Head and neck cancer (HNC) patients are at high risk for developing lymphedema and fibrosis (LEF) following cancer treatment. Once HNC patients develop LEF, they need to conduct life-long self-care to slow LEF progression and reduce associated symptom burden and functional deficits. Data demonstrate that inadequate LEF self-care may be a potentially remediable issue. The objective of this study is to explore the feasibility and preliminary efficacy of an Information-Motivation-Behavioral (IMB) Skills model-driven self-care program (SCP) to improve LEF management and reduce LEF-related symptom burden and functional impairments.This is a three-arm, prospective, randomized controlled clinical trial to compare: Group 1 – Usual Care, Group 2 – Usual Care Plus LEF-SCP, and Group 3 – Usual Care Plus LEF-SCP Plus Follow-Up. Participants will be HNC survivors aged > 18 years of age, who meet predefined inclusion and exclusion criteria. A sample size of 75 participants is targeted. Interventions will be provided by trained staff. The study assessments for all groups will take place at five points: study entry then 3, 6, 9, and 12 months post enrollment. Outcome measures include: (1) feasibility  of the proposed intervention; (2) self-efficacy and adherence to self-care; and (3) preliminary efficacy  of the proposed intervention.This will be the first study to evaluate the feasibility of a LEF-SCP in the HNC population and its impact on self-efficacy and adherence. Furthermore, it will evaluate the potential benefit of routine follow-up on adherence and fidelity to the self-care protocol. We expect that the trial will provide evidence supporting the feasibility of a LEF self-care program. In addition, we anticipate that preliminary data will support improved outcomes including increased adherence and fidelity, and decreased LEF-associated symptoms.NCT 03030859). Registered on 22 January 2017.ClinicalTrials.gov, a service of the US National Institute of Health ( The incidence of head and neck cancer (HNC) has increased rapidly due to the epidemic of human papillomavirus (HPV)-associated oropharyngeal carcinoma , 2. PatiLEF is a trans-tissue process that affects three quarters of HNC patient more than 3 months post treatment . It maniCurrently, head and neck lymphedema is an incurable but manageable, chronic condition , 13, 14.While it is generally agreed that self-care is critical for long-term management in patients with LEF, the self-care component of HNC-associated lymphedema management has not been standardized. There are no evidence-based LEF self-care programs conducted in HNC populations in the literature; thus, in clinical settings, self-care regimens may vary widely based on the experience and training of the lymphedema therapist . Indeed,The Information-Motivation-Behavioral (IMB) Skills model of health behavior change has been used in addressing self-care issues faced by individuals with chronic conditions. A recent systematic review assessed the applicability and effectiveness of interventions based on the IMB model for promoting behavioral changes among individuals with chronic diseases . SpecialThe study was approved by the Institutional Review Board at the University of Pennsylvania. Recruitment will be conducted at the Head and Neck Cancer Clinics in the University of Pennsylvania Health System. The protocol has been developed in accordance with the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines for interventional trials . The SPIThis study is a three-arm, prospective, randomized controlled clinical trial to compare: (1) Usual Care, (2) Usual Care Plus LEF-SCP, and (3) Usual Care Plus LEF-SCP Plus Follow-Up. Outcome measures include: (1) feasibility  of the LEF-SCP (Aim 1); (2) self-efficacy and adherence to self-care (Aim 2); and (3) LEF progression, symptom burden, and functional status (Aim 3). The study assessments will take place at five points in time, enrollment, 3, 6, 9, and 12 months post enrollment.Individuals will be recruited into the study if they meet the following criteria: (1) post - HNC primary treatment; (2) no evidence of cancer; (3) no more than 6 months after completion of initial lymphedema therapy for head and neck lymphedema; (4) > 18 years of age; (5) the ability to understand English in order to complete questionnaires; (6) ability to complete the onsite training and home self-care activities for LEF management; and (7) ability to provide informed consent.Individuals will be excluded if they have any of the following medical conditions that would prohibit the safe implementation of self-care of LEF: recurrent or metastatic cancer; any other active cancer; acute infection; congestive heart failure; renal failure; cardiac or pulmonary edema; sensitive carotid sinus; severe carotid blockage; or uncontrolled hypertension.A minimum of five participants will be recruited per month. Our previous studies in a similar patient population have achieved recruitment goals in a timely manner. This was largely due to the research team’s dedication, support from attending physicians, and a concerted effort on the part of investigators to minimize patient burden. The volume of patients at the Head and Neck Cancer Clinics in the University of Pennsylvania Health System is sufficient to complete this trial without additional sites. The recruitment procedures used will be identical to those used successfully in our prior studies, such as screening at Head and Neck Cancer Clinics. All direct patient recruitment activities will be conducted at the Head and Neck Cancer Clinics where private rooms are available to be used for conducting clinical research projects.n = 25 per group).Participants will have provided written informed consent, been screened, and completed all study baseline measures prior to study group randomization. Seventy-five participants will be randomized via the use of a computer-generated, permuted block program, developed and executed by the study statistician, to one of the three study groups  at a 1:1:1 allocation ratio  session plus a 1-h LEF self-care training session. After completion of the LEF-SCP training, participants will be given the LEF self-care educational manual. Participants with Internet access will be provided a safe website link to watch the self-care videos. Participants without Internet access will be given the Digital Versatile Discs (DVDs) and a DVD player for watching the videos. Participants will be asked to review the manual and videos monthly or more frequently as needed.In addition, however, participants in this group will be scheduled to meet with the study lymphedema therapist for 3-, 6-, and 9-month LEF follow-ups. Please note that these follow-ups will take place after completion of 3-, 6-, and 9-month follow-up data collection. Each LEF follow-up visit with the lymphedema therapist will take approximately 1 h. The lymphedema therapist will check participants’ LEF status, adjust the LEF self-care plan if needed, make new recommendations about LEF self-care, and review and address any potential issues and questions that participants may have about LEF self-care. The lymphedema therapist will document the suggestions given to each participant.After completion of the baseline measures, participants in the Usual Care Plus LEF-SCP Plus Follow-Up group will follow the same procedures as the Usual Care Plus LEF-SCP group. The Demographic Form: this form will be used to collect the following participant data: date of birth, gender, race, ethnicity, education, marital and employment status, area of residence, medical history, alcohol use, tobacco use, and dietary habits. The HNC Clinical Form: a medical chart review will be conducted to collect data on the diagnosis date of HNC, primary site, stage, type and dates of treatment, and complications. The LEF Treatment Form: the form will be used to collect date of diagnosis of lymphedema and/or fibrosis and treatment history.The Recruitment Log includes the number screened, number recruited, and barriers to recruitment. The Implementation Log-MI Session will be used by the study MI instructor to document barriers of implementing MI sessions. The Implementation Log-LEF Self-Care Training Session will be used by the study lymphedema therapist to document the barriers of implementing LEF self-care training sessions. The Common Terminology Criteria for Adverse Events (CTCAE version 5.0) will be used to document any adverse events [The LEF-SCP Evaluation (Satisfaction) will be completed by participants in the two LEF-SCP study groups after their 12-month post-enrollment assessment has been completed.e events . The LEFThe Perceived Medical Condition Self-Management Scale (PMCSMS): an 8-item generic scale that can be adapted to any specific medical condition , will be used to assess self-efficacy in self-managing LEF  [ha 0.84) –28.The LEF Self-Care Checklist: the form asks participants to complete a checklist of LEF self-care activities they have performed during the past 7 days. Participants will complete this checklist at the baseline, 3-, 6-, 9-, and 12-month follow-ups. Monthly Self-Care Survey: participants will receive an email survey during the last week of each month during the study follow-up. They will be asked to answer four to five brief questions about their adherence to the LEF self-care activities during the past 7 days .Digital photographs of the head and neck region will be obtained at baseline and all follow-up visits. A standardized technique for photography has been established by the team in order to ensure constancy. Head and Neck External Lymphedema-Fibrosis (HN-LEF) Grading Criteria will be used to document participants’ external LEF status. Our previous study supports the scale with content/face validity and good interrater reliability  [The Patterson Scale is a validated measure for documenting internal edema in the pharynx and larynx. The scale has good intrarater reliability (weighted kappa = 0.84) and moderate interrater reliability (weighted kappa = 0.54) [< 0.001) . The Pat = 0.54) . This scHead and Neck Lymphedema and Fibrosis Symptom Inventory: a patient-reported outcome measure developed specifically to assess lymphedema symptoms in HNC patients. Content and face validity of the tool was reported during the development and preliminary testing of the tool. The tool has 58 items  [Vanderbilt Head and Neck Symptom Survey Version 2.0 (VHNSS