinting intervention as required.Each child in the study received injections of BoNT-A  by a single, highly experienced rehabilitation specialist (HBR). Muscles injected were determined by the rehabilitation specialist in consultation with the chief investigator (BJH) at the Physical Rehabilitation clinic based on whether they appear to contribute to abnormal limb position and impair functional use of the limb. Injected muscles included Children with increased resistance to passive stretch or who exhibit early signs of muscle shortening were provided with a thermoplastic stretching splint designed for use overnight for a minimum of 6 hours per night. This protocol has been based on scientific support from a small number of animal studies reporting that muscles increase in length when immobilised in a lengthened position ,97 and aOne-month following injection of BoNT-A, children randomised to the experimental group received modified constraint-induced movement therapy (mCIMT). Children randomised to the control group received conventional bimanual occupational therapy (BOT). The individual-based treatment sessions of 45 to 60 minutes were conducted by the same occupational therapist (BJH) twice weekly for 8 weeks in an outpatient paediatric treatment room. In addition, children in the mCIMT experimental group were required to complete 3 hours of home program (with the mitt on), 7 days a week for the 8 week treatment period. Children in the BOT control group were also encouraged to undertake a home program but no time requirements were specified. A checklist was completed after each treatment session, identifying the activities used and general observations. Important general considerations for both groups related to establishing and maintaining rapport, equipment, preparing and implementing the sessions are described below.Building rapport and establishing the therapist-child/therapist-parent relationship was a primary focus for initial treatment sessions. Along with identifying individual movement, hand skill and motor planning abilities of each child, this time allowed for selection of toys to match the demands of the task with the child's developmental level and specific hand skill goals. It was also necessary to establish expectations for future sessions, create an understanding of acceptable behaviour and to establish patterns of on-task behaviour for the child.Most treatment was undertaken with the child sitting at a height adjustable table  were used to redirect their attention to the task. Because task repetition and practice are key components of training, any attempt by the child to prematurely stop task performance (i.e. by focussing on something else in the room) was ignored by the therapist and parent and the child's attention redirected to the task. The child was required to indicate when an activity had been finished through verbalisation or physical signs. If this occurred after only a few repetitions, further repetitions were negotiated before the activity is stopped. This was seen as important for compliance and motivation as the child felt included in the decision making process whilst further task repetition was achieved. No new activities were chosen until all parts of the activity had been packed up. This was crucial as repetitions of movement/hand skills achieved through packing up were often greater than in the activity itself.With novel and/or challenging tasks, an inability to independently and successfully complete the demands of the task often leads to a child becoming disinterested or frustrated after only a few trials or repetitions. To facilitate learning, improve skills and task performance, develop resilience, and for the therapist to assist the child to establish patterns of on-task behaviour it was important not to allow the child to "give up" or avoid tasks. A variety of task-avoidant behaviours can be successfully used by children from a very early age. Initial and ongoing parent/therapist reinforcement can inadvertently condition task-avoidant behaviour, impeding the participation and engagement of the child and therefore the effectiveness of treatment. It was crucial for the therapist to establish a collaborative partnership with the parents to ensure consistent responses to these behaviours. On occasions where task avoidant behaviours became evident, the therapist provided advice and strategies to the parent(s) on appropriate responses to avoid reinforcement. Examples of typical task-avoidant behaviours displayed by children included:• Throwing objects - this often occurs when a child does not understand the requirement of the task (poor motor planning) or the task is too challenging for the child's abilities. In this RCT the therapist always modelled novel task performance to demonstrate task demands. During early attempts at a task, significant physical assistance and/or verbal cuing were provided to ensure successful performance. The amount of assistance was then gradually decreased with future attempts. Any object thrown by the child was not collected by the therapist/parent and the behaviour was ignored. The object remained on the floor to ensure the child did not receive a response that reinforced the behaviour . If the behaviour persisted the therapist pre-empted the throw preventing it from happening. The child's attention was redirected to the task and, if required, additional modelling of task performance or increased physical assistance was provided.• Mouthing objects - young children can learn that mouthing objects leads to a quick and alarmed reaction from the parent/therapist that leads to diversion of attention away from the task. During treatment if a child demonstrated this behaviour, objects small enough for ingestion were avoided in all sessions until the behaviour ceased. To avoid encouraging this behaviour, larger objects were used (to ensure ingestion was not possible) and the behaviour was ignored when displayed. Any direct eye contact with the child from the parent and therapist was also avoided and directed to the task, so the child did not receive a response .• Crying can be an extremely effective strategy for task-avoidance by young children. The reinforcement of this behaviour occurs when the therapist immediately stops the task or parents intervene to provide comfort and reassurance through physical contact or removal of the child from the activity. The child can associate crying with successful task avoidance with resultant disruption to the session. During treatment, it was important for the therapist to quickly evaluate the situation. If the child was obviously distressed, parental comfort and reassurance was used to settle the child. Removal from the seat was avoided if at all possible. If the child was not obviously distressed and crying was deemed by the therapist/parent as task-avoidant behaviour the task was continued. A graded level of response was initiated: 1) child was not removed from position; 2) therapist negotiated with the child for one or two more repetitions prior to task completion; 3) therapist verbalised when task was completed 4) child was required to pack up the activity before choosing next activity; 5) if the child remained uncooperative the therapist and parent engaged in conversation or played the task/game whilst ignoring the child's behaviour; 6) if the child remained uncooperative the therapist asked the child if they would like the parent to leave the room; 7) as a last resort, if the child remained uncooperative the parent left the room until the child settled. As soon as the child settled the parent was invited back into the room. Great care was taken with this final