In addition, MDA levels were significantly decreased, and functional outcomes of the modified Rankin Scale (mRS), functional ability scale of the WMFT, and Stroke Impact Scale  were improved after the rehabilitation. This pilot study emphasizes the relationship among biomarkers, sleep, and functional outcomes after stroke rehabilitation. Oxidative stress markers may be useful predictors of functional outcomes in subacute stroke survivors.This study examined the association of sleep with inflammation and oxidative stress biomarkers, and with functional outcomes, after stroke rehabilitation. The rehabilitation effects on biomarkers and functional outcomes were also evaluated. Twenty subacute stroke survivors received 4 weeks of upper limb rehabilitation. Baseline inflammatory  and oxidative stress biomarkers  were assessed, as were sleep outcomes. Positive correlations were observed between baseline level of sICAM-1 and number of awakenings at post-treatment (ρ = 0.51,  Over the last two decades, the relationship between stroke and sleep disorders has been of growing interest to researchers and practitioners3. Sleep disorders are found in up to 78% of stroke survivors4. These sleep disorders, including sleep apnea, insomnia, hypersomnia, and parasomnia, interfere with normal physical, mental, social, and emotional functioning5. Sleep is associated with a patient’s ability to achieve the full potential for stroke recovery6; therefore, untreated sleep disorders may diminish rehabilitation efforts and result in poor functional prognosis.Stroke, a leading cause of long-term disability, produces immense health and economic burdens globally7. Specifically, Kaneko et al. found that stroke survivors with sleep apnea had lower functional ability, as measured by the Functional Independence Measure (FIM), than did those without sleep apnea7. A more recent study also suggests that sleep disturbances in stroke survivors can detrimentally affect functional status, such as scores on the modified Barthel Index and grip strength8.Evidence suggests that sleep disorders are an independent factor that predicts functional disability and longer hospitalization following stroke10. A meta-analysis of 51 studies found that, compared to control subjects, patients with obstructive sleep apnea (OSA) have higher levels of inflammatory markers such as C-Reactive protein (CRP), tumor necrosis factor alpha (TNF-α), and intercellular adhesion molecule (ICAM)10. These levels are positively correlated to the level of OSA severity10. On the other hand, previous studies have suggested that elevated inflammatory and oxidative markers may be associated with the pathogenesis of cardiovascular and cerebrovascular diseases12. The underlying mechanisms may involve endothelial dysfunction, systemic inflammation, oxidative stress, and metabolic dysregulation resulting from hypoxemia, reoxygenation, and intrathoracic pressure13. Increased production of pro-inflammatory cytokines has been observed in stroke survivors and may be associated with poor stroke outcomes. Specifically, elevated cytokines, such as interleukin-6, presented in the early stroke period are closely associated with worse stroke outcomes assessed by the Barthel Index and modified Rankin scale14. Another study showed that reduced levels of inflammation are negatively correlated with the Barthel Index, suggesting that the decrease of inflammatory markers was beneficial for stroke recovery15. In addition, our previous work demonstrated that the changes of patients in oxidative stress level, measured by 8-hydroxy-2′-deoxyguanosine, were significantly correlated with the upper limb motor function, muscle power, and perception of pain16. Disturbed sleep is associated with an increased level of inflammation10. There is also evidence suggesting that systemic inflammation results in a disruption of sleep18. Chronic inflammation may reduce sleep continuity and depth and induce sleep disorder, such as insomnia, suggesting a relationship between disturbed sleep and inflammation19. Therefore, examining the associations among level of inflammation, level of oxidative stress, and sleep outcomes is particularly relevant after stroke, given that changes in any one of these factors are proposed to be related to functional prognosis and the effectiveness of rehabilitation treatment post-stroke21.The existing literature describing the relationships between sleep disorders and biomarkers, such as inflammatory and oxidative stress pathways, is extensive22 demonstrated that the severity of OSA is positively associated with the total antioxidant capacity (TAC) and CRP in ischemic stroke subjects. Specifically, the results of the study showed that the levels of CRP and TAC were positively correlated with the oxyhemoglobin desaturation index; the TAC levels were negatively correlated with mean arterial oxygen saturation in stroke survivors with severe OSA22. However, what has been lacking to date is examination of the relationships among the biomarkers, sleep, and functional and health-related outcomes after stroke rehabilitation.Recent work by Chen and colleagues24 and task-oriented training26, may improve functional outcomes in stroke survivors. Research supports the efficacy of upper limb robot-assisted therapy for improving motor and functional outcomes in stroke patients24. Moreover, task-oriented training with stroke survivors improves functional outcomes and overall health-related quality of life26. A better understanding of the changes of the inflammation, oxidative stress, and sleep indicators following stroke rehabilitation is crucial not only for predicting treatment-related outcomes but also for monitoring clinical response and adverse effects.Stroke rehabilitation programs, such as robot-assisted therapyThe pilot study aimed to: (1) characterize the relationships among baseline biomarkers , sleep, and functional and health-related outcomes at post-treatment in subacute stroke patients, and (2) investigate the changes in levels of biomarkers, sleep, and functional and health-related outcomes after 4 weeks of rehabilitation intervention. We hypothesized that (1) higher levels of inflammation and oxidative stress at baseline would be associated with poor sleep quality and functional and health-related outcomes post-treatment, and (2) the levels of inflammation and oxidative stress would decrease, and the functional and health-related outcomes would improve, after the intervention.The study enrolled a total of 20 patients with subacute stroke. Table p < 0.05) (Table p’s > 0.05). In addition, there was a significant positive correlation between pre-MDA and post-WMFT-time , and an almost significant correlation between pre-MDA and post-mRS . However, pre-GPx was not correlated with any post-sleep or post-functional outcomes .The Spearman rank correlation coefficient revealed that the pre-sICAM-1 was positively correlated with the post-number of awakenings  Table , indicatp < 0.05), indicating lower oxidant levels of the patients after treatment. No significant pre- or post-treatment changes were noted for the PSQI total score, sleep percentage, and number of awakenings . However, there was a trend of improvement for biomarkers and sleep outcomes from pre-treatment to post-treatment. Furthermore, after treatment, patients improved significantly on most of the functional outcomes. Specifically, significant changes from pre- to post-treatment were found on the mRS , WMFT-FAS , SIS-total , and SIS-physical function .The pre- and post-treatment levels of biomarkers, sleep outcomes, and functional outcomes are reported in Table This study aimed to characterize the relationships of baseline inflammation and oxidative stress biomarkers to sleep and to functional and health-related outcomes after 4 weeks of rehabilitation intervention in subacute stroke patients. First of all, a positive association was observed between the baseline level of inflammation and the number of awakenings at post-treatment. We also found a positive correlation between the baseline oxidant level and post-treatment upper limb motor function . A similar trend was found for baseline oxidant level and post-treatment level of stroke disability . In addition, significant effects were decreased oxidant levels and improved motor function and health-related outcomes after the rehabilitation. Although not statistically significant, there was a trend suggesting promising and beneficial rehabilitation intervention effects on the biomarkers and sleep outcomes.28. In this study, increases in the baseline level of the inflammatory sICAM-1 were found to be associated with higher numbers of awakenings at post-treatment. This result may support the hypothesis that high inflammation at baseline is related to poor sleep quality via the worsening of comorbidities associated with inflammation. This finding is partly consistent with previous findings that higher levels of inflammation reduce non-rapid-eye-movement sleep due to diminished sleep-promoting effects, and thereby increase wakefulness29. Many studies have found that a sleep deficiency induces systematic inflammation, which may be associated with metabolic changes and immunological deregulation31. This study, on the other hand, suggests a reciprocal regulation between inflammatory response and sleep loss. Our finding of a link between higher baseline inflammation and poor sleep quality echoes previous work suggests that pro-inflammatory cytokines induce sickness symptoms such as sleepiness, fatigue, and poor cognition32.The present study expands on previous findings by showing the association between inflammation markers and objectively measured sleep outcomes in stroke survivors. Elevated levels of inflammation have been found to predict an increased risk for adverse health outcomes such as myocardial infarction and diabetes in healthy populationset al. investigated serum MDA levels in patients with acute stroke and found a negative correlation with clinical outcomes measured by the Canadian Neurological Scale33. Although elevated oxidative stress has been reported in stroke patients, few studies have described