v2.0): a validated measure of HNC treatment-related symptoms, 50 items  [80–0.97) , 31. Van = 0.94) –34.Jaw Range of Motion Scale: a validated and reliable device for measuring jaw range of motion [Cervical Range of Motion Device: a validated and reliable device for measuring the degree of neck movement [Goniometry: a validated and reliable instrument for measuring active and passive shoulder range of motion [f motion , 36. Cermovement . Goniomef motion , 39.Assumptions underlying each of the proposed statistical summaries and comparative analyses will be evaluated. Transformation of continuous data distributions will be completed as necessary to meet those assumptions. Randomly missed responses to items within assessment tools will be handled via protocols specified by the instrument developers. We expect that entire assessments  will not be missing at random, thus imputation of missing data is not planned. While this study is a preliminary study of efficacy and effect sizes, a maximum alpha level of 0.05 will be used for the statistical tests.Descriptive statistical and graphical methods will summarize the rates of participation (recruited vs. consented), log data , completion of assessments throughout the study, adverse events (safety) and satisfaction data  within the two groups assigned to LEF-SCP. Qualitative data analysis will be used to summarize the barriers to performing self-care. It is expected that ≥ 80% of participants will complete the study activities, and ≥ 80% will report acceptable levels of satisfaction with the LEF self-care, thereby demonstrating feasibility of the LEF self-care.Descriptive statistical and graphical methods will be used to summarize and visually inspect the PMCSMS scores (self-efficacy), the number and types of self-care activities reported on the LEF Self-Care Checklist within the three study groups throughout the study period. Mixed-effects generalized linear models that correct the standard errors for repeated assessments and incorporate appropriate distributional link functions  will be used to generate estimates of the effect of the LEF-SCP on self-efficacy and rates of self-care. Within the analyses, bias-corrected estimates of the interaction of group assignment with the time of assessment  on the outcome measures will provide the key effects interest .As with the previous aim, descriptive statistical and graphical methods will be used to summarize and visually inspect the measures of LEF, symptom burden, and functional status within the three study groups throughout the study period. Mixed-effects generalized linear models with the appropriate link function for the nature of the specific outcome variable being analyzed will be used to generate estimates of the effect of the LEF-SCP on the progression of head and neck LEF, symptom burden, and function measures. Bias-corrected estimates of the interaction of group assignment with time of assessment will provide the key effects interest .N = 75) will meet the critical assumptions for estimating central tendency and variability in the key outcome values, as well as for generating estimates of the effects of the LEF-SCP on self-efficacy, self-care adherence, progression of LEF, symptom burden, and functional status. Given the emphasis on effect sizes, there is no plan to adjust for the multiple measures included in this study and any conclusions regarding statistical significance will be based on a two-sided-type alpha of 0.05.The primary purpose of this study is to inform the feasibility and potential efficacy of a tailored LEF self-care program among the population of HNC survivors with LEF. The proposed sample sizes are believed sufficient for gleaning this information. Rather than formal statistical tests of hypotheses, effect sizes generated are the most critical statistical outcome. The expected analysis sample of three groups  from implementing these components have been reported in the known current literature review. However, if study staff identify LEF-associated issues  requiring intervention, the principal investigator (PI), study physician, and study lymphedema therapist will be notified immediately. Patients will be referred to their treating physicians and lymphedema therapist for adequately managing their LEF in a timely manner.The PI, study physician, and study lymphedema therapist will review any adverse events and other unanticipated problems. The PI will report any adverse events and other unanticipated problems to the University of Pennsylvania IRB and the American Cancer Society.Participants will be withdrawn from the study if they develop recurrent or metastatic cancer and any other active cancer.All participants will be assigned a study ID number by a trained study staff member. This information will be maintained in a patient-tracking database that is not stored on the hard drive, accessible only to the study team members through a password-protected computer and link to the server at the study site, which is accessible only the study team.More than half a million HNC survivors are alive in the United States . Three -The date recruitment began: 1 June 2018.The approximate date when recruitment will be completed: 31 May 2020.Data are currently being collected. At the time of submission of this paper (28 April 2019), the protocol is version 2.0 (14 January 2019).Additional file 1:. Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) Checklist.


Young adults with type 1 diabetes (T1D) experience a decline in glycemic outcomes and gaps in clinical care. A diabetes education and support program designed for young adults was delivered through group videoconference and mobile Web.The objective of our study was to assess the feasibility, acceptability, and preliminary efficacy of the program as measured by attendance and webpage views, satisfaction, and pre- and postintervention psychosocial outcomes, respectively.t tests.Young adults aged 18-25 years were recruited to attend five 30-minute group diabetes education videoconferences during an 8-week period. Videoconferences included an expert presentation followed by a moderated group discussion. Within 48 hours of each videoconference, participants were sent a link to more information on the study website. Feasibility was assessed using data on videoconference attendance and webpage views. Acceptability was assessed via a Satisfaction Survey completed at the conclusion of the study. Descriptive statistics were generated. Preliminary efficacy was assessed via a survey to measure changes in diabetes-specific self-efficacy and diabetes distress. Pre- and postintervention data were compared using paired samples In this study, 20 young adults (mean age 19.2 [SD 1.1] years) attended an average of 5.1 (SD 1.0) videoconferences equivalent to 153 (SD 30.6) minutes of diabetes education per participant during an 8-week period. Average participant satisfaction scores were 62.2 (SD 2.6) out of a possible 65 points. A total of 102 links sent via text message (short message service) or email resulted in 504 webpage views. There was no statistically significant difference between pre- and postintervention diabetes-specific self-efficacy or diabetes-related distress.Delivery of diabetes education via group videoconference using mobile Web follow-up is feasible and acceptable to young adults with T1D. This model of care delivery has the potential to improve attendance, social support, and patient-reported satisfaction. Nevertheless, further research is required to establish the effect on long-term psychosocial and glycemic outcomes. The timing of website page views was strongly linked to the date the links were pushed to participants; this indicates a high level of engagement in the educational content by participants. This pilot study with young adults with T1D demonstrated high feasibility for providing diabetes education and support via group videoconference and strong participant engagement in Web-based follow-up. Participants reported high levels of acceptability as measured by user satisfaction. In terms of the preliminary efficacy, there was no statistical difference in pre- and postintervention psychosocial outcomes; however, on average, we observed improved scores for both diabetes-related self-efficacy and diabetes-related distress. The findings support results from previous studies that have demonstrated high attendance and satisfaction with individual clinic visits and group medical appointments provided via telehealth to youth and young adults with T1D ,12.The implications of these results should be considered in the context of the study’s limitations. Owing to the brief duration of and limited funding for the study, other efficacy-related outcome measures including glycemic control and diabetes knowledge attainment were not assessed. Recruitment took place at a university-based diabetes clinic, where patients may be more highly motivated to attend diabetes education visits. In addition, despite the ubiquity of mobile technology, patients with limited data plans or access to wireless networks may not find participation as feasible without financial support to cover the cost of a mobile data plan.Nonetheless, the outcomes suggest that this delivery model for diabetes education and support has the potential to increase contact with the clinic, improve access to diabetes education, and provide peer and social support for young adults who have become disconnected from their diabetes network. Future randomized studies that include a control group should explore the intermediate and longitudinal effect of the model on glycemic control, diabetes knowledge attainment, clinic attendance, and psychosocial outcomes. In addition, future studies should measure provider satisfaction and explore the feasibility of reimbursement for telehealth group videoconference education sessions. Convenient, comprehensive, yet tailored diabetes care, education, and support is required to keep young adults engaged in their diabetes management to reduce gaps in care and to mitigate the decline in glycemic control commonly experienced by this patient population. As T1D management becomes more technically complex, videoconference and Web-based models of diabetes care and education delivery can be leveraged to connect patients to providers and educators at a reduced cost with improved convenience and without a decline in patient satisfaction.


