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Objective: To create an immunodeficient mouse model that spontaneously develops hyperglycemia to serve as a diabetic host for human islets and stem cell-derived beta-cells in the absence or presence of a functional human immune system.Research Design and Methods: We backcrossed the Ins2(Akita) mutation onto the NOD-Rag1(null) IL2rgamma(null) strain and determined 1) the spontaneous development of hyperglycemia, 2) the ability of human islets, mouse islets, and dissociated mouse islet cells to restore euglycemia, 3) the generation of a human immune system following engraftment of human hematopoietic stem cells, and 4) the ability of the humanized mice to reject human islet allografts.Results: We confirmed the defects in innate and adaptive immunity and the spontaneous development of hyperglycemia conferred by the IL2rgamma(null), Rag1(null), and Ins2(Akita) genes in NOD-Rag1(null) IL2rgamma(null) Ins2(Akita) (NRG-Akita) mice. Mouse and human islets restored NRG-Akita mice to normoglycemia. Insulin-positive cells in dissociated mouse islets, required to restore euglycemia in chemically diabetic NOD-scid IL2rgamma(null) and spontaneously diabetic NRG-Akita mice, were quantified following transplantation via the intrapancreatic and subrenal routes. Engraftment of human hematopoietic stem cells in newborn NRG-Akita and NRG mice resulted in equivalent human immune system development in a normoglycemic or chronically hyperglycemic environment, with >50% of engrafted NRG-Akita mice capable of rejecting human islet allografts.Conclusions: NRG-Akita mice provide a model system for validation of the function of human islets and human adult stem cell, embryonic stem cell, or induced pluripotent stem cell-derived beta-cells in the absence or presence of an alloreactive human immune system. [ABSTRACT FROM AUTHOR]

In evaluating energy cost (EC) of walking, referred to as walking efficiency, the use of net measurement protocols (i.e. net=gross-resting) has recently been recommended. However, nothing is known about the comparative reproducibility of net protocols and the commonly used gross protocols. Ten minutes of resting and 5 minutes of walking at a self-selected speed were used to determine gross and net EC in 13 children with spastic cerebral palsy (CP; seven males, six females; mean age 8y 7mo [SD 3y 4mo], range 4y 1mo-13y) and in 10 children (three males, seven females) with typical development. In the former, their Gross Motor Function Classification System levels ranged from Level I to Level III; and seven had hemiplegia and six diplegia. There were four repeated sessions on different days, with periods of 1 week between sessions. Reproducibility was assessed for speed, and gross and net EC, by using the standard error of measurement. The results of this preliminary study showed that EC measurements were more variable for children with CP than for children with typical development. Furthermore, in both groups there was considerably more variability in the net measurements than in the gross measurements. We conclude that, on the basis of the methodology used, the use of gross EC, rather than net EC, seems a more sensitive measure of walking efficiency to detect clinically relevant changes in an individual child with CP. [ABSTRACT FROM AUTHOR]

Background: In order for cancers to progress, they must evade elimination by CD8 T cells or other immune mechanisms. CD8 T cells recognize and kill tumor cells that display immunogenic tumor peptides bound to MHC I molecules. One of the ways that cancers can escape such killing is by reducing expression of MHC I molecules, and loss of MHC I is frequently observed in tumors. There are multiple different mechanisms that can underly the loss of MHC I complexes on tumor and it is currently unclear whether there are particular mechanisms that occur frequently and, if so, in what types of cancers. Also of importance to know is whether the loss of MHC I is reversible and how such loss and/or its restoration would impact responses to immunotherapy. Here, we investigate these issues for loss of IRF1 and IRF2, which are transcription factors that drive expression of MHC I pathway genes and some killing mechanisms., Methods: Bioinformatics analyses of IRF2 and IRF2-dependent gene transcripts were performed for all human cancers in the TCGA RNAseq database. IRF2 protein-DNA-binding was analyzed in ChIPseq databases. CRISRPcas9 was used to knock out IRF1 and IRF2 genes in human and mouse melanoma cells and the resulting phenotypes were analyzed in vitro and in vivo., Results: Transcriptomic analysis revealed that IRF2 expression was reduced in a substantial subset of cases in almost all types of human cancers. When this occurred there was a corresponding reduction in the expression of IRF2-regulated genes that were needed for CD8 T cell recognition. To test cause and effect for these IRF2 correlations and the consequences of IRF2 loss, we gene-edited IRF2 in a patient-derived melanoma and a mouse melanoma. The IRF2 gene-edited melanomas had reduced expression of transcripts for genes in the MHC I pathway and decreased levels of MHC I complexes on the cell surface. Levels of Caspase 7, an IRF2 target gene involved in CD8 T cell killing of tumors, were also reduced. This loss of IRF2 caused both human and mouse melanomas to become resistant to immunotherapy with a checkpoint inhibitor. Importantly, these effects were reversible. Stimulation of the IRF2-deficient melanomas with interferon induced the expression of a functionally homologous transcription factor, IRF1, which then restored the MHC I pathway and responsiveness to CPI., Conclusions: Our study shows that a subset of cases within most types of cancers downregulates IRF2 and that this can allow cancers to escape immune control. This can cause resistance to checkpoint blockade immunotherapy and is reversible with currently available biologics., (© 2024. The Author(s).)