response as it had the potential to cause significant anxiety and stress to the child. Over time, with consistent responses to crying from the parent and therapist, it was expected that the child would stop using crying as a technique for task avoidance.The modified constraint-induced movement therapy (mCIMT) protocol incorporated the two fundamental components of CIMT as described by Taub et al. : the useThe implementation of mCIMT was based on the principles of motor-learning theory ,101. EliActivities deemed too difficult by the therapist were avoided to prevent experiences of failure and frustration. This was particularly important in initial sessions where simple cause and effect activities were used (e.g. battery operated switch activated toys). Parents were educated that use of the glove must be perceived by the child as fun and enjoyable. Rewards, such as playing with favourite toys when the glove was used, was highly encouraged. Negative experiences, such as verbal threats and withdrawal of activities, were discouraged. The child was always kept busy and periods without the child actively engaging in play was avoided. If the child became distracted by the glove their attention was immediately redirected to the task by using noise and sensory input . Any discussion regarding the glove was avoided. At the conclusion of each session the child was praised and informed the session had finished and the child was encouraged to independently remove the glove using his or her affected hand.The bimanual occupational therapy (BOT) was underpinned by components of motor learning  and cognmotor learning framework requires implementation of a motor-teaching model whereby the therapist acts as a teacher and the child, a learner. Factors required to facilitate a child's learning of motor skills include: giving attention to the context; motivation and prior knowledge; instructions; modelling; taxonomy and sequencing of tasks; anticipation skills; mental and physical practice; repetition; facilitation-guidance; and feedback [motor skills acquisition frame of reference [Practical application of a feedback . Similareference . These feference ,102, aloeference ,105, for• Initial and ongoing task analysis to identify if the child's performance was limited by execution of movement or motor planning difficulties (i.e. sequencing of movements) ,105,106.• Repetitive whole-task practice of challenging, motivating and purposeful bimanual activities (i.e. toys and games), carefully selected to facilitate learning and development of goal-based skills and independence with task completion .• Use of modelling, physical assistance, verbal cues or environmental adaptation to enable the child to understand the critical features of the task and the environment.• Facilitation of the child's learning and understanding of the role of their assisting hand (i.e. hemiplegic hand) using active problem solving.• Grading of physical and/or verbal assistance provided to complete tasks.• Provision of feedback focusing on the outcome, task and environment rather than specific movement performance.• Provision of opportunities for the child to repetitively practice tasks in a range of contexts and environments.The Assisting Hand Assessment (AHA), designed specifically for children with unilateral impairment, was developed using a Rasch measurement model. This model allowed identification and ordering of the AHA test items on a scale from easiest to hardest. For example, simply approaching an item using the affected hand (easier item) to using in-hand manipulation skills to move objects in the affected hand (more difficult item). Based on the Rasch model, easier items are more likely to be easier to perform for all children than more difficult items. More able children are also more likely to perform better on more difficult items than less able children. The unique construct allows children's bimanual upper limb ability levels to be placed along a continuum of low ability to high ability. This knowledge can be used to design intervention and guide graded task performance . For exaChildren with hemiplegic cerebral palsy have varied abilities to perform bimanual activities. Often children have the underlying motor abilities  to execute the required movements for task performance, however they are unable to independently identify the specific role for each hand, appropriately position the hands and object or sequence the movements or direction of force required to complete the task. These motor planning difficulties may be just as limiting for the performance of activities of daily living as is movement execution in children with cerebral palsy .Cognitive-based approaches ,104 haveIdentifying and breaking down the specific sequences of a task allowed the therapist to assist the child to discover deficient sequences and to prompt them to consider the error before the end of the whole task. This inhibits failure of the task at an incipient stage thereby reducing the likelihood that the child will become frustrated or non-compliant . Typicalp values were used to indicate the strength of the evidence and will be interpreted according to Sterne and Davey Smith [Data were managed and analysed using the Statistical Package for Social Sciences (SPSS version 16.0). Descriptive statistics were calculated to summarise the data set for both groups and to identify potential baseline differences between the groups; ey Smith  DistribuUsing continuous, interval level data from the AHA, differences between the two groups were assessed using a linear regression approach to analysis of covariance (ANCOVA). This controlled for the covariates of baseline AHA scores and the child's age. These variables were included as covariates as they might provide alternative explanations for any observed changes in scores. The size of treatment effect was estimated by comparing differences in group means and their 95% confidence intervals. Regression analyses and scatter-plots were used to investigate the relationship of post-treatment outcome with initial deficit.Between group differences were assessed using ANOVA for continuous data . Outcomes that had non-continuous data or did not meet usual assumptions of linear regression were investigated using non-parametric statistics.The magnitude of treatment effect was evaluated relative to defined criterion of clinical significance.Data related to the number of minutes spent by all children undergoing treatment was extracted from the log book for each child. Associations between the intensity of therapy and outcome and age of children and outcome were determined, while controlling for the covariate of group, using ANCOVA.Each child was monitored throughout the trial period by the chief investigator. Following trial completion, all medical and research records were retrospectively audited. Any adverse events were recorded and classified according to whether they could be attributed to BoNT-A injection, general anaesthesia or movement-based treatment.The study commenced recruitment in May 2003 and achieved target recruitment in September 2008. Participant follow-up was completed in March 2009. Data analysis is currently being undertaken.Approval was granted by Southern Health and La Trobe University Ethics Committees to extend the completion date of the project to June 2010. Approval was requested due to the trial being behind schedule due to slow recruitment rates.In March 2005 approval was granted by Southern Health and La Trobe University Ethics Committees to extend the upper age limit of children included in the trial from 4 to 6 years of age. Modification was requested due to the development of the