Facts and Hopes on Biomarkers for Successful Early Clinical Immunotherapy Trials: Innovative Patient Enrichment Strategies.

Despite the clinical validation and unequivocal benefit to patients, the development of cancer immunotherapies is facing some key challenges and the attrition rate in early phases of development remains high. Identifying the appropriate patient population that would benefit most from the drug is on the critical path for successful clinical development. We believe that a systematic implementation of patient enrichment strategies early in the drug development process and trial design, is the basis for an innovative, more efficient, and leaner clinical development to achieve earlier a clear proof of concept or proof of failure. In this position article, we will describe and propose key considerations for the implementation of patient enrichment strategies as an opportunity to provide decision-enabling data earlier in the drug development process. We introduce an innovative multidimensional tool for immuno-oncology drug development that focuses on facilitating the identification and prioritization of enrichment-relevant biomarkers, based on the drug mechanism of action. To illustrate its utility, we discuss patient enrichment examples and use a case in the field of cancer immunotherapy, together with technical and regulatory considerations. Overall, we propose to implement fit for purpose enrichment strategies for all investigational drugs as early as possible in the development process. We believe that this will increase the success rate of immuno-oncology clinical trials, and eventually bring new and better medicines to patients faster.

Deciphering molecular and cellular ex vivo responses to bispecific antibodies PD1-TIM3 and PD1-LAG3 in human tumors.

BACKGROUND: Next-generation cancer immunotherapies are designed to broaden the therapeutic repertoire by targeting new immune checkpoints including lymphocyte-activation gene 3 (LAG-3) and T cell immunoglobulin and mucin-domain containing-3 (TIM-3). Yet, the molecular and cellular mechanisms by which either receptor functions to mediate its inhibitory effects are still poorly understood. Similarly, little is known on the differential effects of dual, compared with single, checkpoint inhibition. METHODS: We here performed in-depth characterization, including multicolor flow cytometry, single cell RNA sequencing and multiplex supernatant analysis, using tumor single cell suspensions from patients with cancer treated ex vivo with novel bispecific antibodies targeting programmed cell death protein 1 (PD-1) and TIM-3 (PD1-TIM3), PD-1 and LAG-3 (PD1-LAG3), or with anti-PD-1. RESULTS: We identified patient samples which were responsive to PD1-TIM3, PD1-LAG3 or anti-PD-1 using an in vitro approach, validated by the analysis of 659 soluble proteins and enrichment for an anti-PD-1 responder signature. We found increased abundance of an activated (HLA-DR+CD25+GranzymeB+) CD8+ T cell subset and of proliferating CD8+ T cells, in response to bispecific antibody or anti-PD-1 treatment. Bispecific antibodies, but not anti-PD-1, significantly increased the abundance of a proliferating natural killer cell subset, which exhibited enrichment for a tissue-residency signature. Key phenotypic and transcriptional changes occurred in a PD-1+CXCL13+CD4+ T cell subset, in response to all treatments, including increased interleukin-17 secretion and signaling toward plasma cells. Interestingly, LAG-3 protein upregulation was detected as a unique pharmacodynamic effect mediated by PD1-LAG3, but not by PD1-TIM3 or anti-PD-1. CONCLUSIONS: Our in vitro system reliably assessed responses to bispecific antibodies co-targeting PD-1 together with LAG-3 or TIM-3 using patients' tumor infiltrating immune cells and revealed transcriptional and phenotypic imprinting by bispecific antibody formats currently tested in early clinical trials.

Personality traits modulate the neural responses to handwriting processing.

Handwriting is a vital skill for everyday human activities. It has a wealth of information about writers' characteristics and can hint toward underlying neurological conditions, such as Parkinson's disease, autism, dyslexia, and attention-deficit/hyperactivity disorder (ADHD). Many previous studies have reported a link between personality and individual differences in handwriting, but the evidence for the relationship tends to be anecdotal in nature. Using functional magnetic resonance imaging (fMRI), we examined whether the association between personality traits and handwriting was instantiated at the neural level. Results showed that the personality trait of conscientiousness modulated brain activation in the left premotor cortex and right inferior/middle frontal gyrus, which may reflect the impact of personality on orthography-to-grapheme transformation and executive control involved in handwriting. Such correlations were not observed in symbol-drawing or word-reading tasks, suggesting the specificity of the link between conscientiousness and handwriting in these regions. Moreover, using a connectome-based predictive modeling approach, we found that individuals' conscientiousness scores could be predicted based on handwriting-related functional brain networks, suggesting that the influence of personality on handwriting may occur within a broader network. Our findings provide neural evidence for the link between personality and handwriting processing, extending our understanding of the nature of individual differences in handwriting.