The high rate of recurrence after radiation therapy in triple-negative breast cancer (TNBC) indicates that novel approaches and targets are needed to enhance radiosensitivity. Here, we report that neuropilin-2 (NRP2), a receptor for vascular endothelial growth factor (VEGF) that is enriched on subpopulations of TNBC cells with stem cell properties, is an effective therapeutic target for sensitizing TNBC to radiotherapy. Specifically, VEGF/NRP2 signaling induces nitric oxide synthase 2 (NOS2) transcription by a mechanism dependent on Gli1. NRP2-expressing tumor cells serve as a hub to produce nitric oxide (NO), an autocrine and paracrine signaling metabolite, which promotes cysteine-nitrosylation of Kelch-like ECH-associated protein 1 (KEAP1) and, consequently, nuclear factor erythroid 2-related factor 2-mediated (NFE2L2-mediated) transcription of antioxidant response genes. Inhibiting VEGF binding to NRP2, using a humanized mAb, results in NFE2L2 degradation via KEAP1, rendering cell lines and organoids vulnerable to irradiation. Importantly, treatment of patient-derived xenografts with the NRP2 mAb and radiation resulted in significant tumor necrosis and regression compared with radiation alone. Together, these findings reveal a targetable mechanism of radioresistance, and they support the use of NRP2 mAb as an effective radiosensitizer in TNBC.

Omnipresent suppressive myeloid populations in the tumor microenvironment limit the efficacy of T-cell-directed immunotherapies, become more inhibitory after administration of T-cell checkpoint inhibitors, and are overall associated with worse survival of cancer patients. In early clinical trials, positive outcomes have been demonstrated for therapies aimed at repolarizing suppressive myeloid populations in the tumor microenvironment. We have previously described the key role of P-selectin glycoprotein ligand-1 (PSGL-1) in maintaining an inhibitory state of tumor-associated macrophages (TAMs), most of which express high levels of PSGL-1. Here we describe a novel, first-in-class humanized high-affinity monoclonal antibody VTX-0811 that repolarizes human macrophages from an M2-suppressive phenotype towards an M1 inflammatory phenotype, similar to siRNA-mediated knockdown of PSGL-1. VTX-0811 binds to PSGL-1 of human and cynomolgus macaque origins without inhibiting PSGL-1 interaction with P- and L-Selectins or VISTA. In multi-cellular assays and in patient-derived human tumor cultures, VTX-0811 leads to the induction of pro-inflammatory mediators. RNAseq data from VTX-0811 treated ex vivo tumor cultures and M2c macrophages show similar pathways being modulated, indicating that the mechanism of action translates from isolated macrophages to tumors. A chimeric version of VTX-0811, consisting of the parental murine antibody in a human IgG4 backbone, inhibits tumor growth in a humanized mouse model of cancer. VTX-0811 is exceptionally well tolerated in NHP toxicology assessment and is heading into clinical evaluation after successful IND clearance.