upper age limit for the AHA, allowing the effective/valid measurement of children aged 4 to 6 years old. Children in this age range were also considered to potentially benefit from the treatments provided in the trial and therefore would be a clinical population these treatments would be offered to in the future.This paper describes the methodology of a randomised controlled trial comparing the effects of modified constraint-induced movement therapy  versus conventional occupational therapy  on improving bimanual upper limb performance of young children with hemiplegic cerebral palsy following upper limb injection of BoNT-A. The results of the paper will be disseminated through peer reviewed journal publications.AHA: Assisting Hand Assessment; BoNT-A: Botulinum toxin-A; BOT: Bimanual occupational therapy; CIMT: Constraint-induced movement therapy; COPM: Occupational Performance Measure: GAS: Goal Attainment Scaling; HABIT: Hand arm bimanual intensive training; ICF: International Classification of Functioning, Disability and Health; MAS: Modified Ashworth Scale; mCIMT: Modified constraint-induced movement therapy; MTS: Modified Tardieu Scale; PEDI: Pediatric Evaluation of Disability Inventory; PMAL: Pediatric Motor Activity Log; QUEST: Quality of Upper Extremity Skills Test; RCT: Randomised controlled trial.Allergan Australia provided partial support by providing the BoNT-A (Botox(r)) used in the study, by payment of research assistants blind to group allocation, a blinded rater for the QUEST, and video editing services. The authors have no pecuniary interest in Allergan.BJH is an occupational therapist and has received sponsorship from Allergan Australia to attend and teach at conferences and meetings but has no personal financial interest in Botox(r) or any related product.® or any related product.CI is co-investigator of a randomised controlled trial investigating the effect of repeat injections of BoNT-A and occupational therapy in the upper limbs of children with hemiplegic cerebral palsy that has received support from Allergan Australia. In 2008, CI received a grant from Allergan Australia to present results of this trial at the American Academy of Cerebral Palsy and Developmental Medicine in Atlanta but has no personal financial interest in BotoxHBR has received sponsorship from Allergan Australia to attend and teach at conferences and meetings but has no personal financial interest in Botox(r) or any related product.LC - No competing interestsBJH: PhD candidate, designed the RCT and wrote the paper. CI: PhD supervisor, designed the RCT and monitored progress revising the paper critically for intellectual content. HBR: monitored progress revising the paper critically for intellectual content. LC: PhD supervisor, designed the RCT and monitored progress revising the paper critically for intellectual content. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/10/58/prepub


Improving the way artemether-lumefantrine (AL) is provided to patients attending clinics is critical to maximize the benefit of this new medicine. In 2007, a new initiative was launched in one part of Kenya to improve malaria case-management through enhanced in-service training and provision of job aids.An evaluation of the intervention using pre- and post-intervention cross sectional health facility surveys was conducted in Bondo district. The surveys included: audit of government health facilities, health worker structured interviews and exit interviews with caretakers of sick children below five years of age. The outcome indicators were the proportions of febrile children who had AL prescribed, AL dispensed, and four different dispensing and counseling tasks performed.At baseline 33 government health facilities, 48 health workers and 386 febrile child consultations were evaluated. At follow-up the same health facilities were surveyed and 36 health workers and 390 febrile child consultations evaluated. The findings show: 1) no health facility or health worker was exposed to all components of the intervention; 2) the proportion of health workers who received the enhanced in-service training was 67%; 3) the proportion of febrile children with uncomplicated malaria treated with the first-line anti-malarial drug, artemether-lumefantrine (AL), at health facilities where AL was in stock increased from 76.9%  to 87.6% ; 4) there were modest but non-significant improvements in dispensing and counseling practices; and 5) when the analyses were restricted to health workers who received the enhanced in-service training and/or had received new guidelines and job aids, no significant improvements in reported case-management tasks were observed compared to baseline.In-service training and provision of job aids alone may not be adequate to improve the prescribing, dispensing and counseling tasks necessary to change malaria case-management practices and the inclusion of supervision and post-training follow-up should be considered in future clinical practice change initiatives. By 2009, every country in Africa had transitioned to new, effective artemisinin-based combination therapy (ACT) to manage uncomplicated malaria. Despite efforts to expand accessibility to ACT through private and retail sectors, most ACT drugs are only prescribed by health workers who work within the formal government and mission sector. Ensuring that the new ACT is prescribed according to national guidelines with the provision of adequate dispensing and counseling is critical to maximize patients' adherence ,2 and trNon-adherence to malaria case management guidelines has been commonly reported in the past under ACT and non-ACT policies -7. Complharaka upesi' the Kiswahili meaning of prompt action; and b) improving the performance of health workers at facilities, focusing on reinforcement of AL treatment for febrile children and adherence to recommended dispensing and counseling tasks in accordance with national guidelines. This paper considers the second component of this strategy.A combined community and health facility intervention was launched with the aim of addressing the gaps in access to prompt and effective treatment with artemether-lumefantrine (AL), Kenya's recommended first-line treatment for uncomplicated malaria. The intervention was developed and implemented by Population Services International (PSI/Kenya) and the Division of Malaria Control (DOMC), Ministry of Public Health and Sanitation and funded by Pfizer under their Mobilize against Malaria Initiative . The focEnhanced in-service health worker case-management training sessions were conducted in February 2009 and August 2009. Cadres of health staff involved in the training included: nurses, laboratory technicians, public health officers, clinical officers and doctors. The training sessions took place over three days in the form of workshops sited outside of the health workers' working stations. It consisted of several units including: the epidemiology of malaria in Kenya, diagnosis-treatment-counseling-drug dispensing for uncomplicated malaria, diagnosis and treatment of severe malaria, prevention and management of malaria in pregnancy, basic techniques related to stock management of AL, principles of monitoring and evaluation and practical sessions on diagnostic procedures using blood slides and rapid diagnostic tests [RDTs]. One day of the training was devoted to the management of uncomplicated malaria. No follow up of the training was provided.The training was facilitated by the national DOMC trainers of trainers (TOTs) who are specialists in the different training units and have previously undertaken case-management training of health workers. The training format