The aim of this study was to investigate the use of botulinum toxin type A (BoNT-A) injections and their efficacy on gross motor function for lower limb spasticity in children with spastic cerebral palsy (CP). This retrospective study included 919 injection occasions from 591 children with CP who received a lower limb BoNT-A injection between 2006 and 2016. The Gross Motor Function Measure (GMFM-88), the Modified Ashworth Scale, and the Modified Tardieu Scale were administered before and after injections. Injections were predominantly administered to children under the age of 6 years. The most common muscle injection site was the calf muscle for dynamic foot deformity. The second most commonly injected muscle was the hip adductor among 2–3 year olds and the hamstring muscle among 4–6 year olds. Distal injections were predominantly administered to high-functioning children, whereas proximal injections were typically administered to low-functioning children. Multilevel injections were mostly administered to midfunctioning children. GMFM-88 scores significantly increased post-injection for both high- and low-functioning groups. Younger age at injection and distal injection type were associated with larger improvements on the GMFM-88 at both short- and midterm follow-up. The target muscles for injection varied depending on gross motor functioning and age. Younger age at injection and distal injection type were significantly related with greater gain in gross motor function. Botulinum toxin type A (BoNT-A) injections are widely used to control lower limb spasticity in children with spastic cerebral palsy (CP) ,2. AccorDuring physical development in childhood, the contribution of non-neural components to hypertonia increases and leads to joint contracture or deformity. As for equinus of the foot, the increasing muscle stiffness and joint tightness leads to limitation of joint motion (LOM) as the child grows. Cast applications are known to be useful to improve the passive range of motion (ROM) at the ankle . TherefoAlthough numerous previous studies have established that BoNT-A injections are effective in spasticity control and improving ROM , its effIdentification of related factors that allow for the prediction of the effect of BoNT-A injections could help to guide indications and treatment goals for the injection. Therefore, the aim of the present study was to investigate whether the efficacy of BoNT-A injections, or a combined case application and the targeting of different muscles, differed according to age and gross motor function. Additionally, the study aimed to elucidate whether BoNT-A injections could lead to functional gain and identify contributing factors relating to functional gain in children with spastic CP.In total, 919 injection occasions from 591 participants were analyzed in this study. The sample included 340 boys and 251 girls, with a mean age at first injection of 4.68 years old . Among the participants, children classified as ambulatory CP comprised 73.6% of the sample (GMFCS level I/II/III: 210/111/114), and nonambulatory CP comprised 26.4% (GMFCS level IV/V: 91/65). Meanwhile, 454 participants (76.9%) had bilateral involvement, and 137 participants (23.1%) had unilateral involvement. The characteristics of the participants included in this study are described in p < 0.05). Injections were administered unilaterally on 284 occasions (30.9%) and bilaterally on 635 occasions (69.1%). Distal injections for foot deformity were given on 502 (54.6%) occasions, while proximal injections into the muscles of the hip and/or knee joints were administered on 80 (8.7%) occasions. Multilevel injections targeting the muscles of three joints—the hip, ankle, and/or knee—were given on 337 (36.7%) occasions . The aveThe injection type was significantly different according to GMFCS level . ParticiInjections were administered 1–11 times. However, most of the participants for this study received the injection within 3 times. Injection frequencies of 4 or more occurred on 150 occasions 16.3%; .The most commonly injected muscle in all age groups was the calf muscle to target dynamic foot deformity. The second most commonly injected muscle was the hip adductor in children aged 2–3 years and the hamstring muscle in children aged 4–6 years. Injections were predominantly administered to children under the age of 6 years . Significant improvements on the MAS and R1 (the angle of muscle reaction) on the MTS were observed for most of the muscles injected at the first and second follow-ups compared to baseline, but the tone in some of the muscles significantly increased at the second follow-up compared to the first follow-up . As for The frequency of cast application after injection was significantly different according to age group. The frequency of cast application was significantly higher in children aged over 4 years old compared to children aged 2–3 years .Both ambulatory and nonambulatory groups demonstrated significant gains on the Gross Motor Function Measure (GMFM-88) at both short and midterm follow-ups compared to baseline assessment, but only ambulatory children had significantly improved on the GMFM-88 at the second follow-up assessment (within 3−6 months) compared to the first follow-up assessment (within 1−2 months) . In addition, the present study revealed a significant improvement in GMFM-88 scores following multiple injections, but this improvement was significantly smaller compared to children who had received either one or two-to-three injections .A univariate analysis of both short-term and midterm changes revealed that age at injection, injection type, and number of repeat injections were significant factors associated with gains on the GMFM-88. A multivariate analysis revealed that the age at injection and injection type continued to be significant factors associated with changes on the GMFM-88 at short- and midterm follow-up .According to a previous population-based study, BoNT-A injections were predominantly administered to the calf muscle in ambulatory children (GMFCS level I to III) and to the hamstring and adductor muscles in nonambulatory children (GMFCS level IV to V) . The litBoNT-A injections are known to be an effective intervention for dynamic spasticity but not static contracture. Therefore, children with spastic CP usually received the BoNT-A injection before the development of static contracture, which is often the target for orthopedic surgery. In a previous population-based study, BoNT-A injections were most frequently administered to children aged 4–6 years [Equinus foot is the most common deformity in children with CP. A retrospective cohort registry study demonstrated that the gastrocnemius muscle was the most common muscle treated with BoNT-A injections irrespective of age or level of gross motor function . In addiCombined therapy—using a serial or single short leg cast with BoNT-A injections—is thought to achieve optimal improvements for equinus . In our In previous studies, BoNT-A injections, or combined therapy with BoNT-A and physiotherapy, led to greater improvements on the GMFM-88 in children with CP than physiotherapy ,17,18,19Using BoNT-A injections as an intervention has a long-term effect on motor function, even though the effect of the injection on muscle tone had disappeared ,24,25,26The efficacy of BoNT-A injections has been proven in the treatment of spastic equinus in ambulant children with CP (GMFCS levels I to III) with the aim of improving gait pattern ,31. TherThe effect of gross motor functioning in relation to functional gain, as measured by the GMFM-88, has rarely been reported. In a previous study, moderately impaired children (GMFCS level III) showed a tendency to attain higher gains in GMFM-88 scores, but these improvements were not statistically significant . AnotherThis was a retrospective study in a single center, though most of the included children with CP visited our clinic especially for BoNT-A injections. We established the protocol for children with CP who had received BoNT-A injections for the assessment, but some children were missed during the courses. However, this did not appear to significantly impact our results. In addition, the amount of physical therapy received after injection varied to some extent, but we did not expect the outcome to be significantly affected. As the importance of intensive physical therapy after injection was emphasized to the parents of children who had received BoNT-A injections, physical therapy was performed on most of the children. Regardless of these limitations, a strength of this study is that GMFM-88 changes could be assessed in a relatively large number of cases, enabling us to delineate factors associated with changes on the GMFM-88 after injection. Our study revealed that BoNT-A injections are common under the age of six, and combined cast application after injection for equinus foot became increasingly more common from the age of four. The target muscles for injection varied depending on the level of gross motor function and age. The gross motor functional changes, as measured with the GMFM-88, were significantly associated with age at injection as well as injection type in children with spastic CP. This study was conducted in a pediatric spasticity management clinic in a university-affiliated, tertiary-care teaching hospital. In this clinic, children with CP were regularly followed by a multidisciplinary clinic team of three pediatric physiatrists, nurses, physiotherapists, and an occupational therapist.For this study, the medical records of children with spastic CP who had received BoNT-A injections, with or without neurolysis or motor point block, with ethyl alcohol in the lower limb between January 2006 and December 2016 were retrospectively reviewed. The medical records of 2329 injection occasions of 1213 children were reviewed. Of these records, children under 13 with full assessments before injection and a follow up assessment at least once after injection were selected for this study. As a result, the data from a total of 919 injections from 591 children with CP were analyzed.This study was approved by the Institutional Review Board of the Severance Hospital (4-2019-0587). Informed consent for BoNT-A injection was obtained from the parents of all children in this study. In addition, oral or written assent was also obtained from the children over 7 years old according to their understanding and cognitive abilities.At our clinic, children with spastic CP are regularly followed in order to provide appropriate interventions, including BoNT-A injections, in a timely manner. At every visit, spasticity, ROM, gross motor skills, and movement or gait patterns are assessed. In the case of patients receiving BoNT-A injections, the selection of muscles and the dosage to be injected was determined by the physician based