Background: People with plantar flexor weakness generate less ankle push-off work during walking, resulting in inefficient proximal joint compensations. To increase push-off work, spring-like ankle foot orthoses (AFOs) can be provided. However, whether and in which patients AFOs increase push-off work and reduce compensatory hip and knee work is unknown., Methods: In 18 people with bilateral plantar flexor weakness, we performed a 3D gait analysis at comfortable walking speed with shoes-only and with AFOs of which the stiffness was optimized. To account for walking speed differences between conditions, we compared relative joint work of the hip, knee and ankle joint. The relationships between relative work generated with shoes-only and changes in joint work with AFO were tested with Pearson correlations., Results: No differences in relative ankle, knee and hip work over the gait cycle were found between shoes-only and AFO (p>0.499). Percentage of total ankle work generated during pre-swing increased with the AFO (AFO: 85.3±9.1% vs Shoes: 72.4±27.1%, p=0.026). At the hip, the AFO reduced relative work in pre-swing (AFO: 31.9±7.4% vs Shoes: 34.1±10.4%, p=0.038) and increased in loading response (AFO: 18.0±11.0% vs Shoes: 11.9±9.8%, p=0.022). Ankle work with shoes-only was inversely correlated with an increase in ankle work with AFO (r=-0.839, p<0.001) and this increase correlated with reduction in hip work with AFO (r=-0.650, p=0.004)., Discussion: Although stiffness-optimized AFOs did not alter the work distribution across the ankle, knee and hip joint compared to shoes-only walking, relative more ankle work was generated during push-off, causing a shift in hip work from pre-swing to loading response. Furthermore, larger ankle push-off deficits when walking with shoes-only were related with an increase in ankle work with AFO and reduction in compensatory hip work, indicating that more severely affected individuals benefit more from the energy storing-and-releasing capacity of AFOs., Competing Interests: Declaration of Competing Interest None to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Neuromuscular disorders often lead to ankle plantar flexor muscle weakness, which impairs ankle push-off power and forward propulsion during gait. To improve walking speed and reduce metabolic cost of transport (mCoT), patients with plantar flexor weakness are provided dorsal-leaf spring ankle-foot orthoses (AFOs). It is widely believed that mCoT during gait depends on the AFO stiffness and an optimal AFO stiffness that minimizes mCoT exists. The biomechanics behind why and how an optimal stiffness exists and benefits individuals with plantar flexor weakness are not well understood. We hypothesized that the AFO would reduce the required support moment and, hence, metabolic cost contributions of the ankle plantar flexor and knee extensor muscles during stance, and reduce hip flexor metabolic cost to initiate swing. To test these hypotheses, we generated neuromusculoskeletal simulations to represent gait of an individual with bilateral plantar flexor weakness wearing an AFO with varying stiffness. Predictions were based on the objective of minimizing mCoT, loading rates at impact and head accelerations at each stiffness level, and the motor patterns were determined via dynamic optimization. The predictive gait simulation results were compared to experimental data from subjects with bilateral plantar flexor weakness walking with varying AFO stiffness. Our simulations demonstrated that reductions in mCoT with increasing stiffness were attributed to reductions in quadriceps metabolic cost during midstance. Increases in mCoT above optimum stiffness were attributed to the increasing metabolic cost of both hip flexor and hamstrings muscles. The insights gained from our predictive gait simulations could inform clinicians on the prescription of personalized AFOs. With further model individualization, simulations based on mCoT minimization may sufficiently predict adaptations to an AFO in individuals with plantar flexor weakness., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Kiss, Waterval, van der Krogt, Brehm, Geijtenbeek, Harlaar and Seth.)

Objective: To assess the preliminary effectiveness of three-dimensional printed orthoses compared with conventionally custom-fabricated orthoses in persons with chronic hand conditions on performance of daily activities, hand function, quality of life, satisfaction, and production time and costs., Design: Interventional feasibility study., Subjects: Chronic hand orthotic users (n = 21)., Methods: Participants received a new three-dimensional printed orthosis according to the same type as their current orthosis, which served as the control condition. Primary outcome was performance of daily activities (Patient-Reported Outcomes Measurement Information System-Upper Extremity; Michigan Hand Questionnaire). Secondary outcomes were hand function, quality of life, and satisfaction. Furthermore, production time and costs were recorded., Results: At 4 months' follow-up, no significant differences were found between three-dimensional printed orthoses and participants' existing conventional orthoses on activity performance, hand function, and quality of life. Satisfaction with the three-dimensional printed orthosis was significantly higher and the production time and costs for three-dimensional printed orthoses were significantly lower compared with conventional orthoses. The three-dimensional printed orthosis was preferred by 79% of the participants., Conclusions: This feasibility study in chronic hand conditions suggests that three-dimensional printed orthoses are similar to conventional orthoses in terms of activity performance, hand function, and quality of life. Satisfaction, and production time and costs favoured the three-dimensional printed hand orthoses.