and teaching modalities included lectures, group discussions, case scenarios, role plays and question and answers quizzes. Between October and November 2008, a two-day pilot of the training with health workers and national trainers was undertaken in Kisumu East and Kisumu West districts in Nyanza province to establish the duration and feasibility of the intervention training. The feedback given by the health workers from this pilot was that overall duration of the training required more than two days to cover successfully all training units. Following this feedback the DOMC and PSI concluded that three-day training would provide adequate time to cover all training programme according to the curriculum. Furthermore, considering that peripheral health facilities often have only one health worker any training duration beyond three days was deemed unacceptable since it would have resulted in substantial disruption of the service delivery.haraka upesi' intervention - prompt AL treatment for febrile children and health workers' adherence to recommended case management. The manuals included objectives, content and modalities of delivery for each training unit. Alongside the training, the job aids were piloted to assess health workers understanding of the job aids messages. The key recommendation from the pilot, that was included in the final intervention package, was that for ease-of-use health workers requested a booklet encompassing miniature wall charts. Therefore, of relevance for the evaluation of the management of febrile children below 5 years of age, the following training materials were distributed to each health worker who attended the training: 1) revised national guidelines for the diagnosis, treatment and prevention of malaria for health workers; 2) participant's training manual based on revised national guidelines; and 3) five job aids promoting presumptive AL treatment of febrile children and dispensing and counseling tasks for patients with AL prescribed. Of five job aids disseminated, three were in wall chart or poster format, one was a booklet with miniature versions of the wall charts and one was the folder for the training materials with the 'haraka upesi' banner.The training curriculum, facilitators' manual on the diagnosis, treatment and prevention of malaria and job aids were developed by PSI/Kenya in collaboration with the Ministry of Public Health and Sanitation's, Division of Malaria Control (DOMC), case management working group partners and educational specialists. The training materials covered all of the training topics including those where deficiencies in practices were detected in the past and those which were the focus of the 'The target of the training was universal coverage of all front-line health workers providing outpatient services. By the end of August 2009 an estimated 962 health workers had received the enhanced in-service training in 25 separate sessions at different locations across twelve districts within Nyanza province.Bondo district was selected as the evaluation district from the twelve districts located in Nyanza Province. Bondo is on the shores of Lake Victoria where residents experience intense, perennial malaria transmission  and wherThe design adopted for this evaluation was a pre-post intervention comparison of health worker case-management practices for febrile children aged less than five years. The key measured indicators reflected national guidelines, in-service training and job aid recommendations and included the following: 1) proportion of febrile children having AL prescribed; 2) proportion of febrile children having AL prescribed and dispensed; 3) proportion of febrile children with AL prescribed and dispensed who had a) weight measured; b) first dose administered at the facility; c) advice provided on dosing schedule; and d) advice provided on what to do in case of vomiting.Cross-sectional health facility surveys were undertaken at all government health facilities in Bondo district between August and October 2008 (pre-intervention) and November to December 2009 (post-intervention). Data were collected using three main tools: a) health facility audit to assess drug stocks, availability of equipment and training materials; b) health worker structured interview to obtain information on health worker's demographics and exposure to training and guidelines; and c) exit interviews with caregivers of febrile children below five years of age presenting to the outpatient department on the survey day. All health workers attending the febrile children on the survey day were interviewed. All caregivers of children under five years with fever or history of fever who were not referred for hospitalization were interviewed on leaving the health facility. Data were collected at each facility over two consecutive survey days by seven survey teams, each composed of two nurses. Informed written consent was obtained from all interviewed health workers and caretakers of sick children. Training and concordance testing were undertaken over a five day period immediately prior to data collection. Concordance testing for exit and health worker interviews was assessed through role playing at health facilities not included in the survey until 96% concordance was achieved.Data were doubled entered by four independent data entry clerks using Microsoft Access customized software . Data files were subjected to a verification programme to check for data entry errors and these were corrected by referring to the original questionnaires. All analyses were performed using STATA, version 11 .The primary pre- and post-intervention analysis included febrile children weighing five kilograms and above presenting for an initial visit at health facilities where AL was in stock on the survey day. The children with negative test result were excluded from the analyses. This analysis reflected effectiveness of the intervention regardless of the exposure to the intervention. However, due to incomplete coverage of health workers with all intervention components, two post-intervention analyses were additionally performed. The first analysis was restricted to febrile children seen by health workers who received the enhanced training, and the second analysis included children seen by health workers who in addition to the enhanced training had either access to guidelines or the participants' training manual, and at least one of the five job aids promoting the same case management recommendations in different formats. The latter analysis was defined as the minimum intervention package. Since none of the children were seen by health workers who were exposed to all intervention components the analysis reflecting the full intervention package could not be performed. Data are presented as percentages, with corresponding 95% confidence intervals (CIs) adjusted for clustering at health facility level.Ethical approval was obtained from the KEMRI/National ethical review committee (KEMRI SSC number 1375) and the London School of Hygiene and Tropical Medicine Ethics Committee During the pre-intervention survey, conducted between August and October 2008, 33 health facilities, 48 health workers and 599 consultations for febrile children were assessed. After the training sessions, in November 2009, 36 health workers working at the same health facilities and 541 consultations were assessed. No health worker or caretakers refused to participate in the study. The majority of assessed facilities were dispensaries (75.8%) followed by health centres (18.2%) and hospitals (6.1%). During both, pre-intervention and post-intervention surveys, nurses represented the majority of health workers (87.5% and 72.2% respectively), followed by clinical officers (12.5% and 17.0% respectively). Of the 33 health facilities, only one facility during the pre-intervention survey did not have any AL pack in stock and observations from this facility were excluded from the patient level analysis. Paediatric AL formulations were widely available during both surveys: between 94-100% of facilities stocked AL six and AL twelve pack formulations. Malaria diagnostic services were provided at 24.2% and 27.3% of facilities during the pre- and post-intervention survey, respectively.haraka upesi' banner. None of the health workers had received all components of the intervention and only 50% (18/36) were exposed to the minimum intervention package as defined above.Health workers and health facility coverage with the components of the intervention as assessed during the post-intervention survey are presented in Table Of 599 consultations assessed for febrile children during the pre-intervention survey in 2008, 386 children met the inclusion criteria for the case management analysis. The remaining 213 children were excluded from the analysis because they attended the facility for a follow-up visit (156), weighed less than five kilograms (4), had negative routine malaria slide result (14) or were seen at a facility without any AL in stock (39). During the post-intervention survey in 2009, of 541 consultations assessed 390 children met the inclusion criteria while the remaining 151 children were follow up visit (118), weighed less than five kilograms (23) or had negative routine malaria slide result (23). Finally, the restricted post-intervention analyses included 249 children seen by health workers who had received the enhanced in-service training and 174 seen by health workers who received the minimum intervention package.Case management practices for febrile children before and after the intervention are presented in Table During both surveys, nearly all (98.5% and 99.1% respectively) caretakers of children for whom AL was dispensed, reported that the health workers provided advice on completion of the AL treatment regimen. Small post-intervention increases were observed in comparison to the pre-intervention survey in the proportion of children having AL dispensed who were weighed, who were administered the first AL dose at the health facility and who were advised what to do in case of vomiting; however none of these differences were statistically significant Table . During Despite improvements in some indicators post intervention, the analyses restricted to children seen by health workers with enhanced in-service training and those seen by health workers with access to enhanced training, guidelines and job aids, showed no statistically significant differences in any indicator compared to the pre-intervention analyses Table .The MAM initiative was launched to improve coverage of trained health workers and case management practices in Nyanza Province, an area harbouring the highest malaria endemicity in Kenya, but characterized with low access to ACT  and repoharaka upesi' notably AL prescription for febrile children increased by 10% post-intervention (Table Where parts of the intervention had reached health workers and their facilities, there were modest improvements in indicators that were linked to the intervention banner 'on Table . This coon Table . AdminisMalaria case-management training was the key component of the intervention evaluated in Bondo. The training programme in the study area was based on fairly well developed curriculum and implemented to the best of our knowledge according to the planned format facilitated by experienced trainers from the national malaria control programme. Furthermore, indicators measured in this evaluation were relatively basic and change in practices following training could have been expected. Yet, when the analysis was restricted to health workers exposed to the intervention training there was no significant change in any of the outcome indicators. Several health worker performance studies have indicated that single one-off interventions without follow up often do not translate into the desired outcomes , notablyThe intervention in Nyanza province made a special effort to combine training with provision of supporting job aids and wall charts. However, even when the analysis was restricted to health workers who had received the intervention training and had access to some of the support materials no additional improvements were observed compared to the pre-intervention period. The one important case-management intervention not included within the intervention package was on-going supervision and follow-up after the in-service training. This additional component may have enhanced single training or improved use of job aids, wall charts and guidelines.There are several limitations of this study. Not all prescribing, dispensing and counseling tasks recommended in the national malaria treatment guidelines were evaluated in the present report because questions were unfortunately asked differently during pre and post intervention surveys. These include: dosing and advice on second dose after eight hours, administration after the meal and emphasis on full course completion. This limited our ability to examine the range of possible behavioural changes that were contingent on training. However, those reported reflect important features of good clinical practice and are likely to represent reasonable indicators of training and job aide impact. The study was undertaken in only one district of twelve where the intervention was implemented. However those implementing the in-service training and distribution of job aids did know which district would be evaluated and made additional efforts to increase intervention coverage in this district relative to other districts.Even enhanced, well funded in-service training and job aid development packages aimed at improving the way febrile patients are managed in health facilities does not reach all those who provide care within government health sectors. Improving intervention coverage must be a pre-requisite to any significant changes in the proportions of patients managed effectively with new ACT medicines. However, where these interventions reached health workers and facilities only modest improvements were observed in the prescription and dispensing of AL and counseling provided to caretakers. Despite limitations of the present study we would urge caution in future large scale investments in in-service training and provision of job aids without other features of support, such as supervision, to enhance the value of training and printed materials. Finally, it is also important that the quality of the training is optimized to ensure effective content delivery in achieving desired case-management practices.Pfizer Inc. provided funding for this study but were not involved in study design, data collection, and interpretation of the results or decisions to submit the final manuscript. DZ and RWS have participated in the Novartis National Malaria Control Programme Best Practice Workshops in Africa and have received consultative fees for their participation.BW was responsible for study design, data cleaning, analysis, interpretation and writing of the final manuscript. DZ provided design and analysis support. JB provided design and statistical support. JW and CJ provided guidance on survey design and interpretation of the results. RWS was responsible for overall intellectual direction, study interpretation and contributed to the preparation of the final manuscript. All authors have read and approved the final manuscript.