on clinical assessments including the MAS, MTS, the Hypertonia Assessment Tool (HAT) if possible, and the GMFM-88. One vial containing 100 units of onabotulinum toxin-A  was diluted with 2 mL normal saline; 200 units of Neu-BoNT/A  was diluted with 4 mL normal saline. For the children who received multilevel injections, nerve block with 50% ethyl alcohol was added if the total dose per one session exceeded the maximal safe dosage . Obturator nerve block, tibial nerve block to the gastrocnemius muscle, or motor point block to the hamstring muscle was administered depending on whether additional nerve or motor point block was needed. Depending on the size and the severity of spasticity of the target muscles, 1–5 U/kg botulinum toxin was injected per muscle. The total muscle dose was divided between two to four injection sites depending on muscle size. Injections were guided by ultrasonography or by electrical stimulation for the exact identification of target muscles or nerve or motor point blocks. Electrical stimulation for nerve or motor point blocks was administered under general anesthesia. Distal injections were defined as those that were confined to the calf muscle when used to treat a foot deformity, such as equinus with or without varus or valgus foot. For distal injections, the gastrocnemius muscle was the main target muscle on all occasions. Meanwhile, the peroneus muscle or tibialis posterior muscle were also injected on nine occasions when equinus was accompanied by varus or valgus foot. In some hemiplegic children with equinus foot, both the soleus muscle and the gastrocnemius muscle were injected. Proximal injections were defined as those that were confined to the hip muscles, such as the hip adductor or iliopsoas muscle, and/or the hamstring muscle, without the calf muscle injection for foot deformity. Most of the children who had proximal injections had received the injection into both the hip adductor and/or hamstring muscles. Multilevel injections were defined as those that were distributed diffusely from hip to ankle joint muscles. For multilevel injections, the gastrocnemius, hamstring, hip adductor, iliopsoas, and rectus femoris were the target muscles based on the clinical assessment.For children who had LOM at the ankle joint, a short leg cast was applied for one to three weeks (depending on the degree of LOM) to increase the ROM after injection. The short leg casts were applied with the patient in a prone position with the knee flexed to 90° and the ankle dorsiflexed to maximal attainable dorsiflexion and held in the neutral hind foot position. The consecutive cast was applied with a progressively increasing degree of ankle dorsiflexion to stretch the calf. The importance of intensive physical therapy after injection was emphasized to all parents. Subsequently, most of the children in the study received intensive physical therapy. Using the GMFM-88, a physical therapist assessed the gross motor function of children who received a BoNT-A injection in the lower limb. The physiatrist assessed muscle tone using the MAS and MTS by assessing the ROM in each joint and by determining the child’s GMFCS level. All children who received the injection were assessed before and after the injection was administered. Follow-up assessments were conducted 1–2 months post-injection, and again 3–6 months post-injection. The level of gross motor functioning of children with CP can be classified according to the GMFCS, which consists of five levels, from level I (most able) to level V (least able). The GMFCS is the most widely used system to classify gross motor function in children with CP. The GMFCS levels used for this study were determined based on classification at the last visit. High functioning was defined as ambulatory children at GMFCS level I to III, while low functioning was defined as nonambulatory children at GMFCS levels IV to V. Functional gross motor gains were assessed by physical therapists using the GMFM-88. Evidence indicates that the GMFM-88 is a valid and reliable measurement tool . The shot test was used to compare the differences in improvement between the groups. Additionally, univariate and multivariate linear regression modeling were used to identify factors significantly associated with functional change after injection. A p-value less than 0.05 was considered statistically significant for all statistical tests. Statistical analysis was performed using the Statistical Package for the Social Sciences for Windows . Chi-squared tests were used to compare the distribution of injection types according to GMFCS level. Linear mixed or generalized estimating equation models were used to analyze the changes in GMFM-88 score, MTS angle, and MAS grade after intervention in each group. Post hoc Bonferroni correction was used for multiple comparisons. The independent


Despite intensive treatment, 50% of children with high-risk neuroblastoma (HR-NB) succumb to their disease. Progression through current trials evaluating the efficacy of new treatments for children with HR disease usually depends on an inadequate response to induction chemotherapy, assessed using imaging modalities. In this study, we sought to identify circulating biomarkers that might be detected in a simple blood sample to predict patient response to induction chemotherapy. Since exosomes released by tumor cells can drive tumor growth and chemoresistance, we tested the hypothesis that exosomal microRNA (exo-miRNAs) in blood might predict response to induction chemotherapy. The exo-miRNAs expression profile in plasma samples collected from children treated in HR-NBL-1/SIOPEN before and after induction chemotherapy was compared to identify a three exo-miRs signature that could discriminate between poor and good responders. Exo-miRNAs expression also provided a chemoresistance index predicting the good or poor prognosis of HR-NB patients. Neuroblastoma (NB) is the most common pediatric extracranial solid tumor, with an incidence of 10 cases per million individuals, and accounting for about 15% of childhood cancer-related deaths. NB arises in the developing sympathetic nervous system and is characterized by high clinical heterogeneity. Based on the International Neuroblastoma Staging System (INSS) patients can be stratified in different risk groups  ranging from spontaneously regressing to highly malignant and metastatic tumors [MYCN amplification, recurrent segmental chromosome aberrations including losses of chromosome 1p, 3p, 4p, 11q and gains of 1q, 2p, 17q) that together with age and stage are currently employed to define the risk level and treatment burden. Despite multi-modality therapy, almost 50% of HR-NB patients suffer from a refractory or relapsed disease [Half of all NB patients present a high-risk (HR) disease associated with the presence of aggressive genetic features  as intraluminal vesicles. Exosomes exert important functions in the maintenance of the correct physiological state of the cell, but they have been shown to play a role in pathological conditions, promoting tumor initiation, progression, and metastasis [The exosome content is heterogeneous, comprising proteins, DNA, messenger RNA, microRNAs (miRNAs), other noncoding RNAs, and different lipids that compose the plasma membrane. In particular, miRNAs have been broadly described as biomarkers in different pathologies. Recently, it has been shown that the level of specific miRNAs in serum samples of HR-NB patients was associated with disease burden and treatment response .Because of exosome involvement in determining cancer progression and treatment response, we were interested to investigate exosomal (exo)-miRNAs modulation in liquid biopsies from HR-NB patients after frontline induction chemotherapy to assess the presence of early markers of patient response to treatment. We report here the identification of an exo-miRNAs signature that could discriminate between children with HR-NB who are poor and good responders to induction chemotherapy and predict event-free survival. The exo-miRNAs signature could be further investigated in an early time point to assess whether this approach could be useful to quickly identify patients resistant to the drugs employed in induction chemotherapy and in need of a different drug combination. The present exploratory study demonstrates that analysis of exo-miRNAs modulation is feasible in a large cooperative survey and lays the groundwork for a robust prospective study as part of the HR-NB clinical study.n = 47), 4 subjects had a stage 3 disease, and one patient had a 4S stage disease, according to INSS  classification system. MYCN oncogene was amplified in 22 patients, non-amplified in 25 cases, and not evaluable in 5 patients. According to the INRC  classification system, 6 patients reported a minor response (MR) to induction chemotherapy, 8 patients showed a very good partial response (VGPR), and 33 patients had a partial response (PR). For 5 patients, the data of response to treatment is missing because it was not updated in the SIOPEN database, even if the patients completed the induction chemotherapy. A relapse occurred in 22 patients, while 30 patients did not report any progression or relapse. Finally, 40 patients had a favorable overall outcome while 12 patients deceased.Exosomes were purified from peripheral blood samples obtained from 52 HR-NB patients whose clinical characteristics are summarized in n = 9; End of Induction n = 9) using a monoclonal antibody (mAb) recognizing the typical exosomal surface marker, tetraspanin CD9 [+ exosomes corresponded to 50% of the whole exosome population in plasma samples at diagnosis and to 27% after the induction treatment . The results demonstrate that plasma samples of HR-NB patients contain NB-derived exosomes, which decreased upon induction chemotherapy.To investigate the presence of NB-derived exosomes, vesicles collected before and after induction chemotherapy were characterized in terms of size, positivity for the GD2 marker, which is specifically expressed on NB tumor cell surface and barely in normal tissues , and miRanin CD9 , to confreatment B. The diTo investigate exo-miRNAs modulation upon treatment, exo-miRNAs expression profile was compared in HR-NB patients at diagnosis and after induction chemotherapy. We extracted total RNA from the whole population of isolated exosomes and evaluated the content of miRNAs by capillary electrophoresis to ensure a sufficient retrieval of material for the following analyses. The whole population of circulating exosomes is representative of the tumor status, as cancer cells produce a higher number of exosomes than normal proliferating cells . Tumor-dWe then applied a filter based on the number of undetermined (NA) values. Dealing with such values is challenging when interpreting RTqPCR data because missing data can adversely affect downstream