the correlation between this biomarker and functional outcomes after stroke rehabilitation. Hsieh et al. demonstrated that urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage, is a valid predictor of functional outcomes in chronic stroke survivors. Specifically, they showed that the baseline 8-OHdG content was significantly correlated with post-treatment functional outcomes such as the Fugl-Meyer Assessment (FMA) and Medical Research Council scale (MRC)16. In the present study, we measured the MDA level as a marker of oxidative stress and observed that baseline serum MDA levels were significantly correlated with post-treatment motor function measured by the WMFT-time. This relationship may not be attributed to the influence of treatment because WMFT-time scores did not change significantly from pre- to post-treatment. A higher level of baseline MDA content was significantly associated with less favorable functional outcomes  after rehabilitation. This finding indicated that the oxidative stress markers might be valid in predicting functional outcomes in stroke survivors. Our results also found a similar trend for baseline oxidant level and post-treatment level of disability measured by the mRS. However, we did not find any significant correlations between antioxidants  and post-treatment functional outcomes.Oxidative stress has been found to play an important role in acute ischemic stroke pathogenesis. Ozkul 35. Regular rehabilitative exercise including functional and movement training has been shown to promote upregulation of antioxidant capacity and attenuate oxidative stress in stroke patients36. Although our results showed a trend of increasing antioxidant capacity, the significantly decreased oxidative stress may suggest the possibility of the reduced occurrence of oxidative damage, which may partially contribute to improving functional and health-related outcomes.Another key finding is that the 4 weeks of rehabilitation produced significant effects on oxidative stress levels, indicating that the intervention, did not produce oxidative stress in the stroke survivors, but rather alleviated it. In addition, some of the functional and health-related outcomes were significantly improved after the intervention as compared with the baseline. Rehabilitative intervention such as robot-assisted therapy is considered to be an intervention approach for restoring upper limb function and has been examined in large clinical trials in stroke patients8 and cognitive recovery37. Although not designed for this purpose, we noted that stroke survivors with good sleep quality at baseline had lower oxidant levels and greater health-related outcomes  after the treatment. All participants had improved motor function and physical function, but only the bad sleepers showed decreased level of disability following the treatment. Individuals with sleep disturbance had lower level of disability at baseline and may have more room for improvement. Of note, a limitation of the study was the relatively small sample size, especially the good sleepers. Follow-up study using a larger sample for study is needed to validate the findings. In addition, we used a subjective measure, the PSQI, to quantify sleep quality and differentiated between good and bad sleepers. Further research employing an objective sleep monitoring system, such as actigraphy, to record the quality, quantity, and rhythm of sleep may provide better understanding of the relationship of sleep outcomes to functional recovery after stroke. Also, future work could include other potential inflammatory and oxidative stress biomarkers, such as CRP or TAC, to establish a strong correlation between biomarkers of oxidative stress and functional outcomes. Finally, future work could also examine different rehabilitative approaches that activate the biochemical regulations most likely to promote neuronal plasticity and recovery following stroke.Sleep disorders, which are common in stroke patients, are associated with poorer stroke recovery outcomes and increased cerebrovascular morbidity. It has been shown that, in stroke survivors, better sleep quality is associated with better functional statusPatients with subacute stroke were recruited if they met the following criteria: (1) unilateral stroke with onset of less than 6 months; (2) upper extremity score of the Fugl-Meyer Assessment (FMA) > 10; (3) no excessive spasticity in any joints of the affected arm ; (4) ability to follow study instructions and perform tasks ; (5) no other neurologic or major orthopedic diseases. All participants signed informed consent forms approved by the institutional review boards. Ethical approval was provided by the Institutional Review Board for Human Studies of Chang Gung Memorial Hospital. All methods were performed in accordance with the relevant guidelines and regulations.The stroke rehabilitation programs included robot-assisted therapy and task-oriented approach training. All patients received one 90-minute therapy session each day, 5 days per week, for 4 weeks. The training sessions were delivered by certified occupational therapists, and the daily logs of therapeutic activities were recorded.38. This robotic device delivers forearm pronation/supination and wrist flexion/extension movements. Each movement pattern has 3 modes: (1) passive-passive, (2) active-passive, and (3) active-active. In the passive-passive mode, the robot controls movements of both arms. In the active-passive mode, either the affected or non-affected arm moves the robot handle while the other robot handle moves the non-affected or affected arm at the same movement speed and amplitude. In the active-active mode, both arms perform movements by overcoming an initial isometric resistance. The movement speed, amount of resistance, and range of movement can be adjusted individually. In this study, each participant performed an average of 1,200 to 1,600 movement repetitions for each session.Robot-assisted therapy was carried out using the Bi-Manu-Track device  for bilateral arm symmetrical trainingDuring the task-oriented training, participants practiced functional activities in accordance with their functional levels and individual goals. Examples of the functional activities are as follows: (1) walk to a water fountain, fill a glass with water, and drink; (2) wring out a wet rag and wipe a Table All measurements were obtained at baseline (pre-treatment) and 4 weeks after treatment (post-treatment).39. In addition to the subjective measurement of sleep outcome, each participant wore an accelerometer to detect and record arm movements. The data collected from the accelerometers were used as a proxy for assessment of sleep and wake. The sleep percentage  and the total number of awakenings for 3 consecutive days at baseline and 4 weeks post-treatment  were collected using the MicroMini-Motionlogger activity monitor . The accelerometer was set to record hand movement in 60-sec epochs using the Proportional Integrating Mode (PIM) that captures the amplitude of arm movement. The data were further configured using ACT-Millennium software (AMI) and processed in Action4 software. The accelerometer was worn on the non-affected wrist. The accelerometer was removed during the 90-minute therapy session; therefore, the average total wearing time for each participant was approximately 22 to 22.5 hours per day. While wearing the accelerometer, participants completed daily monitoring logs to document the activities they performed , the time they went to bed, and periods when the accelerometer was removed. The accelerometer daily monitoring log was used to identify any discrepancy between self-reported functional use of the non-affected arm and the accelerometric data.Self-reported sleep disturbances were evaluated using the Pittsburgh Sleep Quality Index (PSQI), which includes 7 components: subjective sleep quality, latency, duration, efficiency, sleep disturbances, use of sleep medication, and daytime somnolence. The component scores are summed to produce a global score (ranging from 0 to 21). Higher scores indicate worse sleep quality. A global PSQI score greater than 5 indicates poor sleepers40. The evaluated oxidative stress indicators included glutathione peroxidase (GPx) and malondialdehyde (MDA). GPx is an antioxidant enzyme that can be used to evaluate the body’s defense mechanism against oxidative stress indicators. MDA is a biomarker for tissue damage and reperfusion41. A higher level of GPx indicates a higher antioxidant level, whereas a higher level of MDA reflects a higher oxidant level. Standard operating procedures consistent with expert consensus recommendations were used to collect, process, and store blood samples42. We performed the measurements using colorimetric enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s instructions for sICAM-1  and MDA . Level of GPx was evaluated using the Glutathione Peroxidase assay kit . The units of measurement are expressed as ng/ml for sICAM-1, nmol/min/ml for GPx, and μM for MDA.We evaluated the level of the soluble intercellular adhesion molecule-1 (sICAM-1) as the pro-inflammatory marker. Elevated concentrations of sICAM-1 are associated with local or systemic inflammation43. The mRS score ranges from 0 to 5, with 0 representing “no symptoms” and 5 representing “severe disability.” A change of one or more points indicates meaningful improvement or decline in functional independence over time44. Its validity and inter-rater reliability are well documented45. The WMFT is a laboratory-based measurement for assessing upper-extremity motor function46. The WMFT comprises 17 tasks with 15 function-based and 2 strength-based tasks. We calculated two different scores: the WMFT-time  and WMFT-FAS . The psychometric properties of the WMFT have been validated in patients with stroke48. The SIS is a stroke-specific, self-report, and health status measure that assesses multidimensional stroke outcomes49. Items are scored on a Likert-type scale of 1 (inability to complete the item) to 5 . We collected data from all domains and generated a global score . The subdomains in physical function  and ratings on overall stroke recovery (SIS-stroke recovery) were also reported.We used the modified Rankin Scale (mRS), Wolf Motor Function Test (WMFT), and Stroke Impact Scale (SIS) to evaluate level of stroke disability, motor function, and health outcomes, respectively. Level of stroke disability related to activity performance and participation was assessed using the mRS. The mRS is a six-point ordinal scale commonly used in stroke patientsp < 0.05.We used the Spearman rank correlation coefficient to quantify the correlations among the pre-treatment biomarkers , sleep, and functional and health-related outcomes at post-treatment. We also performed a Wilcoxon signed rank test to examine the statistical significance of changes from pre-treatment to post-treatment in all variables. Statistical analysis was performed in SPSS version 19.0. The level of significance was set at