Therapeutic vaccination against disseminated prostate cancer (PCa) is partially effective in some PCa patients. We hypothesized that the efficacy of treatment will be enhanced by individualized vaccination regimens tailored by simple mathematical models.R2 = 0.972). The model could not account for the inconsistent changes in PSA levels in 3 of the 15 responsive patients at the end of treatment. Each validated personalized model was simulated under many hypothetical immunotherapy protocols to suggest alternative vaccination regimens. Personalized regimens predicted to enhance the effects of therapy differed among the patients.We developed a general mathematical model encompassing the basic interactions of a vaccine, immune system and PCa cells, and validated it by the results of a clinical trial testing an allogeneic PCa whole-cell vaccine. For model validation in the absence of any other pertinent marker, we used the clinically measured changes in prostate-specific antigen (PSA) levels as a correlate of tumor burden. Up to 26 PSA levels measured per patient were divided into each patient's training set and his validation set. The training set, used for model personalization, contained the patient's initial sequence of PSA levels; the validation set contained his subsequent PSA data points. Personalized models were simulated to predict changes in tumor burden and PSA levels and predictions were compared to the validation set. The model accurately predicted PSA levels over the entire measured period in 12 of the 15 vaccination-responsive patients (the coefficient of determination between the predicted and observed PSA values was Using a few initial measurements, we constructed robust patient-specific models of PCa immunotherapy, which were retrospectively validated by clinical trial results. Our results emphasize the potential value and feasibility of individualized model-suggested immunotherapy protocols. Prostate cancer (PCa) is the second most common malignancy in men ex vivo activated antigen presenting cells ex vivo processed autologous antigen presenting cells combined with prostatic acid phosphatase PCa immunotherapy has begun to yield encouraging clinical effects, though not a definitive cure Mathematical modeling has been a valuable tool in describing, quantifying and predicting the behavior of complex systems. In particular, mathematical models have played an important role in providing non-intuitive insights into tumor growth and progression in silico treatment modification (adjustment of the vaccine dose or administration schedule), we have also defined the individualized treatment protocols to be tested for more effective clinical outcomes.Differences in individual responses to PCa vaccination First, we constructed a general mathematical model of the immune response in PCa patients receiving vaccination therapy . The modR2 = 0.972).Next, we tested the ability of the model to describe the PSA course in the patients who initially responded to therapy see . We usedThe initial stepwise increase of the size of the training set improved the prediction accuracy for all patients, but at some point the improvement became negligible . PredictThree patients displayed unusual and inexplicably abrupt changes in PSA levels, or inconsistent PSA trends, towards the end of treatment; the model could not account for this behavior . Howeveri.e., to the change in dose size or administration schedule. We hypothesized that by the use of personalized models, we can suggest treatment modifications to stabilize PSA levels. Consequently, we simulated treatment protocols modified either by an increased vaccine dose or decreased intervals between vaccinations in the individually parameterized models, for the nine patients who completed treatment.Having validated the model, we could test the response to modification of treatment, i.e., 21 days compared to the standard 28 days) was predicted to suffice, while other patients required more frequent vaccination with the standard dose (2.4×107 cells). Patient 20, however, required either the largest among the considered vaccine doses (a 30-fold increase), or daily administration of the standard dose.We found that for each patient, the putative stabilization of PSA levels required different modifications of vaccine dose or interval between vaccinations . For exaTo maintain the suggested regimens within clinical constraints, we studied the effects of reducing the interval between vaccinations to 14 or 21 days, or of doubling or tripling the standard dose, and compared the predicted outcomes to the actually measured effects of the administered standard treatment. Co-evolution of tumors and immunity is complex and not fully understood. The process includes time-dependent interactions that shape both immunity and the tumor, and determine which will prevail. For situations when immunity prevails, we have little information about the factors that determine the outcome; when tumors prevail, we observe only their manifest phase although tumor effects on immunity may have started even at the precancerous phase We have developed a simple mathematical model, individualized it by fitting to PSA values recorded in individual patients before and during vaccination therapy, validated the model by subsequent individual PSA values, and used the results to predict the immediate response of PSA levels to modifications of vaccine dose or administration schedule. The model was remarkably successful in predicting PSA level changes in 12 out of 15 analyzed treatment-responsive patients. The manifested robustness of the fits was not compromised by the model simplicity, encompassing no more than four patient-specific parameters, with other parameters being derived from preclinical and clinical information collected from disparate published sources. Apparently, a generic representation of the interplay of immune activation and suppression suffices to describe clinical responses without the need to consider all individual mechanistic elements participating in immune regulation separately.Derivation of patient-specific parameters from training sets and the successful validation of individualized models ascertain the predictive power of our model. For three patients, validation was unsuccessful because of the non-monotonous behavior of PSA levels at the end of vaccination course. Of note, deviation of the course of PSA levels from monotony could indicate unpredicted significant changes in the dynamic relationships between immunity and the tumor. It is tempting to speculate that this took place because vaccination broke down tumor progression.As responses to vaccination differed among the patients significantly, a major motive for this study was to ascertain the feasibility of improving individualized treatment. Having validated the individually parameterized models of the effect of vaccination, we tested whether the model can suggest modifications in vaccine dose or administration schedule needed to stabilize PSA levels. The suggested changes also differed among patients, a finding emphasizing the potential value of testing individualized vaccination protocols in clinical trials. It is noteworthy that modifications of either the size of vaccine dose or the interval between doses could result in comparable tumor responses, allowing considerable flexibility in the choice of clinically and logistically