analysis . Imputatp value < 0.05) after induction chemotherapy. The list of downregulated exo-miRNAs is reported in + exosomes, the results show that there are tumor-specific exo-miRNA whose modulation is indicative of the response to treatment, as discussed in the next sections.Technical sources of variation, named batch effects, can be introduced in the data when handling and processing samples. Because plasma samples used in our study were collected from different UE centers , we wondered whether the different origin of samples could introduce additional technical variations influencing the results. To test this hypothesis, we performed a Principal Variance Component Analysis (PVCA). PVCA pointed out that the contribution of the origin to variability is negligible, accounting for the 3% of the variance, as can be observed in the PVCA bar chart  see Mat shows thTo determine whether differences in the expression of exo-miRNAs before and after induction chemotherapy could discriminate among patients with different clinical responses, we compared two groups of patients showing clearly different clinical response to the treatment according to the INRC  classification system . Specifip value < 0.05). Enriched pathways included also adherens and gap junctions that, when deregulated, promote metastasis and also the p53 signaling pathway, which is known to play a major role in cell cycle regulation and tumor chemoresistance [Having established that the exo-miRNAs signature discriminated between MR and VGPR patients, we evaluated whether the three exo-miRNAs could predict pathways involved in the chemotherapeutic response. To this aim, we performed pathway analysis of the exo-miRs signature with miRNet. We found that the three exo-miRNAs are involved in pathways that play a major role in cancer development and chemoresistance. The main enriched pathways known to contribute to tumor progression and survival are reported in sistance .We assessed the efficacy of the three exo-miRNA signature in differentiating PR patients into two clusters that could point out PR subjects closer to MR or VGPR patients. The results of this evaluation are reported in p value < 0.05). Cluster 1 showed high CI toward CBDCA, CDDP, and DOXO and was associated with poor responders, while Cluster 2, showing low CI for the same drugs, was associated with good responders. Additionally, we found that Cluster 1 patients were more sensitive to VP-16 and VCR, while cluster 2 patients showed slightly high CI toward these drugs. CBDCA, CDDP, and DOXO seem to have a major impact on the response to treatment, as high CI for these drugs is sufficient to determine worse prognosis, despite the sensitivity/resistance toward VP-16 and VCR. Log-rank test further pointed out the significant difference in survival between the two clusters identified by the CI , strengthening its predicting value of EFS and treatment response.This targeted approach allowed to estimate the probability of chemoresistance for each patient toward each chemotherapeutic drug used in the induction protocol. The resulting chemoresistance matrix was analyzed by unsupervised k-means clustering to identify homogeneous subgroups of response . We perfHR-NB patients do not respond adequately in around 50% of cases to induction therapy, making the exploration of new and more effective strategies for improving risk stratification at diagnosis crucial for the amelioration of the outcome . In the The whole fraction of purified nanovesicles included a significant number of NB-derived exosomes, suggesting that NBs can release exosomes in vivo in agreement with what previously observed in in vitro culture  and enabEvaluation of exo-miRNAs expression was challenging because a standardized RTqPCR analysis approach applied to exo-miRNAs is currently lacking. We defined a novel bioinformatics pipeline, named ExoPIPE , specifically designed to analyze exo-miRNAs expression. Importantly, we observed that the different origin of samples from several countries had a negligible impact on data variability, preventing the need of removing the batch effects introduced by processing plasma samples.We identified a signature of three differentially expressed exo-miRNAs  that could significantly discriminate between VGPR and MR patients. In particular, MR patients showed a considerable downregulation of miR-342-3p, miR-29c, and let-7b upon treatment, whereas in VGPR subjects their expression remained mainly unchanged. Two patients, classified as VGPR by the INRC system, fell into the group of MR patients according to the 3 exo-miRNAs profile. Interestingly, one of these patients relapsed this year, supporting the efficacy of exo-miRNAs signature in determining patient outcome respect to the INRC system. Two of these miRNAs (miR-29c and let-7b) target MYCN gene . The thrCI was able to effectively stratify HR-NB patients into two groups that were significantly associated with a poor and a good response on the bases of EFS, calculated with a 3-year follow up. Poor responders showed high CI for CBDCA, CDDP, and DOXO. High resistance toward these drugs appeared to be sufficient to determine a poor treatment outcome and unfavorable prognosis, as the sensitivity to VP-16 and VCR was not able to overcome CBDCA, CDDP, and DOXO resistance, while good responders showed exactly the opposite CI. Survival analysis confirmed the results. Our cohort included patients with at most 3 years follow-up, and the association analysis would certainly be more robust if we could have had available patients with at least 5 years follow-up. The present exploratory study, however, indicates that the CI calculated on the basis of exo-miRNA modulation could define patient response to specific drugs at a molecular level. The novelty of the CI makes it an important tool in personalized medicine but, at the same time, should be carefully validated in order to define a reliable parameter for tumor chemoresistance evaluation. The CI would improve and complete the information obtained with the three exo-miRNA signature, which can be applied only to a subset of patients. If our results are confirmed, a panel of exo-miRNAs could be customized as an array to assess the response to chemotherapy for each patient. In particular, the exo-miRNA evaluation performed in the middle phase of induction chemotherapy would allow to adjust the treatment according to the specific patient sensitivity/resistance profile. Therefore, both the three exo-miRNA signature and CI could be useful tools for the clinician in the decision-making process to define which drug combination apply for every single patient.Therefore, to perform an analysis considering PR patients and to better define a good or poor chemotherapeutic response, we set up a new approach able to predict treatment outcome at a molecular level. We considered all the exo-miRNAs that were modulated after chemotherapy in the cohort of 52 HR-NB patients for which up or downregulation was shown in the literature to be associated with resistance toward the drugs employed in the induction protocol. Then, we measured exo-miRNAs expression levels in response to induction chemotherapy in each patient of our cohort. We calculated the chemoresistance index (CI) on the basis of the number of exo-miRNAs, related to a specific drug, that were modulated according to a chemoresistant behavior see . A smootSince the present study suffers from an important bias due to the limited cohort of patients analyzed, our proposal is to validate the prognostic value of exo-miRNAs here identified in a prospective study on HR-NB patients who will be enrolled in the next HR-NB study protocol.ClinicalTrials.govidentifier: NCT01704716) were considered for this study. Patients were recruited at enrollment into the HR-NBL SIOPEN study starting in 2016 without being preselected for the analysis. In this protocol, patients were randomized in the induction phase to receive one of two regimens, Rapid-COJEC  or modified-N7 regimen . The procedures were carried out according to the proper guidelines and in adherence with the ethical principles of the Declaration of Helsinki. Written informed consent was provided from the parents or legal guardian of patients enrolled in this study. Blood samples from 52 patients were collected in ACD or EDTA tubes at two time points: before and at the end of the induction chemotherapy, for a total number of 104 samples analyzed. Most blood samples were provided by the Italian AIEOP  centers. Further, frozen plasma samples were from La Fe Hospital , the Leeds Institute of Medical Research , and the Curie Institute . Blood was centrifuged at 1200 g for 10 min at room temperature (RT) to collect plasma. Plasma was stored at −80 °C or used immediately for exosomes isolation. Blood processing was performed within 24 hours from sampling. The study cohort included mainly patients with a stage 4 disease, with different clinical and biological features, as reported in Patients affected by HR-NB enrolled in the HR-NBL Study of SIOP-Europe  to the filter column and spun at 5000 × g for 5 min at RT. The spin column was transferred to a new collection tube and centrifuged at 5000 × g for 5 min at RT after the addition of 700 μL of QIAzol to the membrane to collect the lysate. RNA extraction was then performed according to the manufacturer’s instructions. Quality control and miRNAs evaluation were carried out on the Agilent 2100 Bioanalyzer, using the small RNA assay .Exosomes isolation and RNA extraction were performed with the exoRNeasy Serum/Plasma Midi kit . Briefly, 500 μL of plasma was centrifuged at 16,000 × To collect intact exosomes, the exoRNeasy Serum/Plasma Midi kit protocol was used: in the last step of the process, QIAzol was substituted with 200 μL of Buffer XE, a reagent enabling elution and collection of exosomes. Exosome size was evaluated using the zetasizer nano ZS90 particle sizer . Retrieved exosomes were then analyzed for the presence of CD9 and GD2 markers by flow cytometry after vesicles adsorption onto latex beads, as previously described . BrieflyTM RT primers Human Pool A . Pre-amplification of cDNA was performed with TaqMan® PreAmp Master Mix and MegaplexTM Pre-Amp primers Human Pool A. The pre-amplification product was diluted according to the manufacturer’s instructions and used to perform microRNA profiling on the ViiATM 7 Real-Time PCR System. Briefly, 9 μL of the diluted pre-amplified product was mixed with 450 μL TaqMan® Universal Master Mix II, No UNG , and 441 μL of nuclease-free water. 