Relapse in T-cell acute lymphoblastic leukemia (T-ALL) may signify the persistence of leukemia-initiating cells (L-ICs). Ectopic TAL1/LMO expression defines the largest subset of T-ALL, but its role in leukemic transformation and its impact on relapse-driving L-ICs remain poorly understood. In TAL1/LMO mouse models, double negative-3 (DN3; CD4 - CD8 - CD25 + CD44 - ) thymic progenitors harbored L-ICs. However, only a subset of DN3 leukemic cells exhibited L-IC activity, and studies linking L-ICs and chemotolerance are needed. To investigate L-IC heterogeneity, we used mouse models and applied single-cell RNA-sequencing and nucleosome labeling techniques in vivo. We identified a DN3 subpopulation with a cell cycle-restricted profile and heightened TAL1/LMO2 activity, that expressed genes associated with stemness and quiescence. This dormant DN3 subset progressively expanded throughout leukemogenesis, displaying intrinsic chemotolerance and enrichment in genes linked to minimal residual disease. Examination of TAL/LMO patient samples revealed a similar pattern in CD7 + CD1a - thymic progenitors, previously recognized for their L-IC activity, demonstrating cell cycle restriction and chemotolerance. Our findings substantiate the emergence of dormant, chemotolerant L-ICs during leukemogenesis, and demonstrate that Tal1 and Lmo2 cooperate to promote DN3 quiescence during the transformation process. This study provides a deeper understanding of TAL1/LMO-induced T-ALL and its clinical implications in therapy failure., (© 2024. The Author(s).)

Salmonella serovars Typhi and Paratyphi cause a prolonged illness known as enteric fever, whereas other serovars cause acute gastroenteritis. Mechanisms responsible for the divergent clinical manifestations of nontyphoidal and enteric fever Salmonella infections have remained elusive. Here, we show that S . Typhi and S . Paratyphi A can persist within human macrophages, whereas S . Typhimurium rapidly induces apoptotic macrophage cell death that is dependent on Salmonella pathogenicity island 2 (SPI2). S . Typhi and S . Paratyphi A lack 12 specific SPI2 effectors with pro-apoptotic functions, including nine that target nuclear factor κB (NF-κB). Pharmacologic inhibition of NF-κB or heterologous expression of the SPI2 effectors GogA or GtgA restores apoptosis of S . Typhi-infected macrophages. In addition, the absence of the SPI2 effector SarA results in deficient signal transducer and activator of transcription 1 (STAT1) activation and interleukin 12 production, leading to impaired T H 1 responses in macrophages and humanized mice. The absence of specific nontyphoidal SPI2 effectors may allow S . Typhi and S . Paratyphi A to cause chronic infections., Importance: Salmonella enterica is a common cause of gastrointestinal infections worldwide. The serovars Salmonella Typhi and Salmonella Paratyphi A cause a distinctive systemic illness called enteric fever, whose pathogenesis is incompletely understood. Here, we show that enteric fever Salmonella serovars lack 12 specific virulence factors possessed by nontyphoidal Salmonella serovars, which allow the enteric fever serovars to persist within human macrophages. We propose that this fundamental difference in the interaction of Salmonella with human macrophages is responsible for the chronicity of typhoid and paratyphoid fever, suggesting that targeting the nuclear factor κB (NF-κB) complex responsible for macrophage survival could facilitate the clearance of persistent bacterial infections., Competing Interests: The authors declare no conflict of interest.

Objective: To evaluate the 2-year course of walking adaptability in persons with late effects of polio., Design: Prospective cohort study., Patients: A total of 48 persons with late effects of polio (69% female, mean age 63.1 years) with a fall history and/or fear of falling., Methods: Walking adaptability (i.e. variable target-stepping and reactive obstacle-avoidance) was assessed on an interactive treadmill at baseline, 1 year and 2 years. Further, leg-muscle strength and balance were assessed at baseline. The course of walking adaptability was analysed with linear mixed models. Based on median values, subgroups were defined for low vs high baseline walking-adaptability and for clinical characteristics. Tme by subgroup interactions were analysed., Results: Variable target-stepping and reactive obstacle-avoidance did not change (p > 0.285). Reactive obstacle-avoidance improved for persons with a high balance score at baseline (p = 0.037), but not for those with lower scores (p = 0.531). No other time by subgroup interactions were found (p > 0.126)., Conclusion: Walking adaptability did not change in persons with late effects of polio over 2 years, and walking adaptability course did not differ between subgroups stratified for walking adaptability determinants, except for balance. Since falls are a major problem among persons with late effects of polio, future studies should investigate whether walking adaptability declines over a longer time and which persons are most at risk.