A human receptor occupancy assay to measure anti-PD-1 binding in patients with prior anti-PD-1.

Receptor occupancy (RO) assessment by flow cytometry is an important pharmacodynamic (PD) biomarker in the clinical development of large molecules such as monoclonal therapeutic antibodies (mAbs). The total-drug-bound RO assay format directly assesses mAb binding to cell surface targets using anti-drug detection antibodies. Here, we generated a flow cytometry detection antibody specifically binding to mAbs of the IgG1 P329GLALA backbone. Using this reagent, we developed a total-drug-bound RO assay format for RG7769, a bi-specific P329GLALA containing mAb targeting PD-1 and TIM3 on T cells. In its fit-for-purpose validated version, this RO assay has been used in the Phase-I dose escalation study of RG7769, informing on peripheral T cell RO and RG7769 antibody binding capacity (ABC). We assessed RG7769 RO in checkpoint-inhibitor (CPI) naïve patients and anti-PD-1 CPI experienced patients using our novel assay. Here, we show that in both groups, complete T cell RO can be achieved (~100%). However, we found that the maximum number of T cell binding sites for RG7769 pre-dosing was roughly twofold lower in patients recently having undergone anti-PD-1 treatment. We show that this is due to steric hindrance exerted by competing mAbs masking the available drug binding sites. Our findings highlight the importance of quantitative mAb assessment in addition to relative RO especially in the context of patients who have previously received anti-PD-1 treatment.

Chinese calligraphy handwriting (CCH): a case of rehabilitative awakening of a coma patient after stroke.

INTRODUCTION: This study investigated the efficacy of Chinese calligraphy handwriting (CCH) for the awakening of patients under a vegetative state after stroke. The theories, the instrument, and the treatment protocols were reported. A single case of a severe stroke patient who was in a coma state for 2 years is presented in this study. The objectives were to apply finger writing as a new method to awaken a stroke patient in a coma state and to test the effect of this method in improving the patient's vegetative states over time. CASE PRESENTATION: A 55-year-old man suffered a severe stroke in 2004 which left him in a coma for 2 years without any systematic rehabilitation. A culture-based finger-writing method of visual-spatial intervention was then applied to improve his condition. The writing tasks involved aided viewing and finger tracing of sets of innovative characters with enriched visual-spatial and movement characteristics. Following regular treatment protocols involving diverse movement and sensory feedback, the patient was awakened after 12 months. As a consequence, the patient showed significant behavioral changes favoring enhanced focusing, alertness, visual scan, visual span, and quickened visual and motor responses. The treatment continued for another 12 months. As the treatment progressed, we gradually observed improvements in his attention span and mental concentration. His eye ball movements - the left eye in particular - were quickened and showed wider visual angularity in his focal vision. Currently, the patient can now watch television, engage in improved visual sighting, and focus on visual-spatial and cognitive-linguistic materials. CONCLUSION: This CCH method of training by finger tracking has shown its effectiveness in awakening the patient from his coma state and in producing long-term, clinical outcomes that were similar from those that took place 10 years ago. This finding supports the efficacy of the system for clinical improvement of the patient's conditions.

Anatomical and electrophysiological characterization of a population of dI6 interneurons in the neonatal mouse spinal cord.

The locomotor central pattern generator is a neural network located in the ventral aspect of the caudal spinal cord that underlies stepping in mammals. While many genetically defined interneurons that are thought to comprise this neural network have been identified and characterized, the dI6 cells- which express the transcription factors WT1 and/or DMRT3- are one population that settle in this region, are active during locomotion, whose function is poorly understood. These cells were originally hypothesized to be commissural premotor interneurons, however evidence in support of this is sparse. Here we characterize this population of cells using the TgDbx1Cre;R26EFP;Dbx1LacZ transgenic mouse line, which has been shown to be an effective marker of dI6 interneurons. We show dI6 cells to be abundant in laminae VII and VIII along the entire spinal cord and provide evidence that subtypes outside the WT1/DMRT3 expressing dI6 cells may exist. Retrograde tracing experiments indicate that the majority of dI6 cells project descending axons, and some make monosynaptic or disynaptic contacts onto motoneurons on either side of the spinal cord. Analysis of their activity during non-resetting deletions, which occur during bouts of fictive locomotion, suggests that these cells are involved in both locomotor rhythm generation and pattern formation. This study provides a thorough characterization of the dI6 cells labeled in the TgDbx1Cre;R26EFP;Dbx1LacZ transgenic mouse, and supports previous work suggesting that these cells play multiple roles during locomotor activity.