100 μL of the PCR reaction mix was dispensed into each well of the TaqMan® Array human microRNA A card , enabling the quantification of 381 human miRNAs. The validation was performed with individual qPCR assays based on specific TaqMan miRNAs Assays . Samples were run in triplicate on MicroAmp Fast Optical 96-well reaction plate .Exo-miRNAs were analyzed by the TaqMan Array Card Technology. Briefly, 50 ng of RNA was reverse transcribed with the TaqMan® microRNA Reverse Transcription Kit, using the Megaplexp value < 0.001) . Raw data were normalized by global mean method (GM) . The glo< 0.001) . The boxFeature selection to identify relevant exo-miRNAs was performed with GLMNET  setting p values lower than 0.05 were considered significant.Among the exo-miRNAs modulated after the induction chemotherapy, we identified a “chemoresistance list” including miRNAs known from the literature to be up or downregulated in chemoresistance condition for each chemotherapeutic drug used in the NB induction therapy. By looking at our data, we analyzed the modulation after the induction chemotherapy of the exo-miRNAs in the “chemoresistance list” in each patient. The −ΔΔCT values were used to define the up or downregulation upon treatment. We counted the miRNAs coordinately up or downregulated according to the “chemoresistance list” for each patient and each single drug. This number and the total number of miR in the “chemoresistance list” have been used to estimate the probability of chemoresistance of a patient to a chemotherapeutic drug using the Laplace smoothing method. Heat map visualization and unsupervised k-means clustering analysis were carried out using Morpheus: versatile matrix visualization and analysis software . Z test for proportions was used to estimate whether two patient groups differ significantly on one characteristic. Three-year event-free survival (EFS) curves were compared with the log-rank test using GraphPad Prism version 6.0 for MAC, GraphPad Software, San Diego California USA. ClinicalTrials.govidentifier: NCT01704716) were considered for this study. The study was approved in 2016 by Comitato Etico Regionale . The procedures were carried out according to the proper guidelines and in adherence with the ethical principles of the Declaration of Helsinki. Written informed consent was provided from the parents or legal guardian of patients enrolled in this study.Patients affected by HR-NB enrolled in the HR-NBL Study of SIOP-Europe (Our results demonstrate that the blood of HR-NB patients contains tumor-specific exosomes and that exo-miRNAs can be efficiently isolated from plasma specimens. The possibility of defining patient response on the bases of molecular parameters meets the concept of precision medicine, which enables to distinguish patients with similar clinical presentations but different cellular and molecular responses. In this case, response to induction chemotherapy, currently defined by imaging and clinical parameters, could potentially be determined by blood exo-miRNAs expression. To our knowledge, this is the first study that investigates exosomes in HR-NB plasma samples and identifies exo-miRNAs as indicators of induction chemotherapy response. Our results pave the way for exo-miRNAs application in liquid biopsies as circulating biomarkers of chemotherapeutic response and for the development of NB-targeted treatment.


Herein, a novel strategy to fabricate haze films employing liquid crystal (LC) technology for photovoltaic (PV) applications is reported. We fabricated a high optical haze film composed of low-molecular LCs and polymer and applied the film to improve the energy conversion efficiency of PV module. The technique utilized to fabricate our haze film is based on spontaneous polymerization-induced phase separation between LCs and polymers. With optimized fabrication conditions, the haze film exhibited an optical haze value over 95% at 550 nm. By simply attaching our haze film onto the front surface of a silicon-based PV module, an overall average enhancement of 2.8% in power conversion efficiency was achieved in comparison with a PV module without our haze film. Environmentally friendly energy generation devices such as photovoltaic (PV) cells have attracted much attention because of the depletion of fossil fuels as well as the environmental issues arising from the use of these fuels. Hence, the development of high-performance PV cell technologies is a pressing global need, and many attempts have been made to improve power conversion efficiency (PCE) at low cost ,3,4,5,6.Herein, our novel strategy for combining liquid crystal (LC) technology with a PV cell is proposed. Self-aggregating and self-assembling properties of LCs are very useful for developing novel functional devices ,19,20. Wed light .In this work, we demonstrate PCE enhancement of a PV module using a LC technology-based optical film that exhibits a high haze performance, which is caused by the diffusion of light associated with low molecular micro-segregated LCs (MSLCs) surrounded by a polymer medium. This MSLC is made using a simple mixed system of low molecular LCs and polymers. In general, a polymer-dispersed LC (PDLC) consists of LC droplets dispersed in a polymer matrix. A PDLC can be electrically switched from a light-scattering (opaque) state to a non-scattering (transparent) state for smart windows or display applications ,23,24. HOur MSLC film elongates the optical path length of light and hence the light absorption in the active layer of the PV module is boosted through diffusion of the incident light ,25. We oWe fabricated a MSLC haze film composed of LC/polymer composite with haze properties by employing a spontaneous phase separation process between low molecular LC and a polymer medium ,27. Figuen = 1.706 , and refractive index of the prepolymer is n = 1.56. To fabricate MSLC film, commercially available low molecular LC  and photo-curable prepolymer  were employed. Extraordinary refractive index of the LC used here is The TT and DT spectra of the haze films were measured using a UV-Vis-NIR spectrophotometer  with an integrating sphere (inner diameter: 60 mm). Optical haze was calculated using Equation (1). The bidirectional transmittance distribution function (BTDF) is used when specifically referring to transmitted scatter through a medium as a function of the angular positions of the incident and scattered beams. The BTDF was evaluated using SDR-300S .−2). External quantum efficiency (EQE) measurements were obtained as functions of wavelength with incident photon-to-current conversion equipment .The J–V curves of the PV cells were obtained using a Keithley model 2400 source measurement unit under AM1.5G simulated illumination  from the homogenous phase to the inhomogeneous phase on cooling, and plotted a phase diagram of the LC/prepolymer mixtures with different mixing ratios, as shown in hi. hi. hi. By means of UV irradiation, LCs came out from the homogeneous phase and formed LC droplets a few micrometers in size. The spontaneous phase segregated state, MSLC, was kept at RT because the prepolymer solidified such that the LCs were trapped. When light passed through the MSLC film, these micro-LC droplets acted as light scattering centers due to the refractive index-mismatch between the LCs and the polymer [Typical polarized optical microscopy (POM) images of a LC/prepolymer mixture (30:70 wt%) before UV irradiation are shown in the inset to  polymer .hi. We found that the LC droplet size is an important parameter influencing the optical haze in our films. It was revealed that the MSLC film with small LC droplets (a few micrometers) exhibited a higher haze value than the film with large LC droplets (a few tens of micrometers). In the case of PDLC for switchable smart windows (displays), small LC droplets are unfavorable because they increase the driving voltage ,30. A smn = 1.55), was the reference module without the MSLC film. PV-30 was a silicon-based PV module that had MSLC-30 film integrated onto its front surface via refractive index matching fluid (n = 1.56). The PV performance of PV-30 was investigated against that of PV-R using a solar simulator under AM 1.5 G conditions (1000 Wm−2). The typical current-voltage (J–V) curves are presented in ocV), short circuit current density (scJ), fill factor (FF), and power conversion efficiency (PCE) of the two PV modules are summarized in scJ. The EQE curves of the two modules are also presented in the inset to scJ values of the two PV modules also showed similar trends as the experimentally evaluated ones. Since the only difference between the two PV modules was in the attached film, the higher EQE values for the MSLC-attached PV cells indicate enhanced harvesting of the incident solar light due to the haze behavior of the MSLC films. To demonstrate the applicability of the MSLC film with optical haze behavior in a PV module, we integrated the film with a commercially available silicon-based PV module (itemSchool). To investigate the performance improvement caused by the integration of the MSLC film, we prepared two modules; PV-R and PV-30. Schematic illustrations of the two modules are depicted in To explain PCE enhancement of the PV-30 compared to that of the PV-R, we investigated the BTDF of transmitted light through MSLC-30 and bare glass. As presented in The haze properties MSLC-20 exhibited were similar to those of MSLC-30. Thus, the PCE enhancement that the PV module with MSLC-20 showed was similar to that of PV-30. On the other hand, the PV module with MSLC-10 (MSLC-50) was inferior to the PV module without MSLC films because the optical haze was decreased in MSLC-10 (MSLC-50).We evaluated haze films using LC technology for PV applications. A new kind of high-performance haze film, namely the MSLC film, which improves the PCE of PV cells is reported herein. Our MSLC film provided a simple, effective, and low-cost method of further enhancing PV cell efficiency employing LC technology. MSLC film was made from a simple mixed system of low molecular LCs and polymers, and fabricated by polymerization-induced phase separation between the LCs and polymers. We optimized the optical performance of our haze film by adjusting the mixing ratio of LC and polymer, and UV curing temperatures. It was revealed that the MSLC film with small LC droplets (a few micrometers) showed higher haze value than that with large LC droplets (a few tens of micrometers). Under optimal conditions, the MSLC exhibited a high optical haze value up to ~97% (at 550 nm). By simply attaching this MSLC film onto the front surface of a silicon-based PV modules, an overall PCE enhancement of 2.8% in comparison with the bare PV module was achieved. The improvement in the PV performance was attributed to enhancement of the light absorption in the active layer of the PV cell through diffusion of the incident light via the haze film. Our results demonstrate the promising potential of MSLC films, which can be easily fabricated using simple and inexpensive LC technology for all types of PV devices. We expect that our approach will be widely used for developing high-performance and cost-effective PV devices in the future.