most feasible protocols. Thus, the benefit of the method is that it could identify the patients who will not respond to therapy and enhance treatment efficacy for those who will.et al., in preparation).To obtain an accurate predictive model, we found that each patient required a different number of PSA measurements to complete his personal training set. This raises the question whether one can determine, during treatment, the number of measurements sufficient for evaluation of personal model parameters, so that the model can be used for individualized modification of the subsequent course of treatment. Hence, we recently developed an algorithm by which one can determine the number of accumulated measurements that suffices for completion of the training set . The initial three doses were spaced two weeks apart, followed by 11 doses spaced four weeks apart, so that the treatment period lasted approximately one year. Circulating PSA was measured prior to treatment, at vaccine injections, and sporadically between injections and after completion of treatment. The number of pre-treatment, in-treatment and post-treatment PSA measurements varied among the patients.We collected the de-identified data from a Phase 2 clinical trial of an To classify the patients by response to treatment, we evaluated individual PSA velocity (linear change of logarithm of PSA levels) before therapy and during the first four cycles of therapy. In 15 patients vaccination reduced the PSA velocity, while in others it did not. We studied the PSA data solely from the responding patients.We modeled the basic interactions of PCa and immunity by a system of seven ordinary differential equations accounting for interactions of the vaccine, the immune system and cancer cells within the skin, the lymph nodes and other tissues (disseminated tumor cells). The model is based on the assumption that the vaccine stimulates cancer-specific immunity, but also that normal regulatory mechanisms and the tumor suppress this immunity. The model is fully detailed in The model and curve-fitting algorithms were implemented on a MATLAB® programming platform . We solved the model equations by the numerical ordinary differential equation solvers available in MATLAB. Fitting of the model to the data was performed using constrained optimization procedures.in vitro and in vivo data and were assumed to be similar for all patients (r), CTL killing activity (pa), as well as A and B, the two parameters correlating tumor burden and PSA levels, were considered patient-specific, based on the observations that tumor growth rates, PSA secretion rates and intensity of vaccine-induced immune response vary significantly among individuals. To avoid over-parametrization, we attributed the intensity of immune response to the single parameter pa.To individualize the model, we evaluated selected parameters for individual patients. Most model parameters were evaluated from published patients . Tumor gTo estimate patient-specific parameters in individual models, we fitted the model by the least-squares method to the pertinent “training set” that included all pre-treatment and several initial in-treatment PSA values for each patient; the number of training data points could differ among the patients. Next, we used the results to simulate the subsequent course of PSA change and compared the simulation with PSA measurements recorded following measurements in the training set . If prediction accuracy was low, the size of the training set was iteratively increased by a subsequent PSA measurement, subtracting the point from the validation set.R2, between the predicted and observed PSA values.To predict PSA dynamics beyond the training set, we simulated each individualized model under the personal vaccination schedule . For each patient, predictions were compared with the clinically observed PSA levels in the validation set. Goodness-of-fit was evaluated pooling together all the validation data points from all the patients. To compare predictions with measurements, we calculated the coefficient of determination, We probed whether intensifying treatment could improve vaccine efficacy in individual patients. Hence, based on the validated individual models for the nine patients who completed treatment, we simulated the effects of many intensified vaccination protocols for each patient. Intensification included a graded 10 percent increase above the standard vaccine dose or graded one-day reduction of administration interval relative to standard schedule. We singled out individual vaccine administration schedules that should lead to stabilization of PSA levels at the end of treatment, at concentrations not more than 10 percent above the pre-treatment level. For each patient, minimal increase in vaccine dose and minimal reduction in dosing interval that meet the above PSA stabilization criterion are reported in Material S1This file includes (a) the mathematical model of prostate cancer therapeutic vaccination; (b) parameter estimation.(DOCX)Click here for additional data file.


Introduction. Cesarean section is an independent risk factor for peripartum hysterectomy. As a method of delivery, cesarean section may interfere with a number of molecular changes that occur at the maternal-fetal interface during the course of labor. Methods. The level of CD3, CD56, CD25, and CD69 antigen immunoreactivity was assessed by immunohistochemistry in 26 decidual tissue samples. The tissue samples were obtained from 18 women who underwent cesarean sections at term and from 8 women who underwent cesarean hysterectomies. Results. An increase in the activity and infiltration of immune cells in the decidua sampled during the spontaneous beginning of labor was observed. The further progression of labor was accompanied by a decrease in the number and activity of immune cells. The number of CD56+ and CD3+cells in the decidua was statistically significantly lower in patients who had undergone cesarean hysterectomies than in those who had had cesarean sections at term. Conclusion. Abnormal immune response during labor may increase the risk for peripartum hysterectomy. Peripartum hysterectomy, a life-savingprocedure, is performed more often on women who have undergone cesarean sections(cesarean hysterectomy) than on those who experience normal labor (postpartum-hysterectomy). Most stElective cesarean section (cesareansection without labor) is performed before the physiological alteration of immunetolerance associated with pregnancy and the beginning of spontaneous labor hasoccurred. The level of immune tolerance during newborn expulsion at term incases of elective cesarean section appears to differ from the level found in casesof spontaneous vaginal delivery at term. Molecular changes at the maternal-fetalinterface are the pivotal mechanism in the alteration of the immune tolerancephenomenon. These changes lead to an increase in the activity of the maternaland fetal immunological systems –14. DuriIn the present study we have aimedto assess the number and activity of immune cells in the decidua using samplescollected from patients who have had term cesarean sections and from patientswho have had postcesarean hysterectomies.The decidual tissue samples evaluated in our study were obtained from 26 pregnantwomen who underwent cesarean sections. 