Probing diversity within subpopulations of locomotor-related V0 interneurons.

The V0 interneuronal population is derived from Dbx1 expressing progenitors. Initial studies on these interneurons in the mouse spinal cord demonstrated that they project commissural axons and are involved in coordinating left-right alternation during locomotion. Subsequent work has indicated that the V0 population can be divided into genetically distinct ventral (V0V) and dorsal (V0D) subpopulations, and experimental evidence suggests that each is responsible for left-right alternation at different locomotor speeds. In this study, we perform a series of experiments to probe the location and connectivity of these subpopulations in neonatal mice and demonstrate that they are more diverse than previously predicted. While the distribution of either subpopulation remains consistent along the extent of the lumbar spinal cord, a cluster of V0D cells lateral to the central canal receive substantial input from primary afferents. Retrograde tracing and activity dependent labeling experiments demonstrate that a group of V0 interneurons located in this same region preferentially project axons towards contralateral motoneurons via an oligosynaptic pathway, and are active during fictive locomotion. Our results suggest that this subset of V0 interneurons may be primarily responsible for coordination of left-right alternation during locomotion. Furthermore these experiments indicate that while genetic identity is one determinant of the function of a neuron during locomotion, the specific position in which the cell is located may also play a key role.

Effect of calligraphy training on hyperarousal symptoms for childhood survivors of the 2008 China earthquakes.

BACKGROUND: This study investigated the treatment effects of calligraphy therapy on childhood survivors of the 2008 Sichuan earthquakes in the People's Republic of China. METHODS: In experiment 1, 129 children participated in a 30-day calligraphic training, and 81 children were controls. The Children's Revised Impact of Event Scale was adopted to assess behavioral effects. Experiment 2 involved 41 treatment subjects and 39 controls, with the same procedure as in experiment 1 except that salivary cortisol level was also measured as a physiological indicator. RESULTS: After 30 days of calligraphy treatment, the arousal symptoms and salivary cortisol levels in the experimental group decreased from 5.72±0.31 and 13.34±2.88 to 4.98±0.31 and 9.99±2.81, respectively. In the control group, there was not a significant decrease from pretest to post-test. In addition, the arousal scores in posttest (4.98±4.39) were significantly lower than midtest (5.71±4.14) for girls; in contrast, for boys, posttest (4.90±4.24) showed little change compared with midtest (5.04±4.36), but both were significantly lower than pretest (6.42±4.59). CONCLUSIONS: Calligraphy therapy was effective in reducing hyperarousal symptoms among child survivors.

Calligraphy and meditation for stress reduction: an experimental comparison.

BACKGROUND: Chinese calligraphic handwriting (CCH) has demonstrated a new role in health and therapy. Meanwhile, meditation is an traditional and effective method for coping with stress and staying healthy. This study compared the effectiveness of CCH and meditation as distinctive and parallel stress reduction interventions. METHODS: Thirty graduate students and academic staff members in Taiwan who suffered from stress were selected by the General Health Questionnaire and randomly assigned to one of three treatment groups, ie, a CCH group, a meditation group, or a control group, for 8 consecutive weeks. Changes in physiological parameters were measured before, during, and after treatment. RESULTS: CCH and meditation showed their strength in the respective indices of stress. There was a significant difference in respiratory rate, heart rate, and electromyographic scores between the groups. Comparing pre- and post-effects, a decrease in heart rate and an increase in skin temperature was seen in subjects who practiced CCH. Increased skin temperature and decreased respiratory rate were also seen in subjects who practiced meditation, along with reduced muscle tension and heart rate. CONCLUSION: CCH and meditation have good effects in stress reduction. CCH is a particularly promising new approach to reducing stress.

Cognitive-neural effects of brush writing of chinese characters: cortical excitation of theta rhythm.