Recent research has shed light on the plethora of mechanisms by which the gastrointestinal commensal microbiome can influence the local immune response in the gut . However, an area that is much less well explored but of tremendous therapeutic interest is the impact the gut microbiome has on systemic cell-mediated immune responses. In this commentary, we highlight some key studies that are beginning to broadly examine the different mechanisms by which the gastrointestinal microbiome can impact the systemic immune compartment. Specifically, we discuss the effects of the gut microbiome on lymphocyte polarisation and trafficking, tailoring of resident immune cells in the liver, and output of circulating immune cells from the bone marrow. Finally, we explore contexts in which this new understanding of long-range effects of the gut microbiome can have implications, including cancer therapies and vaccination. Functions include those associated with digestion and nutrient status, but sensing of the gut microbiome is also understood to have profound effects on the immune system.The human intestine houses a tremendous quantity and remarkable diversity of microbes, including bacteria, fungi, viruses, and protozoa. Such organisms, collectively termed the gut microbiome, form complex ecosystems capable of performing a diverse array of functions that have a wide spectrum of effects on their host’s physiology and hence health5  production by various lymphocyte subpopulations, including T helper type 17 (Th17) cells, γδ T cells, and type 3 innate lymphoid cells (ILC3s), stimulates antimicrobial protein release by epithelial cells9.Much of this understanding is centred on the effects of the microbiome on the development of local immune responses in the gut, particularly those related to the crucial tasks of maintaining a healthy complex microbial composition and preventing microbes from breaching the simple (one-cell-thick/unilayered) epithelium5 . For exa10 , is associated with immune-mediated inflammation in distal organs, including the joints, skin, and eyes14. However, even in the healthy gut, microbial products constantly transit into the circulation17, and it has been suggested that in health the gastrointestinal microbiome acquires a rheostat-like function, tuning the systemic immune system1.It is increasingly recognised that the gut microbiome can influence not only these local effects on the mucosal immune system but also cell-mediated systemic immune responses10 . Evidenc19, but owing to the recent dramatic advances in the microbiome field these systemic effects are now becoming an area of tremendous research interest. In this commentary, we will focus on three actions that are emerging as keystone mechanisms by which the gut microbiome impacts systemic cell-mediated immunity and their implications for therapies. We will specifically discuss (1) lymphocyte polarisation, trafficking, and cross-reactivity; (2) direct effects of bacterial ligands on distal organ immune cell development and function; and (3) modulation of immune cell output during haematopoiesis.In the 1980s and 1990s, studies using antibiotic treatment and germ-free (GF) mice highlighted effects of the microbiome on systemic immunity21. Indeed, in a number of animal models (described below), it has been established that modifying gut T cells can impact systemic disease either in a non-antigen-specific manner through bystander effects or in an antigen-specific fashion as a result of molecular mimicry by commensal factors.At this time, perhaps the best-characterised mechanism by which the gut microbiome is known to influence systemic immune responses is via its influences on the adaptive immune system, particularly the T-cell compartment22. This is linked to decreased germinal centre formation systemically in the animals with depleted microbiota, hence explaining the lower serum autoantibodies. When the gut of GF animals was recolonised with Th17-inducing segmented filamentous bacteria (SFB), autoimmune arthritis incidence was restored. In this setting, activated Th17 cells from the gut trafficked to the spleen, where they supported germinal centre formation and ultimately increased production of disease-mediating autoantibodies22. Of note, this germinal centre formation is also dependent upon T follicular helper (Tfh) cells as in K/BxN mice it has also been shown that Tfh cells generated in the Peyer’s patches in response to SFB can transit to the spleen and support autoantibody production23.In a mouse model of spontaneous autoimmune arthritis (K/BxN), GF or antibiotic-treated animals have lower serum autoantibody titres (that are associated with disease development) and ameliorated disease24. Once again, when mice were mono-colonised with SFB, this induced Th17 cells in the gut and resulted in enhanced neurodegeneration in the CNS25.Similarly, in experimental autoimmune encephalomyelitis (EAE), a murine model for multiple sclerosis, altering the gut microbiome has been shown to modulate central nervous system (CNS) autoimmunity in a T-cell-dependent manner. In a model of spontaneous EAE, SJL/J mice raised in GF conditions were protected against developing the disease while the introduction of commensal microbiota into the gut restored susceptibility26. SCFAs promote the differentiation of peripherally induced regulatory T cells (Tregs) and in this manner are capable of shifting the balance of effector T cells to Treg cells to limit the development of systemic inflammation27. Although butyrate and propionate are dominantly restricted to the gut and hepatic portal circulation28, acetate can be found in the circulation, implying that SCFAs could be directly sensed by circulating T cells to alter their function.The impact of the gut microbiome on peripheral T-cell subsets can also have positive effects on inflammatory diseases. Through its capacity to induce regulatory populations, the microbiome can also support the suppression of inflammatory responses. One particularly important mechanism is via the production of short-chain fatty acids (SCFAs), including butyrate, propionate, and acetate. SCFAs are generated by the microbiota as a result of its capacity to break down fibre and are now recognised as a keystone metabolite sensed by the immune system and capable of immunomodulation29. Additionally, in autoimmune uveitis, T cells specific for self-antigens are first activated in the gut before trafficking to the eye30. Conversely, myelin oligodendrocyte (MOG)-specific intraepithelial lymphocytes (IELs) were shown to transit from the gut to the CNS, where they were able to suppress neuroinflammation locally via a LAG3-dependent mechanism31, again demonstrating, as is the case for the bystander effect of the microbiome on T cells, that gut commensals can be important in balancing systemic immune responses.Antigen-specific T-cell responses, as opposed to the bystander effects already described, have also been shown to play both positive and negative roles in immune-mediated diseases. Of particular note, myelin basic protein (MBP)-specific T cells can respond to structurally related microbial peptides that can result in neurodegeneration34. The composition of the immune compartment in the liver, as at other sites, is highly tailored to the specialised physiologic and immunologic requirements of the organ. Of note, the immune system of the liver, compared with that of other organs, is particularly enriched in unconventional lymphocyte populations, including invariant natural killer T (iNKT) cells and γδ T cells. These cells, which can respond to microbial lipids, are important in the protection against infections that manage to enter the circulation, but their aberrant activity can also lead to liver pathology35. A number of studies have implicated the gut microbiota in determining the establishment and function of these dominant cell populations.Independently of activation and T-cell trafficking from the gut-associated lymphoid tissue (GALT) to peripheral sites, resident immune cell function in organs distal to the gut can also be directly impacted by their sensing of circulating commensal-derived factors. This is particularly well exemplified by the immune populations of the liver, an organ that receives about 80% of its blood via a major tributary associated with the gut, the portal vein36. Additionally, iNKT cells can exhibit functional alterations in the absence of a commensal microbiome, as they have a less-mature phenotype and are hyporesponsive to stimulation with the lipid α-galactosylceramide 37. A study by Liet al. showed that, alongside effects on iNKT cells, IL-17A-producing liver γδ T cells are also supported by the commensal microbiota35. Notably, GF or antibiotic-treated animals had reduced numbers of hepatic IL-17A-producing γδ T cells; complete restoration of this population was possible through recolonisation with a complex microbiota, whilst partial restoration occurred upon the addition ofEscherichia coli alone in a dose-dependent manner35.iNKT cell numbers have been described to be positively or negatively regulated by the commensal microbiome depending on the strain of animal and type of microbiota present38. These cells are responsive to microbial signals via their expression of various Toll-like receptors (TLRs)39. In the early 1990s40, it was suggested that KCs can recognise and respond to intestine-derived bacterial endotoxins; more recently, it has been demonstrated that KC proliferation and major histocompatibility complex II (MHC II) expression are controlled by a live gut microbiome41. Indeed, aberrant changes to the gut microbiome are associated with increased hepatic inflammation, mediated partly by KC recognition of intestinal microbiota-associated molecular patterns via TLR-4/9 signalling and their subsequent upregulation of tumour necrosis factor-alpha (TNF-α)42.The liver is home to not only iNKT cells