Emerging research proposes the imbalance between microvascular supply and metabolic demand as a contributing factor in the pathophysiology of mild traumatic brain injury. Prolonged effects on the dysregulation of neurovascular coupling may explain persistent symptomatic models such as Post-Concussion Syndrome.Increased knowledge of what we refer to as neurovascular uncoupling provides a template for establishing a new concussion treatment standard in the assessment and therapeutic guidance of concussion.TM was used to establish pre-treatment benchmarks and guide neurotherapy. Conventional and functional neurocognitive imaging-directed measures were used to evaluate post-rehabilitative outcomes.The degree and localization of neurovascular uncoupling were statistically contextualized against a normative-based atlas in 270 concussed patients. Functional NeuroCognitive ImagingFunctional neurocognitive imaging was successful in identifying regions of Neurovascular uncoupling unique to each patient’s brain and concussion profile. Longitudinal objective outcome measures demonstrated timely and lasting improvement of neurovascular coupling functioning in a significant majority of patients.We present practice-based evidence supporting the clinical administration of functional neurocognitive imaging with particular efficacy in the neurorehabilitation of concussion. We advocate the reliability of functional neurocognitive imaging in assessing severity and localization of neurovascular uncoupling, and promote its use in the therapeutic guidance and neurorehabilitation of mild traumatic brain injury. We further support the continual exploration of other potential pathophysiological alterations resulting from concussion. ConsCurrent guidelines for treatment of acute mTBI center on limiting physical and cognitive activity while reducing exposure to symptom-aggravating stimuli . For mor1.2Recent scientific investigations on the effects of concussion suggest that disruptions to neuronally-triggered regional cerebral blood flow (rCBF) may account for a significant portion of PCS symptoms , 9. AnytTM (EPIC) treatment, which employs a novel, evidence-based system of atypically aggressive NVC recovery therapies, and advocate its methodologies for developing new, pathologically-based standards of care for post-concussion treatment.Given the key role that NVU is likely to play in PCS, effective treatment should include 1) an objective neuroimaging method for detecting NVU in the brain, and 2) an informed rehabilitation protocol with advanced techniques for restoring normal NVC. We present here data from a large sample of mTBI patients assessing the effectiveness of one specific treatment program, Enhanced Performance in Cognition22.1healthy brain between neural activity and the blood oxygen level dependent (BOLD) signal that is detected by fMRI [unhealthy brains, specifically as found in PCS.Functional magnetic resonance imaging (fMRI) has been used extensively over the last two decades as the dominant research tool for understanding human brain function. fMRI is often described as a method that detects ‘brain activity.’ However, a more accurate description of fMRI would be that it detects efficiency in NVC. Although fMRI does not directly measure neural activation, it has nonetheless proven to be a highly reliable tool in brain function mapping, given the precise coupling that normally exists in the  by fMRI . HoweverGiven the special nature of fMRI with regard to mTBI dysfunction, many high-quality research studies over the last decade have used fMRI to investigate neurocognitive effects of concussion -21, and,2.1.1TM (fNCI) assessment protocol combines the validity of conventional neuropsychologic testing standards with the reliability and objectivity of informational data output provided by fMRI. The testing battery underwent iterative pilot testing to ensure concurrent validity, reliability, objectivity, and suitability for the scanning environment [Our unique Functional NeuroCognitive Imagingironment , 25.Notus NeuroCogsTM, is comprised of six neuropsychologic test adaptations: the functional Matrix Reasoning TestTM (f-MRT), the functional Trail Making Test-BTM (f-TMTB), the functional Picture Naming TestTM (f-PNT), the functional Face Memory TestTM (f-FMT), the functional Verbal Memory TestTM (f-VMT), and the functional Verbal Fluency TestTM (f-VFT). Each of the six tasks includes eight Test Phases presented in alternating fashion with Rest Phases, in which the subject is asked to silently count from 1 to 10. Compliance monitoring is performed at intervals during each task. Operative descriptions are outlined in Table 1 below:The functional task battery, 2.1.2All scanning was performed at the same location using the same standardized administration protocol and functional task battery to limit platform-generated variability.Functional images were acquired with a 1.5-T GE scanner using an EPIBOLD sequence with the critical parameters TR = 2000 ms; TE = 40 ms; Flip Angle = 90. Images were acquired at 23 contiguous axial locations with a slice thickness of 5 mm, 0 mm interslice gap, with a 3.75 x 3.75 mm in-plane resolution and a 64 x 64 matrix of individual sample points, producing a total of 64 x 64 x 23 voxels for entire brain coverage. Preprocessing procedures included acquisition time realignment, using sinc interpolation, followed by motion correction with echo-planar imaging (EPI) distortion unwarping. No head movement exceeded 1 mm translation or 1° rotation displacement. Images were spatially smoothed with an 8-mm FWHM Gaussian kernel. A high-resolution 3D SPGR was coregistered to each individual’s mean functional image in order to perform subject-specific functional region analyses that take into account individual variability in cortical landmark organization, for the purposes individual activation extraction requisite for normative reference atlas construction described below.For each test condition, a time-series analysis of covariance (ANCOVA) implemented in SPM8 was used to test each voxel, for each subject, against the null-hypothesis that changes in BOLD signal in that voxel, over the duration of the experiment, did not significantly correlate with the temporal sequencing of the cognitive task of interest. A boxcar waveform convolved with a synthetic Hemodynamic Response Function (HRF) with a 4-s lag-to-peak was used to model task-related activation. The data were high-passed-filtered in time, using a set of discrete cosine basis functions with a cut-off period of 128 s, and conditioned for temporal autocorrelations using AR1 correction.2.1.3In order to further support the reliability of our testing battery and employ its clinical application, we utilized a group-summary analysis approach, where single-subject NVC is compared against normative datasets.Methods for deriving normative fMRI data follow a two-stage process -26. First-value spikes within peak clusters.The procedure for designating and identifying anatomical region boundaries essentially follows the automated anatomical labeling scheme described by Tzourio-Mazoyer and colleagues . Howevert-value assigned to each voxel) were overlain for identification of region peaks. Regions were then inspected for cluster peaks, following these guidelines: If the maximum value within a region belonged to a cluster with a centroid in an adjacent region , the region was determined to not have a peak. When more than one peak was identified in a region, the locations of the peaks were catalogued and, if consistently found across subjects (>30%), used to motive further functional region boundary divisions for the cognitive task protocol being analyzed. However, only those regions (or subregions) with peak clusters present in at least 70% of control subjects were included in the normative data set for each protocol . Following t-value extraction from each subject, means and standard deviations were computed for each region and used to derive normalized z-scores. For each of the six protocols, 8-12 regions met the above requirements for inclusion in the database, for a total of 57 regions. The distributions of t-values in each anatomical region were assessed for normality prior to z-score transformation, on the basis of 59 independent control subjects. Normative reference demographics are outlined in Table 2 below. Anderson–Darling sample-size-adjusted tests for normality determined the distributions of each of the 57 regions to be sufficiently normal, with estimates ranging from moderate  to high .After anatomical parcellation, individual functional activation maps  across all target regions associated with a given biomarker within an individual and multiplying it by the positive predictive value for that biomarker. The SIS, then is the sum of this computation for all 5 biomarkers. The SIS was found to have an approximate range of 3-8, for PCS patients. For healthy control subjects, the PCS range was approximately 1-4 , with a mean of 2.04. As the SIS is intended to be used primarily with patients who are independently diagnosed with probable PCS, as opposed to healthy controls, a patient-based SIS scale was developed (the one reported in this study) in which the value 0 was set to the healthy control mean, such that PCS