Purpose: Brain metastases are associated with high morbidity and are often resistant to immune checkpoint inhibitors. We evaluated whether CDK4/6 inhibitor (CDKi) abemaciclib can sensitize intracranial tumors to programmed cell death protein 1 (PD-1) inhibition in mouse models of melanoma and breast cancer brain metastasis., Experimental Design: Treatment response was evaluated in vivo using immunocompetent mouse models of brain metastasis bearing concurrent intracranial and extracranial tumors. Treatment effect on intracranial and extracranial tumor-immune microenvironments (TIME) was evaluated using immunofluorescence, multiplex immunoassays, high-parameter flow cytometry, and T-cell receptor profiling. Mice with humanized immune systems were evaluated using flow cytometry to study the effect of CDKi on human T-cell development., Results: We found that combining abemaciclib with PD-1 inhibition reduced tumor burden and improved overall survival in mice. The TIME, which differed on the basis of anatomic location of tumors, was altered with CDKi and PD-1 inhibition in an organ-specific manner. Combination abemaciclib and anti-PD-1 treatment increased recruitment and expansion of CD8+ effector T-cell subsets, depleted CD4+ regulatory T (Treg) cells, and reduced levels of immunosuppressive cytokines in intracranial tumors. In immunodeficient mice engrafted with human immune systems, abemaciclib treatment supported development and maintenance of CD8+ T cells and depleted Treg cells., Conclusions: Our results highlight the distinct properties of intracranial and extracranial tumors and support clinical investigation of combination CDK4/6 and PD-1 inhibition in patients with brain metastases. See related commentary by Margolin, p. 257., (©2023 American Association for Cancer Research.)

Viral myocarditis is characterized by infiltration of mononuclear cells essential for virus elimination. GPR15 has been identified as a homing receptor for regulatory T cells in inflammatory intestine diseases, but its role in inflammatory heart diseases is still elusive. Here we show that GPR15 deficiency impairs coxsackievirus B3 elimination, leading to adverse cardiac remodeling and dysfunction. Delayed recruitment of regulatory T cells in GPR15-deficient mice was accompanied by prolonged persistence of cytotoxic and regulatory T cells. In addition, RNA sequencing revealed prolonged inflammatory response and altered chemotaxis in knockout mice. In line, we identified GPR15 and its ligand GPR15L as an important chemokine receptor-ligand pair for the recruitment of regulatory and cytotoxic T cells. In summary, the insufficient virus elimination might be caused by a delayed recruitment of T cells as well as delayed interferon-γ expression, resulting in a prolonged inflammatory response and an adverse outcome in GPR15-deficient mice., (© 2023. The Author(s).)

Background: Polio survivors often exhibit plantarflexor weakness, which impairs gait stability, and increases energy cost of walking. Quantifying gait stability could provide insights in the control mechanisms polio survivors use to maintain gait stability and in whether impaired gait stability is related to the increased energy cost of walking., Research Question: Is gait stability impaired in polio survivors with plantarflexor weakness compared to able-bodied individuals, and does gait stability relate to energy cost of walking?, Methods: We retrospectively analyzed barefoot biomechanical gait data of 31 polio survivors with unilateral plantarflexor weakness and of 24 able-bodied individuals. We estimated gait stability by calculating variability (SD) of step width, step length, double support time, and stance time, and by the mean and variability (SD) of the mediolateral and anteroposterior margin of stability (MoS ML and MoS AP ). In addition, energy cost of walking (polio survivors only) at comfortable speed was analyzed., Results: Comfortable speed was 31% lower in polio survivors compared to able-bodied individuals (p < 0.001). Corrected for speed differences, step width variability was significantly larger in polio survivors (+41%), double support time variability was significantly smaller (-27%), MoS ML (affected leg) was significantly larger (+80%), and MoS AP was significantly smaller (affected leg:-17% and non-affected leg:-15%). Step width and step length variability (affected leg) were positively correlated with energy cost of walking (r = 0.502 and r = 0.552). MoS AP (non-affected leg) was negatively correlated with energy cost of walking (r = -0.530)., Significance: Polio survivors with unilateral plantarflexor weakness demonstrated an impaired gait stability. Increased step width and step length variability and lower MoS AP could be factors related to the elevated energy cost of walking in polio survivors. These findings increase our understanding of stability problems due to plantarflexor weakness, which could be used for the improvement of (orthotic) interventions to enhance gait stability and reduce energy cost in polio survivors., Competing Interests: Declaration of Competing Interest The authors report no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Stance control knee-ankle-foot orthoses (SCKAFOs) ensure knee stability by locking during stance while allowing knee flexion during swing. Differences in function of the knee joints and building principles between devices may affect their effectiveness., Objective: To investigate the preliminary effectiveness of a NEURO TRONIC on safety outcomes, net energy cost (EC), and user experiences in individuals already using an E-MAG Active SCKAFO., Study Design: Prospective uncontrolled intervention study., Methods: A convenience sample of 10 subjects with flaccid lower extremity muscle weakness, including the quadriceps, due to neuromuscular disorders already using an E-MAG Active SCKAFO were provided with a newly fabricated NEURO TRONIC SCKAFO. Outcomes included knee joint locking failures and unlocking failures (ULFs) (i.e., percentage of steps the knee joint failed to lock/unlock) when walking under challenging conditions on an instrumented treadmill while wearing a safety harness; net EC (J/kg per meter) assessed with a 6-min walk test at comfortable speed; 3D gait kinematics and kinetics; and patient-reported outcomes., Results: No differences between devices were found for knee joint locking failures (both devices 0%) and ULFs (9.9% for the NEURO TRONIC vs. 13.9% for the E-MAG Active SCKAFO). The mean (standard deviation) net EC with the NEURO TRONIC SCKAFO was 8.2% (from 3.68 [0.81] to 3.38 [0.75] J/kg per meter, p = 0.123) lower, although not significantly, compared with that with the E-MAG Active SCKAFO. Significant improvements with the NEURO TRONIC SCKAFO were found for ankle power ( p = 0.003), perceived walking effort ( p = 0.014), and reported falls ( p = 0.034)., Conclusion: Both the NEURO TRONIC SCKAFO and the E-MAG Active SCKAFO were safe in terms of knee joint locking, while ULFs were frequent with both devices. The net EC with the NEURO TRONIC SCKAFO decreased, although not significantly, by 8.2%, likely due to insufficient power. Perceived walking effort was in favor of the NEURO TRONIC SCKAFO., (Copyright © 2023 The Authors. Published by Wolters Kluwer Inc. on behalf of The International Society for Prosthetics and Orthotics.)