18of the tissue samples were obtained during cesarean sections at term, and 8from cesarean hysterectomy specimens. Those patients undergoing cesareansection at term (18 cases) were selected from 650 women undergoing cesareansections in the Department of Gynecology, Obstetrics and Oncology, JagiellonianUniversity, Krakow, Poland, in 2005. Patients with multiple pregnancies orexisting complications of pregnancy, such as preterm deliveries, hypertension,diabetes mellitus, and cases of fetal demise, were excluded from the presentstudy. For the purpose of this study, thehistories of patients who had undergone emergency cesarean hysterectomiesbetween 1999 and 2004 were analyzed and 8 were selected. Women on whomperipartum hysterectomies had been performed due to uterine cancer duringpregnancy were also excluded from our analysis. The tissue samples derived fromthe women undergoing cesarean section at term and obtained during surgicalprocedures were thereafter immediately fixed in 10% buffered formaldehydesolution and sent to the Pathomorphology Department, Jagiellonian University. The decidual tissue samples from the patients havingundergone emergency hysterectomies were obtained from the archive files of theDepartment of Pathomorphology of the Jagiellonian University. The experiencedpathomorphologist  evaluated the routinely stained(hematoxylin and eosin) slides prepared from paraffin-embedded tissue materialand selected the material adequate for further analysis. Chosen paraffin blockswere cut and used for immunohistochemistry.The patient';s consent was obtained ineach case.Immunohistochemical analysis wasperformed in the Pathomorphology Department of the JagiellonianUniversity. Four-micrometerslides from each case, encompassing the decidua, prepared routinely forimmunohistochemistry, were stained to visualize the CD3, CD69, CD25,CD56-positive cells (lymphocytes).In all instances,immunohistochemistry was performed by applying theEnvision method using Dako Autostainer. The following antibodies were applied: CD56  in dilution The distribution of variables in the groups of women studied checked with the use of the Shapiro-Wilk test showedthat all of the women were different from normal. Therefore, non-parametrictesting was employed. The differences between the groups were determined by theKruskal-Wallis analysis of variance (ANOVA) test. The Mann-Whitney U test wasthen used as applicable.Since cesarean section is performed at different times during pregnancy, it seems important to compare theparameters characterizing the course of pregnancy and labor in the respective groupsof patients studied see .When compared, the basic differencebetween the groups was related to the parity of the pregnant women studied. Multiparityis an independent risk factor for emergency hysterectomy  that couCD3 positive cells were identifiedin all the decidual tissue samples derived from patients who had undergone cesarean sections at term and in 25% of the decidual tissue samples derived frompatients having had cesarean peripartum hysterectomies. CD56 positive cellswere observed in 60% of the decidual tissue samples derived from those patientshaving had cesarean sections at term and in 13% of decidua tissue samples derivedfrom patients having had cesarean peripartum hysterectomies. CD69 antigenimmunoreactivity was observed in 40% of the tissue samples derived from those havingundergone cesarean sections at term and in 38% of the tissue samples derivedfrom those having had cesarean peripartum hysterectomies. CD25 antigenimmunoreactivity was weak in the samples derived from the group who hadundergone cesarean sections at term; it was not detected in the samples fromthe group who had had cesarean peripartum hysterectomies see .The changes in the number and activity of immune cells were identified in the decidua and correlated with theprogression of labor. The study group of patients who underwent cesareans atterm was divided into three subgroups according to the degree of uterinecervical ripening and to the presence of uterine contractions during the surgical procedure. Group A consisted of patients not experiencing uterinecontractions and with a closed uterine cervical ostium ; group B consisted of patients with irregularuterine contractions or the beginning of regular uterine contractions andcervical dilation between 1 and 3 cm ; group C consisted of patientswith regular uterine contractions and cervical dilation of more than 3 cm . The comparison of the number and activity of immune cellsbetween subgroups is presented in We found a statisticallysignificantly higher number of CD3 positive cells and a higher CD69 antigenimmunoreactivity level in the decidua from those on whom cesarean sections wereperformed after the spontaneous beginning of labor (B) in comparison to whatwas found in the decidua from those on whom cesarean sections were performedwithout labor (A), and from those on whom cesarean sections were performedduring advanced labor (C) , when taking intoconsideration such factors as the progression of labor during surgery and thegestational age both in the group of patients with cesarean peripartumhysterectomies and in the group with the cesarean sections at term, did notreveal any significant differences. In the group of patients having had cesareanperipartum hysterectomies, significantly higher parity was observed. Also, theaverage age of these women was significantly higher . Thenumber and activity of immune cells in the decidua of the group of cesarean peripartumhysterectomies and of the control group were compared. The results obtained arepresented in An increase in the activity andinfiltration of immune cells in the decidua sampled during the spontaneousbeginning of labor was observed.Moreover, the further progression of labor was accompanied by a decreasein the number and activity of immune cells. The number of CD56 positive and CD3positive cells in the decidua was statistically significantly lower in thesamples from those having had cesarean hysterectomies than in the samples ofthose having had cesarean sections at term.Risk factors for emergencyperipartum hysterectomy include cesarean delivery, prior cesarean, placentapraevia, placenta acreta, and grand multiparity , 7, 27. Abnormal placentation has recentlybeen demonstrated to be a common indication for cesarean section and has beenfound possibly to lead to peripartum hysterectomy. A previous cesareanincreases the risk of abnormal placentation , 31. TheSindram-Trujillo et al. has observed that the course ofspontaneous vaginal delivery is accompanied by an increase in the number ofimmune cells as well as alterations in their distribution (NK CD56+CD16+ cellsand T cell subpopulation) , 18. BulAlthough the presence of uterineleiomyomas was found to increase the risk of peripartum hemorrhages ,SheinerWomen with grand multiparity havebeen identified as more predisposed to postpartum hemorrhage , 42, andThe present study has had certain limitations. First,the small number of patients involved does not allow us to posit that abnormalimmune cell activity is a risk factor for emergency hysterectomy. We can only suggest,on the basis of trends, that a probable association exists between an immuneresponse disturbance during labor and the need for emergency hysterectomy. Thisprobable correlation will have to be explored in more detail in furtherinvestigations.Abnormal immune response duringlabor may increase the risk for peripartum hysterectomy.