Chinese calligraphy has been scientifically investigated within the contexts and principles of psychology, cognitive science, and the cognitive neuroscience. On the basis of vast amount of research in the last 30 years, we have developed a cybernetic theory of handwriting and calligraphy to account for the intricate interactions of several psychological dimensions involved in the dynamic act of graphic production. Central to this system of writing are the role of sensory, bio-, cognitive, and neurofeedback mechanisms for the initiation, guidance, and regulation of the writing motions vis-a-vis visual-geometric variations of Chinese characters. This experiment provided the first evidence of cortical excitation in EEG theta wave as a neural hub that integrates information coming from changes in the practitioner's body, emotions, and cognition. In addition, it has also confirmed neurofeedback as an essential component of the cybernetic theory of handwriting and calligraphy.

Cognitive effects of calligraphy therapy for older people: a randomized controlled trial in Hong Kong.

BACKGROUND: This pilot study investigated the effects of calligraphy therapy on cognitive function in older Hong Kong Chinese people with mild cognitive impairment. METHODS: A single-blind, randomized controlled trial was carried out in a sample of 31 adults aged 65 years or older with mild cognitive impairment. They were randomly assigned to receive either intensive calligraphy training led by a trained research assistant for eight weeks (calligraphy group, n = 14) or no calligraphy treatment (control group, n = 17). Participants' cognitive function was assessed by the Chinese version of the Mini-Mental State Examination (CMMSE) before and after calligraphy treatment. Repeated measures analysis of variance and paired samples t-tests were used to analyze the data. RESULTS: A significant interaction effect of time and intervention was detected [F (1, 29) = 9.11, P = 0.005, η(2) = 0.24]. The calligraphy group was found to have a prominent increase in CMMSE global score, and scores in the cognitive areas of orientation, attention, and calculation after two months (ΔM = 2.36, P < 0.01), whereas their counterparts in the control group experienced a decline in CMMSE score (ΔM = -0.41, P < 0.05). CONCLUSION: Calligraphy therapy was effective for enhancing cognitive function in older people with mild cognitive impairment and should be incorporated as part of routine programs in both community and residential care settings.

Postoperative acquired coagulopathy: a pilot study to determine the impact on clinical and economic outcomes.

STUDY OBJECTIVE: To characterize the clinical factors associated with postoperative acquired coagulopathy, and to estimate the economic impact of resources used to treat postoperative patients with this coagulopathy compared with postoperative patients who did not develop the coagulopathy. DESIGN: Case-control study. SETTING: Academically affiliated public hospital and level I trauma referral center. PATIENTS: Twenty-six patients (mean age 53.9 yrs) who experienced acquired coagulopathy after undergoing an index surgery (cases), and 26 patients (mean age 50.8 yrs) matched to these case patients by index surgery, age, and sex (controls). MEASUREMENTS AND MAIN RESULTS: Data were collected from a database of 5367 adult surgical admissions over 6 months during 2008, corresponding inpatient electronic health records, billing data, and Medicare Resource-Based Relative Value Scale payments. Case patients had a minimum of two postoperative consecutively drawn episodes of prothrombin time (PT) or activated partial thromboplastin time (aPTT) elevated to greater than 20% above the upper limit of normal. Patients with inherited clotting disorders or other identifiable causes of coagulopathy were excluded. Case patients underwent the following surgeries: 12 orthopedic (46%), six cardiovascular (23%), four gastrointestinal (15%), and four neurosurgical (15%). Mean values of the first elevated PT and aPTT were 19.7 and 50.8 seconds, respectively. Mean postoperative stay was 31.5 days for cases versus 9.8 days for controls (p<0.05). Mean cost (2008 U.S. dollars) of resources used was $112,280 for cases versus $38,357 for controls (p<0.001). Costs incurred between the onset of coagulopathy and discharge constituted 67% of postoperative costs. Physician reimbursement expenditures were minimal. CONCLUSION: Postoperative acquired coagulopathy was associated with stays that were 3 times longer and resource use costs that were 3 times higher than those of controls. This type of coagulopathy may be an under-recognized and underappreciated event. The case-control design is limited to exploring associations and does not establish causality. Prospective studies need to be conducted to establish the causes of acquired coagulopathy and methods for screening and diagnosing this condition.