scores tend to fall within the range 0-6. Furthermore, the standard deviation from the healthy controls (0.85) was used to establish approximate severity range labels  for convenience in communicating results with patients and health care practitioners.The fNCI Severity Index Score (SIS) was developed in order to represent the overall presence of PCS biomarkers in an individual with a single summary score. The score is computed by taking the average activation deviation —an existing neurocognitive assessment standard in the form of a self-report survey completed before, during, and after treatment—to supplement our measurement of the effectiveness of fNCI-directed treatment .Pre-EPIC SIS were computed to customize therapeutic priorities based on patient need, and provided baselines with which to objectively measure therapeutic effectiveness, post-treatment. Post-EPIC evaluations were carried out upon completion of individualized rehabilitation routines averaging 4.1 days between scans. In addition, these evaluations utilized different versions of 2.3TM is incorporated into therapy as a means of treating visuospatial deficits and to enhance multitasking and cognitive processing. A destimulation period of varying duration follows to allow for recovery before another round of therapy will begin [In addition to standardized test administration, normative-based assessment, and individual, image-guided measurements, our EPIC treatment exploits the latest knowledge of NVC and its role in PCS by integrating three fundamental rehabilitation facets for each patient: Prepare, Activate, Rest. Specific therapeutic activities included in each of these three phases of EPIC treatment are the product of research, clinical experience, screening, and empirical testing -34. EPICll begin . This cyThese therapeutic approaches address a wide range of cognitive, sensory and physical functions. The key to success, however, is not necessarily the therapies themselves, but the manner in which the therapies are applied . For example, a carefully guided rotation between physical/cardio challenge and cognitive or sensory processing challenge is a central feature in restoring the timed pairing between rCBF and regional neural firing. Our multidisciplinary team was successful in restoring pre-traumatic NVC through the cyclical application of these rehabilitative principles supplied in tandem with the benefit of appropriately timed neurocognitive exercise , 36.A multi-method approach was also employed to avoid overexertion of brain regions with severe NVU. Accordingly, cyclical brain training aimed to strengthen surrounding neural correlate systems, which, in turn, support the more severely uncoupled regions. Sustained therapy over a multi-day period was shown to promote accelerated and longitudinal restoration of pre-traumatic NVC.33.12).Data collection and analysis confirmed the effectiveness of fNCI-directed treatment in accelerated concussion rehabilitation. Quantitative improvement was observed on both objective fNCI measurements and subjective symptom report measurements after an average of 4.1 days of EPIC treatment. Average pre- and post-treatment measurements are reported for a sample of 270 concussed patients .Considering a wide variety of concussion profiles with regards to age, sex, mode of injury, and time since injury, our EPIC treatment has also been shown to be extremely effective under both objective and subjective measures .Improvement, in a broad sense, was been observed in 99.6% and 99.2% of our patients under objective and subject measurements, respectively. But the degree of improvement varied across a wide spectrum of percent differences (Range = 3.24 to 157.0%). Consequently, to address this complication, we established incremental improvement thresholds and report the total percentage of our patients that meet and/or exceed such thresholds. Average improvement is also graphically visualized , observing improvement in 100% of patients, with an average of ~70% improvement amongst the treated patient sample  .A random sample of 27 patients who underwent EPIC treatment was generated to visually characterize the original pool of 270 patients. This sample was reported against a group of 27 patients who, at the time of initial scanning, opted to delay treatment and were rescanned at a later date. Additionally, it was comparatively reported against a sample of 13 healthy controls that were scanned in order to assess test-retest reliability of the fNCI method. The random sample accurately represents improvement trends reported above .Recovery in acute models of mTBI is often unpredictable. For those injuries that progress into persistent cases, research findings allude to the pathophysiological misalignment in default NVC efficiency. Accordingly, a rehabilitative approach that promotes the restoration of default NVC efficiency should, therefore, expect longevity of positive outcomes in PCS. As expected, longitudinal outcomes for fNCI-directed treatment report sustained rehabilitative outcomes in follow-up scans averaging 8.8 months post-treatment  is both unreliable and irreproducible , 40. To protocol , 41, 42.The other well known, reoccurring argument made against clinical fMRI questions the appropriateness and sufficiency of statistical corrections made during fMRI data analysis . NotablyUsing the same standardized administration presented to normative references, each patient was scanned using fNCI to establish pre-treatment benchmarks used in measuring ultimate therapeutic effectiveness. Activation data output was projected against the normative atlas to statistically contextualize degree of NVU present in any given brain region for each patient. Furthermore, regional NVU breakdown provided target regions of interest for neurorehabilitative efforts. This individualized localization of neurovascularly-uncoupled regions supports the clinical appropriateness of fNCI-directed treatment.4.2The normative reference atlas makes it possible to search for and verify biomarkers for specific pathologies , 45—that4.2.1z-score was computed  for each of the 57 regions in 69 initial PCS patients. Prespecified functional-cognitive systems were targeted for analysis based on well-established fMRI experimental findings [Biomarker candidate search was performed with a sample of 69 PCS patients (not included in the main study of this paper). As described above, the normative reference atlas identified 8-12 qualifying regions per each of the six fNCI exams for a total of 57 regions of stable activation in the initial sample of healthy control subjects. For the initial biomarker search, a findings . For exa4.2.2Independent samples validation for true positive rates was performed with a new sample of 120 PCS patients. Resulting sensitivity calculations were consistent with the initial discovery finings to within +/- 5%. Assessment of false positive rates was performed using an additional sample of 52 healthy control subjects. Resulting specificity ranged from 96-100 for the 5 biomarkers. Thus, for example, the final sensitivity/specificity values for the frontal attentional biomarker (1 of the 5 biomarkers), following independent samples validation were 88/100, for that biomarker alone.4.2.3In order to address issues of false-positive bias with multiple biomarkers, a further multivariate base rate discovery procedure was carried out using additional samples of 70 PCS patients and 62 healthy control subjects, respectively. This method was adopted from Iverson and colleagues , in whic4.3Longitudinal data collection has observed a maintained SIS within normal limits, signifying the probable restoration of pre-traumatic NVC and re-establishment of default NVC efficiency. Continued longitudinal data collection will likely explain the mechanistic observation of these positive outcomes.At present, the dynamic approach of fNCI-directed EPIC treatment is unique with its intricate methodologies and implementation of multiple therapeutic elements. As noted previously, many reports demonstrate the effectiveness of physical, cognitive, and traditional rest independently in the rehabilitation of PCS , 48, 49.Due to the unique nature of our methodologies, one obvious limitation of our study is the ability to appropriately compare its effectiveness to other programs of similar treatment modalities. The lack of congruency in objective measurements fails to provide more accurate and thorough comparisons. However, when individually compared to exercise-, neurocognitive-, or rest-centered studies, EPIC treatment similarly reports improvement of acute symptomatology on subjective measurements -50. But Though our positive treatment outcomes support the role of NVC in PCS, the complex interconnectedness of vast neural correlate systems found within the brain advocate the etiological potential of multiple mechanical failings . Therefore, we support continual exploration of other advanced neurorehabilitative therapies and their relationships to persistent models of mTBI. However, standardized application of this evidence-based practice in general neurocognitive assessment and rehabilitation may be key to establishing clinical fMRI practice standards specifically in guiding neurorehabilitative outcomes.We present practice-based evidence for the standardized application of fNCI, which is currently used in directing neurorehabilitation, with particular efficiency in mTBI. We indicate the clinical appropriateness of this image-guided approach through iterative pilot testing, standardized administration, and individualized, normative-based contextualization. We further demonstrate the rehabilitative guidance capability of fNCI in establishing EPIC treatment standards that prioritize neurovascularly-uncoupled regions of interest unique to each patient, and support the targeted, multi-method, cyclical, and sustained approach employed by our multidisciplinary team. Positive patient outcomes—graphically represented using conventional and fNCI-directed measures—advocate fNCI’s efficacy in the accelerated and longitudinal neurorehabilitation of mTBI across a variety of concussion profiles.