Aim: To obtain insights into the effects of fatigue on the kinematics, kinetics, and energy cost of walking (ECoW) in children with cerebral palsy (CP)., Method: In this prospective observational study, 12 children with CP (mean age 12 years 9 months, SD 2 years 7 months; four females, eight males) and 15 typically developing children (mean age 10 years 8 months, SD 2 years 4 months; seven females, eight males) followed a prolonged intensity-based walking protocol on an instrumented treadmill, combined with gas analysis measurements. The protocol consisted of consecutive stages, including a 6-minute walking exercise (6MW) at comfortable speed, 2 minutes of moderate-intensity walking (MIW) (with a heart rate > 70% of its predicted maximal), and 4 minutes walking after MIW. If necessary, the speed and slope were incremented to reach MIW. Outcomes were evaluated at the beginning and end of the 6MW and after MIW., Results: With prolonged walking, Gait Profile Scores deteriorated slightly for both groups (p < 0.01). Knee flexion increased during early stance (p = 0.004) and ankle dorsiflexion increased during late stance (p = 0.034) in children with CP only. Negligible effects were found for kinetics. No demonstrable change in ECoW was found in either group (p = 0.195)., Interpretation: Kinematic deviations in children with CP are progressive with prolonged walking. The large variation in adaptations indicates that an individual approach is recommended to investigate the effects of physical fatigue on gait in clinical practice., (© 2023 The Authors. Developmental Medicine & Child Neurology published by John Wiley & Sons Ltd on behalf of Mac Keith Press.)