The effects of Chinese calligraphy handwriting and relaxation training in Chinese Nasopharyngeal Carcinoma patients: a randomized controlled trial.

BACKGROUND: Chinese calligraphy handwriting is the practice of traditional Chinese brush writing, researches found calligraphy had therapeutic effects on certain diseases, some authors argued that calligraphy might have relaxation effect. OBJECTIVES: This study was to compare the effects of calligraphy handwriting with those of progressive muscle relaxation and imagery training in Chinese Nasopharyngeal Carcinoma patients. DESIGN AND PARTICIPANTS: This study was a randomized controlled trial. Two hundred and eighty-seven Nasopharyngeal Carcinoma patients were approached, ninety (31%) patients were recruited and randomized to one of the three treatment groups: progressive muscle relaxation and guided imagery training group, Calligraphy handwriting group, or a Control group. Seventy-nine (87.8%) completed all of the outcome measures. OUTCOME MEASURES: The primary treatment outcome was the changes of physiological arousal parameters measured by pre- and post-treatment differences of heart rate, blood pressure and respiration rate. The secondary outcomes included: modified Chinese version of Symptom Distress Scale, Profile of Mood State-Short Form, and Karnofsky Performance Status measured at baseline, during treatment (after the 2-week intervention), post-treatment (after the 4-week intervention) and after a 2-week follow-up. Effectiveness was tested by repeated measure ANOVA analyses. SETTING: Cancer centre of a major university hospital in Guangdong, China. RESULTS: Results showed that both of calligraphy and relaxation training demonstrated slow-down effects on physiological arousal parameters. Moreover, calligraphy practice gradually lowered participants' systolic blood pressure (simple main effect of time at pre-treatment measure, p=.007) and respiration rate (p=.000) at pre- and post-treatment measures as the intervention proceeded, though with a smaller effect size as compared to relaxation. Both of calligraphy and relaxation training had certain symptom relief and mood improvement effects in NPC patients. Relaxation was effective in relieving symptom of insomnia (p=.042) and improving mood disturbance, calligraphy elevated level of concentration (p=.032) and improved mood disturbance. CONCLUSIONS: Similar to the effects of relaxation training, calligraphy demonstrated a gradually build-up physiological slow-down, and associated with heightened concentration and improved mood disturbance. Calligraphy offered a promising approach to improved health in cancer patients.

Vitreous VEGF clearance is increased after vitrectomy.

Purpose. Pars plana vitrectomy (PPV) has been reported to reduce macular thickness and improve visual acuity in patients with diabetic macular edema (ME). The hypothesis for the study was that after PPV, clearance is accelerated and VEGF concentrations are reduced. To test this hypothesis, hVEGF(165) injections were performed in rabbit eyes, with and without PPV, and vitreous VEGF levels were measured as a function of time. Methods. The PPV group rabbits had a bilateral 25-gauge PPV, and in the no-PPV group, rabbits had intact vitreous. Intravitreal injections of hVEGF(165) were performed, and the animals were euthanatized at time points up to 7 days. The vitreous was isolated and an enzyme-linked immunosorbent assay was used to measure the VEGF levels. Pharmacokinetic parameters were determined in a noncompartmental analysis approach. Results. Mean vitreous VEGF levels decreased more rapidly in eyes subjected to PPV than in no-PPV eyes. The vitreous VEGF half-life (t([)(1/2)(])) in PPV eyes was 10 times shorter than that in normal eyes. In addition, mean clearance and mean area under the curve (AUC) increased and decreased, respectively, in eyes that underwent PPV. Conclusions. VEGF clearance is increased after PPV. Reducing VEGF concentrations in the vitreous post-PPV may partially explain the improvement in macular thickness in some patients with ME. Unexpectedly, the half-life of VEGF in the vitreous, even in no-PPV eyes, was <3 hours, whereas compounds of similar molecular weight typically have longer vitreous half-lives. The back of the eye may be uniquely adapted with rapid-clearance mechanisms to regulate vitreous VEGF levels. Further study is suggested.

Regulated movement of CD4 in and out of the immunological synapse.