To understand immunogenic potential of these glycans, we developed a validated method to detect plant specific glycan antibodies in human serum. Using this assay, low prevalence of pre-existing anti-plant glycan antibodies was found in healthy humans (13.5%) and in glucocerebrosidase-deficient Gaucher disease (GD) patients (5%). A low incidence (9% in naïve patient and none in treatment experienced patients) of induced anti-plant glycan antibodies was observed in GD patients after up to 30 months replacement therapy treatment with taliglucerase alfa, a version of human glucocerebrosidase produced in plant cells. Detailed evaluation of clinical safety and efficacy endpoints indicated that anti-plant glycan antibodies did not affect the safety or efficacy of taliglucerase alfa in patients. This study shows the benefit of using large scale human trials to evaluate the immunogenicity risk of plant derived glycans, and indicates no apparent risk related to anti-plant glycan antibodies.Plants are a promising alternative for the production of biotherapeutics. Manufacturing  Genetically engineered plants or plant cells can now be used to express plant-derived pharmaceutical proteins  or vaccines on an industrial scale , 2. The Although plants, in common with other eukaryotic organisms, produce glycoproteins with N-glycans attached to asparagine residues, these glycans differ in structure from those of mammals . Plant gThe immunogenic effect of the plant glycan moieties has been the basis of much debate in the literature. Following identification of IgE antibodies in allergic patients that cross react with these structures on glycoproteins from a variety of species, the β xylose and α fucose structures have been designated as cross-reactive carbohydrate determinants . HoweverConcerns have been raised that these unique glyco-epitopes could elicit unwanted immunogenic responses when plant derived biopharmaceuticals and vaccines are administered to humans –15. ThusTaliglucerase alfa (TGA) , an acid β-glucosidase Enzyme Replacement Therapy (ERT) for treatment of Gaucher Disease (GD), is the first plant cell-expressed biotherapeutic approved for use in humans . The varIn the case of TGA, a tri-mannose glycoform with the addition of β xylose and α1,3) fucose is present at above 90% of the total glycan pool . The pla fucose iIn previously described studies of anti-plant glycan antibody immunogenicity , 29, 30,All materials were purchased from Sigma, unless otherwise indicated. Flat-bottomed Nunc MaxiSorp ELISA plates were purchased from Nalge Nunc International . TGA was provided by Protalix Ltd. Mouse monoclonal anti-TGA antibodies were prepared by Green Mountain Antibodies . Rabbit anti-horse radish peroxidase (HRP) antibodies Cat #AS09-549 and Cat #P7899 were purchased from Agrisera and Sigma, respectively, and used as a positive control. Monoclonal anti-TGA antibody produced in mice was a gift from Pfizer Inc. and was used as a negative control. HRP (Cat #P8150 and #P8250) was purchased from Sigma. Trypsin was acquired from Roche Diagnostics GmbH, Mannheim, Germany and peptide N-glycosidase A from Europa Ltd, Cambridge, UK. Pooled and individual normal human sera were obtained from Bioreclamation . GD sera positive for anti-TGA antibodies were obtained from the clinical trials detailed below.Individual normal human serum samples were acquired from Bioreclamation IVT . A total of 52 adult samples  were analysed to determine the assay cutpoint, which was used to distinguish between positive and negative samples to anti-plant glycan antibodies.®) Enzyme Replacement Therapy.’ Adult and pediatric patients  with a total treatment duration of 9 months (study started on December 2008 and ended January 2013) ClinicalTrials.gov: NCT00712348 [https://clinicaltrials.gov/.Patient serum samples were obtained, following written consent of adults and obtained consent from parents or guardians of the minors included in the various clinical studies indicated below. The consent forms were signed and dated by the subjects or parent/guardian before he/she was exposed to any protocol-specific procedure. Samples that were tested positive for ADA  were further analysed for anti-plant glycan antibodies. The samples originated from the following clinical trials: Study PB-06-001, ‘A Phase III Multicenter, Randomized, Double-Blind Trial to Assess the Safety and Efficacy of Two Parallel Dose Groups of Plant Cell Expressed Recombinant Human Glucocerebrosidase (prGCD) in Patients with Gaucher Disease’. ERT-naïve adult patients (N = 32) with a total treatment duration of 9 months (study started on August 2007 and ended September 2009) ClinicalTrials.gov NCT00376168 [00712348 . Study P00712348 . For allThe plant glycoprotein HRP contains glycans with a high similarity to the plant glycan structures that are found on TGA. Since HRP has a distinct protein backbone that differs from TGA, but has glycans similar to TGA, antibodies that react both with HRP and TGA are considered specific for the common plant glycan structures. To verify that the HRP used in this testing contains glycans similar to TGA, we analysed several lots of HRP for their glycan composition. Glycan analysis of HRP was based on the method described by Tekoah et al. , which wA competitive ELISA based assay was developed in order to detect antidrug antibodies (ADA) specific for the glycan moieties on taliglucerase alfa. In brief, serum samples are diluted at minimal required dilution  and pre-incubated in the presence or absence of 200 μg/mL HRP, for 1 hour. Following the pre-incubation step, samples are loaded on microtiter plates, pre-coated with taliglucerase alfa and blocked with 2% bovine serum albumin (BSA), and incubated for an additional 2 hours at RT. Following a wash step, a recombinant Protein A/G-Alkaline Phosphatase (AP) Conjugate  is added at a 1:10000 dilution and incubated for 1.5 hours at RT. After an additional washing step, a phosphatase substrate  is added and incubated for approximately 20 min. Following the addition of a stop solution  the intensity of the color produced is measured by OD at 630 nm and is proportional to the level of the bound antibody. All dilution steps are performed using 1% BSA.The population distribution, log transformation and outlier identification was performed using JMP software. Cut-point calculation was performed according to standard recommendations  on MicroA multi-tiered approach was used for immunogenicity evaluation of patient samples. Specifically, all samples from the clinical trials were first screened for total anti-drug antibodies (ADA) using a screening assay, then confirmed using a separate assay. The screening and the confirmatory assays could detect all ADA, but did not distinguish between antibodies to the protein backbone or to the glycan moieties. The confirmed anti-TGA ADA positive samples were further characterized using a competitive assay, with HRP as a competitor, in order to evaluate the presence of antibody specificity to plant glycans.All the assays for immunogenicity testing were validated according to recommendations of Shankar et al and the FDA Guidance for Industry Assay Development for Immunogenicity Testing of Therapeutic Proteins recommendations , 43.HRP is a plant glycoprotein that has been reported to contain glycans with a high similarity to the plant glycan structures that are found on TGA. Furthermore, since HRP has a distinct protein backbone that differs from TGA, but has glycans similar to TGA, antibodies that react both with HRP and TGA may be concluded to be specific for the common plant glycan structures. To verify that the HRP used in this testing contains glycans similar to TGA, we analysed several lots of HRP for their glycan composition. A competitive ELISA format was implemented using HRP as a competitor for the detection of antibodies specific to plant glycans moieties on TGA. Aliquots of each serum sample were pre-incubated, either with or without the HRP competitor, and then loaded on a TGA-coated ELISA plate. During the pre-incubation step, the antibody population targeted against the plant glycans on TGA could bind to the HRP glycans. The antibodies associated with HRP were then blocked from binding to TGA on the pre-coated wells and washed out in the wash step, leading to a reduction in the OD response, compared to the parallel aliquot pre-incubated without HRP . The redAbout 40 different anti-glucocerebrosidase polyclonal and monoclonal antibodies and commercially available anti-plant glycan antibodies were tested as candidates for negative controls (NC) and positive controls (PC) for the assay. NCs were defined as TGA-binding antibodies that interact with the TGA backbone, but do not recognize the plant glycan structures on the protein. PCs were defined as TGA-binding antibodies that interact with the plant glycan structures of TGA, but do not recognize the protein backbone.Following the PC screening process a commercially available anti-HRP antibody see  producedThe PC and NC were spiked into a normal serum pool (NSP) sample, in order to simulate the patient serum samples. For evaluation of background response, we tested the reactivity of un-spiked NSP with TGA and its inhibition by HRP. As can be seen in The assay cut-point is defined as the level of response at or above which a sample is defined to be positive and below which it is defined to be negative . In ordeantilog(Mean − 1.645 × SD)) and found to be 24.98% immunodepletion.The assay cut-point was calculated based on the method described by Shankar et al.  for calcAssay precision, robustness and sensitivity were measured as part of the method validation. Assay precision was estimated for both intra-run and inter-run and was measured as percent coefficient of variation (% CV). For intra-run, 6 duplicate sets of the assay controls  were each tested in a single validation run. For inter-runs precision testing, 1 duplicate set of each of the above assay controls was tested in all validation runs. Intra-run precision was 12.24% CV or lower and the overall precision of the inter-runs  was calculated as 23.86% CV or below.Robustness was assessed by comparing different plate readers, plate washers and performance by different analysts. All robustness measurements of the PC and NC mean OD values met the acceptance criteria of within ±30% difference set for the assay. Results indicated that the assay was robust under the conditions tested.The assay sensitivity is the lowest concentration at which antibodies specific to plant glycans present on TGA are still detectable . Assay sensitivity was assessed by testing serial dilutions of the high PC from an initial concentration of 1000 ng/mL down to 7.8 ng/mL; each series was tested in 3 independent runs. The sensitivity of the assay was calculated for each run and the mean value of 64.27 ng/mL (ranging between 55.85–77.04 ng/mL) was determined as the assay sensitivity based on the PC.TGA was bound to the ELISA plate and HRP, which contains similar plant glycan structures as in TGA, was used as a competitor in an immunodepletion step; antibodies specific to plant glycans bound to the competitor (HRP) in a pre-incubation step, which prevented these antibodies from binding to the TGA coated plate.Serum samples were first diluted at minimal required dilution (1:30), using 1% bovine serum albumin (BSA) in PBS, and then separate aliquots were pre-incubated in the presence or absence of 200 μg/mL HRP for 1 hour at room temperature. Following the pre-incubation step, samples were loaded and incubated for 2 hrs at RT on ELISA plates that were previously coated overnight with 0.5μg/mL TGA (in 50 mM carbonite/bicarbonate buffer) and blocked with 2% bovine serum albumin (BSA). Following a wash step, a recombinant Protein A/G-Alkaline Phosphatase (AP) Conjugate  was added at a 1:10,000 dilution and incubated for 1.5 hours at RT. After an additional wash step, a phosphatase substrate  was added and incubated for approximately 20 min. Following the addition of a stop solution  the intensity of the colour produced was measured by optical density (OD) at 630 nm and was proportional to the level of the plate-bound antibody.A competitive inhibition assay format was implemented using HRP as a competitor for the detection of antibodies specific to plant glycan moieties on TGA, since it was found to have similar glycans to TGA. Assay details can be found in the Methods section. Negative controls (NC) and positive controls (PC) for the assay were established  and the Using this assay, the data from 52 healthy individuals was evaluated by 3 independent runs . Seven sSamples from 3 different phase III clinical studies were evaluated for the presence of anti-plant glycan antibodies. For the analysis, 2 categories were used, ERT-naïve and ERT-experienced patients, since these separate groups may have a different antibody pattern . Two studies, PB-06-001 and PB-06-005, included ERT-naïve patients  and a third switchover study, PB-06-002, included ERT-experienced patients that were previously treated with another β-glucocerebrosidase drug, imiglucerase.Patients were considered positive for ADA if they had at least one sample positive for ADA. The ADA positive samples were further assessed for anti-plant glycan antibodies. In clinical trials of ERT-naïve patients (n = 43: 32 adults and 11 pediatric), 3 tested positive for ADA at baseline, prior to TGA treatment  of whichThe clinical trial of patients who switched from imiglucerase to TGA therapy included 31 patients (26 adult and 5 pediatric), of which, 4 tested positive for ADA (any TGA epitopes) at baseline, prior to treatment with TGA . FurtherOf a total of 74 patients analysed for ADA, 71 were evaluated for the clinical safety and efficacy to assess the impact of anti-plant glycan antibody positive samples. The data of the additional 3 ERT-experienced pediatric patients were not available at the time of safety and efficacy analysis, but these patients were evaluated separately.The treatment-related and all causality adverse events (AEs) experienced by patients who were found to be positive for anti-plant glycan antibodies (n = 8) were compared with patients who were negative for anti-plant glycan antibodies (n = 63), i.e. those who were negative for anti-TGA ADA or had no detected antibodies to plant-glycans.In patients found negative for anti-plant glycan antibodies, treatment-related AEs were Hypersensitivity (n = 4), Infusion related reaction (n = 4), Headache (n = 4), Pruritus (n = 3) and Arthralgia (n = 2), but, there were no treatment-related AEs in the patients who were found positive to anti-plant glycan antibodies. Therefore, the presence of detectable anti-plant glycan antibodies was not associated with a discernible trend in treatment-related AEs.All causality AEs experienced by the patients who were found positive to anti-plant glycan antibodies, were in greater frequency than amongst patients considered to be negative to anti-plant glycan antibodies. These AEs included Diarrhoea , Vomiting , Influenza , Arthralgia , Pain in extremity  and Headache .Although the presence of detectable anti-plant glycan antibodies appeared to be associated with a greater frequency of certain all causality events, the low number of patients in anti-plant glycan antibody positive group and the low number of events makes comparison challenging because a single occurrence of an AE has a large effect on the calculation of frequency. With regards to treatment related AEs, it should be noted that the presence of detectable anti-plant glycan antibodies was not associated with a discernible trend in treatment-related AEs and no clear demonstrated correlation between the appearance of anti-plant glycan antibodies and an increased clinically relevant risk associated with immunogenicity was observed.The safety data of the three additional patients, whose data were not available at the time of safety and efficacy vs. anti-plant glycan antibodies analysis, show a very small number of AEs  in 2 out of the three patients and all were reported as non-treatment related.GD clinical symptoms include hepatosplenomegaly, anaemia, thrombocytopenia and skeletal deterioration which resulted from glucosylceramide accumulation in the lysosomes of macrophages found in the liver, spleen and bone marrow , 47. TheA post-hoc assessment of absolute and percent change from baseline in the 4 efficacy endpoints  was performed. Although, the group of patients tested positive for anti-plant glycan antibodies was rather small in size (n = 8), it was still clear, as shown in The assessment of the major clinical efficacy endpoints versus anti-plant glycan antibodies status at end of study was done for the ERT-naïve adults  and pediatric  patients. The clinical efficacy data was evaluated versus the anti-plant glycan status. The anti-plant glycan positive group included 6 patients (4 adults and 2 pediatric) and the negative group included 37 patients (28 adults and 9 pediatric).The results show improvement (reduction) of the spleen and liver volume in pediatric and adult patients in both the positive (n = 6) and negative  anti-plant glycan groups. In the adult patients the reduction of spleen and liver volume was similar for both groups. In the ERT-naïve pediatric patients positive for anti-plant glycan antibodies, a higher reduction in spleen and liver volume was shown compared to the negative group .Hemoglobin levels show a similar improvement (increase) in pediatric and adult patients in both the positive and negative anti-plant glycan groups.Improvement of platelet count (increase) was shown in pediatric and adult patients in both the positive and negative anti-plant glycan groups. In the adult patients, positive for anti-plant glycan antibodies, the increase was lower than the negative group. The pediatric patients, positive for anti-plant glycan antibodies were found to have a higher increase compared to the negative group.A similar assessment was done for the ERT-experienced adults (n = 26) and pediatric (n = 5) patients (9 months). The anti-plant glycan positive group included 3 patients (1 adult and 2 pediatric) and the negative group included 28 patients (25 adults and 3 pediatric).The results show stability, with changes of less than 10%, of the spleen volume, liver volume, hemoglobin and platelet count in ERT-experienced patients in both the positive and negative anti-plant glycan groups.The efficacy data of the three additional patients, whose data were not available at the time of safety and efficacy analysis versus anti-plant glycan antibodies status, show stability to slight improvement in hematological parameters (platelet count and hemoglobin) and organ volume (spleen and liver).Different factors may elicit unwanted immunologic responses to biotherapeutic drugs. Immunogenicity of non-mammalian plant glycans on therapeutic glycoproteins and vaccines produced in plants was studied and discussed in previous articles , 29, 30.We identified a negative control antibody that specifically binds the TGA backbone and does not recognize the plant glycan structures on the protein and a positive control (anti-HRP) that interacts only with the plant specific glycans and not the protein backbone of TGA. For demonstrating specificity, we used different plant derived proteins (HRP and TGA) as competitors and different β-glucocerebrosidase molecules lacking plant glycan structures to capture anti-TGA antibodies, in order to confirm that the method only detects antibodies specific to the plant glycans. Rigorous statistical methods were used to establish the cut-point for distinguishing positive and negative samples. In doing so, we were able to demonstrate that the optimized assay is robust, precise and sensitive and is appropriate for the measurement of anti-TGA antibodies directed against the plant glycan structures on TGA.In the healthy population, we were able to show, using the validated method, that 13.5% of the subjects were positive for the presence of anti-plant glycan antibodies. Our data are consistent with some previous publications, showing that less than 20% of the normal healthy population has detectable levels of pre-existing antibodies to plant glycan motifs , 30, butIn the ADA results analysis, we considered the different population groups , and whether or not the patient was ADA positive pre- or post-treatment with TGA.The observation of baseline ADA assay positive results in some subjects prior to exposure to biotherapeutic drugs is not unusual, especially when sensitive assays are used; however, the impact of pre-existing antibodies on post-treatment immune response, as well as on safety and efficacy is not consistent across different biotherapeutics , 50.For 5 out of 74 GD patients (6.8%) that were positive for ADA and anti-plant glycan antibodies at baseline, post-treatment ADA titters were either stable or reduced following exposure to TGA. In an additional evaluation of clinical outcomes and AEs, these 5 patients did not show different trends of safety and efficacy parameters, compared to the remaining study population. Although based on a small number of patients with pre-existing antibodies, these results suggest that some pre-existing antibodies are specific for plant glycan structures on TGA. This result (6.8%) is somewhat lower than what would be expected, since the healthy population evaluated in this study showed a total of 13.5% of the total subjects having pre-existing anti-plant glycan antibodies. This difference could result from immune dysregulation shown in GD patients . In addiSamples from 23 patients were found positive for treatment-induced ADA, of which 1 also had pre-existing ADA. Out of these patients, only 4  were positive at one or more time points for antibodies to plant glycans. These were mostly associated with ADA titers lower than the median titer of 215.Despite these low numbers, we tried to correlate safety and efficacy data with the presence of anti-plant antibodies. TGA shows equivalent, or better safety, when comparing between the three alternative β-glucocerebrosidase ERT treatments . The preTo conclude, we developed a validated method for detection of antibodies directed specifically against plant glycans to assess the abundance of anti-plant glycan antibodies in the healthy human population. Furthermore, we were able to assess the presence of these antibodies in ADA positive TGA-treated GD patients, and correlate the results with safety and efficacy parameters. Based on these results, the prevalence of anti-plant glycan antibodies in the total population was found to be under 15% and does not increase after exposure to plant derived biotherapeutics. Furthermore, this response appears to be limited in magnitude and impact on safety and efficacy.The data that was collected in taliglucerase alfa studies in general –39 and iLimitations of this study are due to the small sample size, including a total of 74 patients. Of which, only few were positive for anti plant glycan antibodies (pre- and post- treatment). Therefore, further evaluation of plant glycan immunogenicity risk using a validated analysis method is needed for other biotherapeutics. Albeit, our results suggest that plant glycan structures do not pose a greater immunogenicity risk than any other epitope. Thus, plant derived biotherapeutics can be efficiently and safely given to humans.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file.