Understanding the cell biological mechanisms that enable tumor cells to persist after therapy is necessary to improve the treatment of recurrent disease. Here, we demonstrate that transient receptor potential channel 6 (TRPC6), a channel that mediates calcium entry, contributes to the properties of breast cancer stem cells, including resistance to chemotherapy, and that tumor cells that persist after therapy are dependent on TRPC6. The mechanism involves the ability of TRPC6 to regulate integrin α6 mRNA splicing. Specifically, TRPC6-mediated calcium entry represses the epithelial splicing factor ESRP1 (epithelial splicing regulatory protein 1), which enables expression of the integrin α6B splice variant. TRPC6 and α6B function in tandem to facilitate stemness and persistence by activating TAZ and, consequently, repressing Myc. Therapeutic inhibition of TRPC6 sensitizes triple-negative breast cancer (TNBC) cells and tumors to chemotherapy by targeting the splicing of α6 integrin mRNA and inducing Myc. These data reveal a Ca 2+ -dependent mechanism of chemotherapy-induced persistence, which is amenable to therapy, that involves integrin mRNA splicing., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Approaches to study human pharyngeal foregut endoderm-a developmental intermediate that is linked to various human syndromes involving pharynx development and organogenesis of tissues such as thymus, parathyroid, and thyroid-have been hampered by scarcity of tissue access and cellular models. We present an efficient stepwise differentiation method to generate human pharyngeal foregut endoderm from pluripotent stem cells. We determine dose and temporal requirements of signaling pathway engagement for optimized differentiation and characterize the differentiation products on cellular and integrated molecular level. We present a computational classification tool, "CellMatch," and transcriptomic classification of differentiation products on an integrated mouse scRNA-seq developmental roadmap confirms cellular maturation. Integrated transcriptomic and chromatin analyses infer differentiation stage-specific gene regulatory networks. Our work provides the method and integrated multiomic resource for the investigation of disease-relevant loci and gene regulatory networks and their role in developmental defects affecting the pharyngeal endoderm and its derivatives., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: The stiffness of a dorsal leaf AFO that minimizes walking energy cost in people with plantarflexor weakness varies between individuals. Using predictive simulations, we studied the effects of plantarflexor weakness, passive plantarflexor stiffness, body mass, and walking speed on the optimal AFO stiffness for energy cost reduction., Methods: We employed a planar, nine degrees-of-freedom musculoskeletal model, in which for validation maximal strength of the plantar flexors was reduced by 80%. Walking simulations, driven by minimizing a comprehensive cost function of which energy cost was the main contributor, were generated using a reflex-based controller. Simulations of walking without and with an AFO with stiffnesses between 0.9 and 8.7 Nm/degree were generated. After validation against experimental data of 11 people with plantarflexor weakness using the Root-mean-square error (RMSE), we systematically changed plantarflexor weakness (range 40-90% weakness), passive plantarflexor stiffness (range: 20-200% of normal), body mass (+ 30%) and walking speed (range: 0.8-1.2 m/s) in our baseline model to evaluate their effect on the optimal AFO stiffness for energy cost minimization., Results: Our simulations had a RMSE < 2 for all lower limb joint kinetics and kinematics except the knee and hip power for walking without AFO. When systematically varying model parameters, more severe plantarflexor weakness, lower passive plantarflexor stiffness, higher body mass and walking speed increased the optimal AFO stiffness for energy cost minimization, with the largest effects for severity of plantarflexor weakness., Conclusions: Our forward simulations demonstrate that in individuals with bilateral plantarflexor the necessary AFO stiffness for walking energy cost minimization is largely affected by severity of plantarflexor weakness, while variation in walking speed, passive muscle stiffness and body mass influence the optimal stiffness to a lesser extent. That gait deviations without AFO are overestimated may have exaggerated the required support of the AFO to minimize walking energy cost. Future research should focus on improving predictive simulations in order to implement personalized predictions in usual care. Trial Registration Nederlands Trial Register 5170. Registration date: May 7th 2015.  http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=5170., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Orthosis satisfaction is an important outcome in assessing quality of care. However, no measurement specifically assessing orthosis satisfaction is available in the Dutch language. Therefore, the aim of this study was to translate the Client Satisfaction with Device (CSD) module of the Orthotics and Prosthetics Users' Survey (OPUS) into Dutch, and to assess its content validity, structural validity and reliability in persons with chronic hand conditions., Methods: The CSD was translated and cross-cultural adapted according to respective guidelines. To determine content validity, 10 chronic hand orthotic users and two professionals judged the relevance, comprehensibility, and comprehensiveness of the Dutch CSD (D-CSD). Thereafter, in a cross-sectional study, 76 persons were asked to complete the D-CSD twice, with a 2-week interval. Dimensionality of the D-CSD was examined by principal component analysis (PCA), and factor model fit was assessed by confirmatory factor analysis (CFA). Reliability was assessed as internal consistency and test-retest reliability, including the 95% limits of agreement (LoA), the standard error of measurement (SEM) and smallest detectable change (SDC)., Results: The D-CSD items and response options were deemed relevant and comprehensible. After adding an item on cleaning the orthosis, content validity was judged sufficient. PCA indicated a one-factor model, which was confirmed by CFA. We found good internal consistency (Cronbach's alpha = 0.82; 95%CI 0.75-0.87), and moderate to good test-retest reliability (ICC = 0.81; 95%CI 0.71-0.87). There was no difference between the mean D-CSD score at test (26.8 points) and retest (25.9 points) (mean (SD) difference: 0.86 points (4.00); 95%CI -0.06-1.79; p = 0.07). The 95% LoA were -6.99 to 8.71, and the SEM and SDC were 2.88 and 7.98 points, respectively., Conclusions: Based on sufficient content and structural validity, and good reliability, we consider the D-CSD a useful tool to evaluate orthosis satisfaction in persons with chronic hand conditions on group level. Because of a relatively high SDC, sensitivity to detect changes over time on individual level is limited., Study Registration Number: NCT05320211., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Innate lymphoid cells (ILCs) are a diverse population of cells that include NK cells and contribute to tissue homeostasis and repair, inflammation, and provide protection from infection. The interplay between human blood ILCs, as well as their responses to HIV-1 infection, remains poorly understood. This study used transcriptional and chromatin profiling to explore these questions. Transcriptional profiling and flow cytometry analysis support that there are four main ILC subsets found in human blood. Unlike in mice, human NK cells expressed the tissue repair protein amphiregulin (AREG). AREG production was induced by TCF7/WNT, IL-2, and IL-15, and inhibited by TGFB1, a cytokine increased in people living with HIV-1. In HIV-1 infection, the percentage of AREG + NK cells correlated positively with the numbers of ILCs and CD4 + T cells but negatively with the concentration of inflammatory cytokine IL-6. NK-cell knockout of the TGFB1-stimulated WNT antagonist RUNX3 increased AREG production. Antiviral gene expression was increased in all ILC subsets from HIV-1 viremic people, and anti-inflammatory gene MYDGF was increased in an NK-cell subset from HIV-1-infected people whose viral load was undetectable in the absence of antiretroviral therapy. The percentage of defective NK cells in people living with HIV-1 correlated inversely with ILC percentage and CD4 + T-cell counts. CD4 + T cells and their production of IL-2 prevented the loss of NK-cell function by activating mTOR. These studies clarify how ILC subsets are interrelated and provide insight into how HIV-1 infection disrupts NK cells, including an uncharacterized homeostatic function in NK cells., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