and γδ T cells but also various antigen-presenting cell populations, including dendritic cells (DCs) and the major liver-resident macrophage, the Kupffer cell (KC)44, various scavenger receptors46, and lymphocyte adhesion molecules such as DC-SIGN47. Along with KCs, LSECs prime liver-localised CD8+ and CD4+ T cells in response to the recognition of microbe-associated ligands passing through the sinusoids that can originate from the intestine44. Thus, overall, the cellular composition and gross structure of the liver seem to co-operatively enable resident immunological subsets to respond to microbe-derived ligands derived from the intestinal microbiota.Though not a haematopoietic immune population, liver sinusoidal endothelial cells (LSECs) can also present antigens recognised in the sinusoidal space because of their expression of MHC I and MHC II+ and CD8+ T cells by LSECs49 and IL-10 secretion by KCs and conventional DCs51. However, these immunologic subsets remain capable of responding to high LPS concentrations, and it is possible that increased LPS stimulation (greater than the baseline levels), or LPS exposure in conjunction with additional pathogen-associated molecular patterns (PAMPs)/metabolites, acts as a means to signal alterations to the commensal microbiome or intestinal barrier breach or both. This was previously proposed by Belkaid and Naik, who suggested that the liver may sense a commensal microbiome ‘molecular fingerprint’ and that changes to this ‘fingerprint’ could act as an alarm to the periphery10. Perturbations to this dialogue between the gut and liver are exemplified by the pathological progression of alcoholic liver disease (ALD). ALD is associated with increased gut permeability  and in turn endotoxemia52, mediated by increased ethanol consumption and commensal outgrowth53, where the recognition of increased LPS titres by TLR-4 and CD14 leads to hepatic inflammation and steatosis54.As discussed, even in the absence of intestinal inflammation, the liver is chronically exposed to intestinally derived microbial products such as lipopolysaccharide (LPS). Owing to this baseline LPS exposure, endotoxin tolerance is observed in the liver and is associated with the priming and entrapment of tolerogenic CD455. Whether this is mediated by direct effects of microbial ligands on mature immune populations or is due to alterations in haematopoietic development (as discussed in the next section) is unclear. As in the liver, these effects are just beginning to be explored and hold much potential for understanding systemic complications associated with shifts in the commensal microbiome.It is clear that immune populations in highly vascularised organs in addition to the liver can be impacted by the gut microbiome. In particular, non-mucosal mononuclear phagocytes have been shown to have altered methylation patterns at key genes associated with type I interferon (IFN) production in GF animals, leading to impaired priming of natural killer cells in the spleen57. Studies in the 1980s of GF animals, alongside specific pathogen-free animals treated with the antibiotic polymyxin, implicated Gram-negative commensal bacteria in promoting the development of bone marrow granulocyte-monocyte progenitor cells18. In line with this dependency of granulocyte-monocyte progenitors on the gut microbiota, more recent investigations established deficiencies in differentiated myeloid cell populations in both the spleen and the bone marrow of GF mice56.The ability of microbiome-derived ligands and their metabolites to enter the circulation allows resident bacteria in the gut to modulate the immune system from the earliest times of immune cell development during haematopoiesis60. In humans, signalling via TLR-2 and TLR-7 directs haematopoietic differentiation towards a myeloid cell fate62. After myeloid differentiation, microbiome-derived ligands can also augment the release of myeloid populations from the bone marrow. Sensing of low-level changes of the TLR-4 ligand LPS, which reflects fluctuations in circulating microbial molecules after their absorption from the gut, supports the release of mature monocytes from the murine bone marrow in a CCL-2-dependent fashion63.Human and mouse haematopoietic stem cells express TLRs, providing a mechanism by which circulating microbiome-derived ligands could instruct haematopoiesis64. Gastrointestinal helminth infections can lead to alterations in the gut microbiota such that systemic SCFA levels are increased65. In line with these increases in SCFA during helminth infection, DC precursors are also modulated in the bone marrow65. Ultimately, this altered bone marrow output can have implications for inflammation at other mucosal sites. For example, following the administration of propionate in drinking water, the development of allergic responses in the lung is reduced. This alteration is associated with the presence of DCs that have impaired T-cell-activating capacity in the lung64. This finding highlights the possibility that manipulating the factors, such as metabolites, that gut microbiota produce limits inflammation at distal sites.Another mechanism by which the gut microbiome can influence haematopoiesis is via exposure to commensal-dependent metabolites. Systemic increases of the SCFA propionate, by administration in drinking water, led to alterations in bone marrow haematopoiesis characterised by enhanced DC precursor production67.As highlighted, better characterisation of the gut microbiota and understanding of its mechanisms of action on systemic immune responses hold tremendous opportunities for the development of therapeutics and also patient stratification. Two current areas of particular research interest are cancer therapy and vaccination responses69, while recolonisation with specific bacterial species can lead to a restored anti-tumour efficacy70. Recently, immunotherapy has revolutionised cancer treatment, particularly with regard to checkpoint blockade. In this context, T-cell pathways that are associated with regulatory checkpoints, such as PD-1 and CTLA-4, are inhibited to augment anti-tumour responses71. Remarkably, the capacity of CTLA-4 blockade to have anti-tumour effects was reliant onBacteroides species72. Both a mouse model and studies in patients revealed that specific T-cell responses toBacteroides thetaiotaomicron andBacteroides fragilis correlate with the efficacy of CTLA-4 blockade72. In a similar vein,Bifidobacterium was shown to improve melanoma control alongside PD-L1-specific antibody therapy. Indeed, in tandem, the presence ofBifidobacterium with PD-L1 resulted in tumour clearance73.GF and antibiotic-treated mice show reduced tumour regression and impaired survival following treatment with chemotherapeutic agents compared with controls74. This effect is dependent upon the capacity of the microbiome to act as an adjuvant via TLR-5-mediated sensing of bacterial flagellin. Oral reconstitution of GF mice with flagellated strains ofE. coli (but not aflagellated forms) restored vaccine responses. Interestingly, this effect may be specific to certain types of vaccine, as the seasonal influenza vaccine is a non-adjuvanted vaccine74. Studies of human cohorts, alongside murine models, have demonstrated effects of the microbiome on vaccine responses. For example, the relative abundance of specific bacterial species in stool microbiota of a small cohort of Bangladeshi infants was correlated with vaccine-specific IgG and T-cell proliferation towards vaccinations, including Bacillus Calmette–Guérin and hepatitis B vaccine75.It is becoming clear that the precise gut microbiome of an individual also has implications for the development of vaccine-mediated protection. This has perhaps been best demonstrated in GF animals or animals treated with antibiotics, where an absence or reduced gut microbiome is associated with impaired IgG and IgM responses to the seasonal influenza vaccine76. It is clear that, irrespective of the exact mechanisms, improved understanding of the key pathways and bacterial species involved in systemic instruction of the immune system holds promise to inform the development of novel therapeutic strategies to modify immune function. One such evolving strategy is faecal microbiota transplantation (FMT), in which the faecal material from a healthy donor is transferred to a patient with suspected microbial dysbiosis to restore diversity of the commensal microbiota77. FMT has been used successfully to treat recurrentClostridium difficile infection in patients without IBD78, and studies suggest that FMT may be beneficial to some patients with IBD79. Whether FMT can also promote the resolution of systemic disease symptoms associated with gut inflammation and microbial alterations is just beginning to be explored, and studies are underway in diseases, including psoriatic arthritis80.Researchers are just beginning to understand the variety of mechanisms by which the gut microbiome can influence systemic immunity and the implications of this for human health. This review has highlighted three distinct types of mechanism that are already being explored . Another82. In rodent models, probiotics can improve systemic inflammatory disease, such as joint inflammation83. However, a recent publication has suggested that, following antibiotic treatment, the use of probiotics may compromise gut mucosal recovery, demonstrating that such therapies need to be employed with caution84. These early studies of microbiome-modifying therapies reveal that much future work is required to translate our rapidly advancing knowledge of how the gut microbiome impacts systemic immunity into altered patient outcomes. Even so, there is no doubt that such research does hold much promise for improving treatments in diverse disease states from cancer to autoimmunity.Another widely promoted approach for modulating the gut microbiome is the use of probiotic therapies