Non-muscle-invasive bladder cancer (NMIBC) is categorized into high-risk and low-risk groups. Although, bacillus Calmette-Guerin (BCG) is the recommended adjuvant therapy of high-risk bladder tumor, optimal schedule (induction versus maintenance) of this therapy is a subject of debate. The objective was to evaluate outcomes of induction BCG in high-risk NMIBC patients at Shaukat Khanum Memorial Cancer Hospital & Research Centre, Pakistan and retrospective cohort study conducted in the department of urology, Shaukat Khanum Memorial Cancer Hospital & Research Centre, Pakistan. Three-year disease-free survival and progression-free survival was the main outcome measure. Data of 68 high-risk (Ta and T1 with G3 or high-grade subtype) bladder cancer patients who underwent transurethral resection followed by six-weekly intravesical BCG instillation was included in the study. Recurrence was described as biopsy-proven bladder cancer; whereas the presence of muscle invasion was considered as progression. Disease-free survival and progression-free survival were defined as time intervals elapsed between the starting date of BCG instillation and recurrence or progression, respectively. Kaplan-Meier curve was employed to estimate the three-year study end-points. Disease-free survival at three years was observed to be 66.2% and progression-free survival at 86.8%. The use of induction BCG alone for high-risk patients of NMIBC is a viable option both in terms of effective disease-free and progression-free survival rates. Transitional cell carcinoma (TCC), the commonest type of bladder cancer, has a heterogeneous clinical spectrum, disease progression, therapeutic modalities and prognosis . ApproxiAlthough, transurethral resection is a well-recognized initial treatment modality for all NMIBC, institution of subsequent adjuvant intravesical therapy to prevent tumor recurrence and progression to muscle invasive category depends on risk groups derived from numerous prognostic factors . It has The archetypal schedule, known as induction therapy, in high-risk group bladder cancer patients includes six-weekly intravesical instillation of BCG . In ordeSeveral studies also demonstrate the efficacy of BCG induction therapy alone both to delay tumor progression and to improve the five-year disease-free survival ,15. A stHitherto, a large body of controversial reports and contradictory discursive evidence has been observed in the literature regarding the optimal regimen of adjuvant BCG therapy in high-risk bladder cancer. Although, published guidelines recommend maintenance BCG for one to three years ,15, subsThe purpose of this study was to evaluate the outcomes of induction BCG in high-risk NMIBC patients at Shaukat Khanum Memorial Cancer Hospital & Research Centre, Pakistan. Both progression-free and disease-free survivals were estimated prospectively by analyzing retrospective data. The data will provide supporting evidence in scientific literature about the ongoing debate of optimal BCG schedule but also valuable in the revision of protocol for high-risk NMIBC patient’s management at local level. Informed consent was obtained from the patient for this studyThis retrospective review of the database of patients having bladder cancer at Shaukat Khanum Memorial Cancer Hospital & Research Centre, Pakistan, was accomplished from January 2008 to Dec 2012 after approval of Institutional Review Board (IRB).Clinical information of all patients of both sexes who underwent transurethral resection followed by intravesical BCG induction therapy between January 2008 to December 2012 having following parameters were included in the study: Ta and T1 tumor categories, high-grade, between 50 to 80 years of age. Patients who underwent transurethral resection followed by intravesical instillation of chemotherapy or combination of both chemotherapy and BCG, previously treated with BCG therapy, had recurrent tumors, isolated carcinoma in situ (CIS), associated upper urinary tract urothelial tumors, and non-urothelial bladder cancer and patients with inconclusive histopathology due to absence of muscle layers in specimen were excluded.The included patients had undergone transurethral resection of bladder tumor by a consultant urologist. After two weeks, TICE BCG in a dose of 500 mIU diluted in 50 ml 0.9% saline was given intravesically once a week for six weeks. The instilled amount was retained in bladder for an hour. Tumor recurrence and progression were evaluated on check cystoscopy scheduled at every three months for three years after primary resection of bladder cancer.All clinical records were recorded into proforma designed for the study. Data collection process included: socio-demographic status, date of first cystoscopy and transurethral resection, tumor category, tumor extent, tumor size, associated CIS, BCG therapy compliance, and surveillance status. The tumor surveillance incorporated regular check cystoscopies after primary resection and BCG induction. The occurrence of biopsy-proven bladder cancer was considered as recurrence, whereas presence of muscle invasion on histopathology was described as progression. Disease-free survival and progression-free survival were defined as time intervals elapsed between the starting date of BCG instillation and recurrence and progression, respectively. Outcome end point was three-year disease-free and progression-free survival. Data which showed no events throughout the surveillance period, lost to follow-up patients and those who discontinued BCG were considered as censored events.The Statistical Package for the Social Sciences (SPSS) version 19 was utilized for all statistical analysis. Mean SD was employed for quantitative variable. Frequencies and percentages were utilized to summarize qualitative variables. Times to events (i.e. recurrence and progression) were calculated considering the start date of BCG as time zero. Kaplan-Meier curve was employed to estimate the disease-free survival and progression-free survival. Multivariate cox proportional hazard model was used to adjust for the potential confounding effect of explanatory variables on end-point variables with the p-value < 0.05 taken as statistical significance. The results were also described in hazard ratio (HR) and 95% confidence interval (CI).a disease and 65 (95.6%) had T1 disease. Forty (58.8%) patients had a single lesion and the same number had a tumor size less than 3 cm.  Associated CIS was found in three (4.4%) of the patients  of the study group was 62.66 + 12.05 years. 41.2% were < 60 years of age, and 88.2% were male. Three patients had TBladder cell carcinomas are the fourth most common cancer affecting the American males and accounts for 60,000 new cases in the United States alone -19. ApprA varied range of treatment options has been proposed, tested and rejected over the course of many years for optimal management of this clinical entity. Transurethral resection, combination of resection with chemotherapy and intravesical instillation of immunotherapeutic agents have all been proposed to varying degrees of success and the debate still rages on.Bacillus Calmette-Guerin (BCG) was initially developed to treat tuberculosis. Pearl in 1929 was the first to report its anti-neoplastic activity . Since tThe current debate focuses on whether induction therapy of BCG alone is sufficient or should the maintenance therapy be pursued. One of the main limitations associated with the use of this agent is the local and systemic side effects which range from cystitis, epididymitis, prostatitis to lung infection, liver toxicity and sepsis, these side effects are dose dependent . Hence aWe at our institute, therefore decided to follow induction therapy as the methodology for treatment of high risk NMIBC. Using this regimen protocol we found the three-year disease-free survival in our patients was 66.2% and the progression-free survival for the same period was 85%. Herr, et al. in their study of 816 patients who got induction chemotherapy showed a two year and five year disease-free survival of 73% and 46% respectively. Their calculated progression-free survival was 89% . DespiteA recent study was published evaluating maintenance BCG therapy . The autA recently published multi-centre study that randomly allocated patients to no maintenance versus maintenance arm showed 33.5% and 38.5% recurrence rates. This study had 195 patients in the former arm and 202 in the latter. They concluded that maintenance therapy did not lead to a decrease in recurrence or progression. They reported that 20 patients in the maintenance arm had to stop treatment because of side effects and only five faced this situation in the no maintenance arm .The following factors should be considered while interpreting our results: this study is based on retrospective review of database. Although, attention was given to methodological rigor by incorporating explanatory variables and analyzing confounding effect of these on endpoint measures, various biases related to operative and BCG instillation techniques cannot be controlled. Secondly, the study has a relatively short follow-up time. Therefore, both the disease-free and progression-free survival rates may not be as precise as other longer surveillance published series -14.The use of induction BCG alone for high-risk patients of NMIBC is a viable option. The disease-free and progression-free survival rates at our institution using this option is comparable with the internationally published data.