The mechanism underlying the transient accumulation of CD4 at the immunological synapse (IS) and its significance for T cell activation are not understood. To investigate these issues, we mutated a serine phosphorylation site (S408) in the cytoplasmic tail of murine CD4. Preventing phosphorylation of S408 did not block CD4 recruitment to the IS; rather, it blocked the ability of CD4 to leave the IS. Surprisingly, enhanced and prolonged CD4 accumulation at the supramolecular activation cluster in the contact area had no functional consequence for T cell activation, cytokine production, or proliferation. Protein kinase C theta (PKCtheta)-deficient T cells also displayed enhanced and prolonged accumulation of wild-type CD4 at the IS, indicating that theta is the critical PKC isoform involved in CD4 movement. These findings suggest a model wherein recruitment of CD4 to the IS allows its phosphorylation by PKCtheta and subsequent removal from the IS. Thus, an important role for PKCtheta in T cell activation involves its recruitment to the IS, where it phosphorylates specific substrates that help to maintain the dynamism of protein turnover at the IS.

Protein kinase C-theta is required for efficient positive selection.

Protein kinase C-theta (PKCtheta) is critical for TCR-initiated signaling in mature T cells, but initial reports found no requirement for PKCtheta in thymocyte development. Thymocytes and peripheral T cells utilize many of the same signaling components and, given the significant role of PKCtheta in peripheral T cells, it was surprising that it was not involved at all in TCR signaling in thymocytes. We decided to re-evaluate the role of PKCtheta in thymocyte development using the well-characterized class II-restricted n3.L2 TCR-transgenic TCR model. Analysis of n3.L2 PKCtheta(-/-) mice revealed a defect in thymocyte-positive selection, resulting in a 50% reduction in the generation of n3.L2 CD4 single-positive thymocytes and n3.L2 CD4 mature T cells. Competition between n3.L2 WT and n3.L2 PKCtheta(-/-) thymocytes in bone marrow chimeras revealed a more dramatic defect, with a >80% reduction in generation of n3.L2 CD4 single-positive thymocytes derived from PKCtheta(-/-) mice. Inefficient positive selection of n3.L2 PKCtheta(-/-) CD4 single-positive cells resulted from "weaker" signaling through the TCR and correlated with diminished ERK activation. The defect in positive selection was not complete in the PKCtheta(-/-) mice, most likely accounted for by compensation by other PKC isoforms not evident in peripheral cells. Similar decreased positive selection of both CD4 and CD8 single-positive thymocytes was also seen in nontransgenic PKCtheta(-/-) mice. These findings now place PKCtheta as a key signaling molecule in the positive selection of thymocytes as well as in the activation of mature T cells.

An antagonist peptide mediates positive selection and CD4 lineage commitment of MHC class II-restricted T cells in the absence of CD4.

The CD4 coreceptor works together with the T cell receptor (TCR) to deliver signals to the developing thymocyte, yet its specific contribution to positive selection and CD4 lineage commitment remains unclear. To resolve this, we used N3.L2 TCR transgenic, RAG-, and CD4-deficient mice, which are severely impaired in positive selection, and asked whether altered peptide ligands can replace CD4 function in vivo. Remarkably, in the presence of antagonist ligands that normally deleted CD4+ T cells in wild-type mice, we induced positive selection of functional CD4 lineage T cells in mice deficient in CD4. We show that the kinetic threshold for positive and negative selection was lowered in the absence of CD4, with no evident skewing toward the CD8 lineage with weaker ligands. These results suggest that CD4 is dispensable as long as the affinity threshold for positive selection is sustained, and strongly argue that CD4 does not deliver a unique instructional signal for lineage commitment.

Cognitive facilitation associated with Chinese brush handwriting: the case of symmetric and asymmetric Chinese characters.

Chinese calligraphic handwriting involves a process of visual spatial structuring of the characters as well as the motor control of the brush to follow specific character configurations through a projection of the cognitive images of the character. Previous research has shown that Chinese calligraphic handwriting facilitates perceptual and cognitive abilities. Reading Chinese characters with geometric features also facilitates cognitive processing. This study investigated Chinese calligraphic handwriting effect on cognitive facilitation measured by responses to asymmetric and asymmetric Chinese characters. 11 participants performed Chinese calligraphic handwriting for 30 min. and had their reaction time and error rate taken before and after the writing session. The reaction time to asymmetric characters was significantly shortened after Chinese calligraphic handwriting. These results confirm the general Chinese calligraphic handwriting effect on cognitive facilitation, but the asymmetric characters seem to result in greater such effects than the symmetric characters.