In this study, we describe for the first time Besides phenotypic antimicrobial susceptibility testing, molecular strain typing and sequencing was performed to describe the phylogenetic relation of the Ethiopian isolates in detail in relation to published isolates from all over the globe.blaNDM-1-positive Acinetobacter baumannii isolates, most likely a local clonal diffusion, were isolated. Two of the three isolates described within this study were untreatable with the locally available antimicrobials and were only susceptible to polymyxin B and amikacin. The genome sequences confirmed the isolates to be distinct from the outbreak strains reported from Kenya, the only other characterized blaNDM-1 positive Acinetobacter baumannii strains in East Africa so far. Up to date, no other bacterial species were found to harbour the gene cassette in Jimma and conjugation to E. coli was not successful under laboratory conditions. However, natural transmission to other bacteria seems likely, given the evident lack of hygienic precautions due to limited resource settings.Three multi-resistant, The detected isolates could solely be the tip of the iceberg regarding the presence of NDM-1 producing organisms in the region, as only a limited number of bacterial isolates were evaluated so far and until recently, susceptibility testing and isolation of bacteria could hardly be performed in clinical patient care. These multi-drug resistant organisms pose a serious threat to antimicrobial treatments in Jimma, Ethiopia.The online version of this article (doi:10.1186/s12879-017-2289-9) contains supplementary material, which is available to authorized users. Multidrug-resistant Gram-negative bacterial infections are recognized as one of the major threats to global health, the leading cause of nosocomial infections both in industrialized as well as developing countries and leading to a high economic burden –4. TheseAcinetobacter baumannii producing either extended-spectrum ß-lactamases (ESBL), carbapenemases or a combination of different factors [A. baumannii resistant to carbapenems (CRAB) are regarded as sentinels for emerging antimicrobial resistance [One of the most common and feared multidrug-resistant organisms responsible for hospital-acquired infections are  factors . Strainssistance . The mossistance , 14. So sistance , 15. In sistance , 15, 16.Jimma University Specialized Hospital (JUSH) is located in southwest Ethiopia and constitutes the only referral hospital for over 15 million people in the catchment area . Until 2Acinetobacter baumannii isolates in Ethiopia, which were obtained from patient samples in JUSH. Besides phenotypic antimicrobial susceptibility testing, molecular strain typing and sequencing was performed to describe the phylogenetic relation of the Ethiopian isolates in relation to published isolates from all over the globe.This report describes to our knowledge the first analysis of NDM-1 producing 2. Although the hospital is rated as the local referral center, running water and hand sanitizer were not easily obtainable at the time of the study. Within 17 months, between January 2014 and June 2015 a total of 224 pure Gram-negative isolates were obtained from clinically apparent infections from routine clinical specimens in Jimma. Out of these, 14 could be identified as Acinetobacter baumannii. Three of these isolates were found to be meropenem resistant and NDM-1 positive. The samples originate from three independent inpatients (A–C) attending the surgical department of JUSH. Basic demographic and medical data were documented along with the specimens using standard clinical and laboratory record forms.Jimma University Specialized Hospital (JUSH) is located in Jimma city, Oromiya regional state, situated at a distance of more than 350 km southwest of the Ethiopian capital city Addis Ababa. At the time of the study, JUSH had a capacity of 450–500 inpatient beds providing curative and preventive services for over 15 million people within the catchment area of approximately 15–16,000 km2. After isolation, identification was performed by Gram-stain and using standardized biochemical reactions .The specimens were inoculated onto appropriate media according to standardized protocols. For isolation, 5% Columbia sheep blood agar as well as MacConkey II agar was used. Cultivation was performed in incubators at 37 °C for 24 and 48 h respectively. Blood agar plates were incubated at 5% COIsolates were tested against a common panel of antibiotics for their susceptibility pattern by disc diffusion method as outlined by Kirby-Bauer at JUSH bacteriology laboratory  and thenAll strains were shipped to the Department of Bacteriology, Max von Pettenkofer-Institute (LMU), Munich, Germany for further investigation of their genetic traits using molecular methods. Upon receipt, all isolates were sub-cultured to ensure viability as well as purity and identified again to species level by MALDI-TOF mass spectrometry  to exclude errors occurring during transport, labelling and reading the biochemical differentiation reactions. Bacterial DNA was isolated from a single colony using the High Pure PCR Template Preparation Kit  for genetic typing of resistance mechanisms and from 10 ml of stationary culture using the NucleoBond PC 20  kit as outlined by the manufacturers.Acinetobacter spp., real time PCR reactions were performed to detect OXA-23-like, OXA24/40-like, OXA-51-like and OXA-58-like genes [in silico using the whole genome sequence data according to the Oxford-scheme [in silico MLST are detailed in the supporting information . The strain information and the accession numbers are listed in the supporting information  following the manufacturer’s instructions, allowing for the detection of CTX-M ESBL enzyme subgroups, TEM Mutations, SHV Mutations, AmpC alleles CMY, DHA, FOX, MOX, ACC, MIR, ACT as well as Carbapenemases such as KPC, NDM-1, VIM, IMP, OXA-48-like. To also test for the carbapenemases common in ke genes . Whole gd-scheme  and the The draft genomes of the three isolates A, B and C have been deposited at DDBJ/ENA/GenBank under the accession numbers LWSM00000000, LWSN00000000 and LWSO00000000 respectively.blaNDM-1-carrying bacteria or multidrug-resistant A. baumannii have been described in Ethiopia so far. In Kenya, an outbreak with NDM-1 producing A. baumannii was recently described, and strain types of the isolates have been determined [blaNDM-1 could also be demonstrated in Klebsiella in neighbouring Kenya [A. baumannii harbouring the blaNDM-1 gene within one hospital in Ethiopia, the Sequence Types were different than the isolates published from Kenya. We did not perform anal swab screening to explicitly search for blaNDM-1 positive bacteria; only clinically relevant materials were examined. However, the lack of other blaNDM-1-positive isolates is surprising, given the reports about the ease of horizontal transfer of NDM-1 containing plasmids [In general, data on the distribution of antimicrobial resistance genes on the African continent is scarce, especially regarding carbapenemases. To our knowledge, no termined . Years bng Kenya . We deteplasmids , 23.Acinetobacter baumannii culture from putrid pleural fluid. She was treated with high dose trimethoprim/sulfamethoxazole after the antimicrobial susceptibility test result was available and improved with the therapy. This patient could be treated and survived the infection, whereas patient B and C did not offer any treatment option given the available drugs in JUSH. Patient C developed peritonitis after surgical intervention and grew predominantly Acinetobacter baumannii from peritoneal fluid. He died due to overwhelming systemic infection, whereas the otherwise young and healthy patient B growing pure Acinetobacter baumannii culture from an inguinal abscess was able to fight off the infection. During the course of infection, the process remained localized and treatment was given with antimicrobials which were tested resistant in the isolate.Table blaNDM-1-carrying isolates can be seen in Table TM, Bio-Rad Laboratories GmbH, Munich, Germany). Growth of the three isolates was detected without any inhibition zone for chloramphenicol, ciprofloxacin, erythromycin, amoxicillin, cefoxitin, ceftazidime, cefepime and gentamicin. One isolate (B) showed an inhibition of 1 mm around the meropenem disc, the others did not. The raw values are detailed in Table A summary of the antimicrobial susceptibility test results of the three Enterobacteriaceae, conjugation experiments against three different polymyxin B resistant E. coli mutant strains were conducted. Polymyxin B resistant mutants were chosen to allow for efficient selection of transformant E. coli strains, as the donor organism was resistant to almost all other substances. Conjugation was performed by co-cultivation of the two organisms in high density on solid Columbia 5% blood media  for different time periods, as well as in liquid media. Counter selection was performed with Polymyxin B. To enhance transformation efficiency, F+ strains were also employed. However, up to now, the blaNDM-1 gene could not be transferred to E. coli. This observation is obviously not sufficient to conclude that transfer might not occur between Acinetobacter strains or in accordance with the limited spread within the isolates encountered in Jimma. It might also be explained by low mobilization rates and chromosomal location of the gene. On the other hand, transposases were found in silico adjacent to the NDM-1-cassette and plasmid mobilization as well as pilus assembly genes showed up in the sequence data of the strain.To assess the transferability of the NDM-1 carrying genetic element to other http://pubmlst.org/abaumannii/). Already published A. baumannii strains from Kenya had different STs indicating a different origin [Upon sequencing, the three isolates exhibited an identical pattern regarding their MLST profile. It was identified to be ST957  could be assembled from the sequences of all three strains and was highly similar to the blaNDM-1-cassettes of previously published strains. The NDM-1 (813 bp) and the blaMBL-gene (366 bp) showed 100% sequence identity with the respective genes on the NDM-1-plasmids of the published A. baumannii strains as well as from Acinetobacter lwoffi strain Iz4b (NCBI accession number NC_025000) and Providencia rettgeri strain 09ACRGNY2001 (NCBI accession number KF295828). This again demonstrates the global distribution of the NDM-1 gene with vastly identical sequence. The genetic environment of blaNDM-1 is shown in Fig. Unfortunately, we were unable to isolate or sequence the full length of the larger  strains , 26. TheblaNDM-1 gene carrying organisms from Ethiopia for the first time. The three Acinetobacter baumannii isolates from patients in Jimma were genetically analysed and were found to be distinct from the outbreak strains isolated in Kenya, the only characterized blaNDM-1 positive Acinetobacter baumannii strains in East Africa so far. This argues strongly against the regional spread of blaNDM-1 positive organism but rather suggests the independent, repetitive import of such strains from other regions. Clinicians should be aware of the possible emergence of outbreaks with different multi-resistant Acinetobacter baumannii strains. Although, no other bacterial species were found to harbour the gene cassette in Jimma so far, and conjugation was not successful under laboratory conditions, natural transmission to other bacteria seems likely given the evident lack of hygienic precautions due to limited resource settings. It is further likely, that the detected isolates are solely the tip of the iceberg regarding the presence of NDM-1 producing organisms in the region, as only a limited number of bacterial isolates could be evaluated. The three isolates were genetically very closely related and might represent clonal diffusion within the surgical department of the hospital.In this study, we describe