γδ T cells are important tissue-resident, innate T cells that are critical for tissue homeostasis. γδ cells are associated with positive prognosis in most tumors; however, little is known about their heterogeneity in human cancers. Here, we phenotyped innate and adaptive cells in human colorectal (CRC) and endometrial cancer. We found striking differences in γδ subsets and function in tumors compared to normal tissue, and in the γδ subsets present in tumor types. In CRC, an amphiregulin (AREG)-producing subset emerges, while endometrial cancer is infiltrated by cytotoxic cells. In humanized CRC models, tumors induced this AREG phenotype in Vδ1 cells after adoptive transfer. To exploit the beneficial roles of γδ cells for cell therapy, we developed an expansion method that enhanced cytotoxic function and boosted metabolic flexibility, while eliminating AREG production, achieving greater tumor infiltration and tumor clearance. This method has broad applications in cellular therapy as an 'off-the-shelf' treatment option., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

To maximize effects of dorsal leaf ankle foot orthoses (AFOs) on gait in people with bilateral plantarflexor weakness, the AFO properties should be matched to the individual. However, how AFO properties interact regarding their effect on gait function is unknown. We studied the interaction of AFO bending stiffness with neutral angle and footplate stiffness on the effect of bending stiffness on walking energy cost, gait kinematics and kinetics in people with plantarflexor weakness by employing predictive simulations. Our simulation framework consisted of a planar 11 degrees of freedom model, containing 11 muscles activated by a reflex-based neuromuscular controller. The controller was optimized by a comprehensive cost function, predominantly minimizing walking energy cost. The AFO bending and footplate stiffness were modelled as torsional springs around the ankle and metatarsal joint. The neutral angle of the AFO was defined as the angle in the sagittal plane at which the moment of the ankle torsional spring was zero. Simulations without AFO and with AFO for 9 bending stiffnesses (0-14 Nm/degree), 3 neutral angles (0-3-6 degrees dorsiflexion) and 3 footplate stiffnesses (0-0.5-2.0 Nm/degree) were performed. When changing neutral angle towards dorsiflexion, a higher AFO bending stiffness minimized energy cost of walking and normalized joint kinematics and kinetics. Footplate stiffness mainly affected MTP joint kinematics and kinetics, while no systematic and only marginal effects on energy cost were found. In conclusion, the interaction of the AFO bending stiffness and neutral angle in bilateral plantarflexor weakness, suggests that these should both be considered together when matching AFO properties to the individual patient., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Humanized mouse models, created via transplantation of human hematopoietic tissues into immune-deficient mice, support a number of research applications, including transplantation immunology, virology and oncology studies. As an alternative to the bone marrow, liver, thymus humanized mouse, which uses fetal tissues for generating a chimeric human immune system, the NeoThy humanized mouse uses nonfetal tissue sources. Specifically, the NeoThy model incorporates hematopoietic stem and progenitor cells from umbilical cord blood (UCB) as well as thymus tissue that is typically discarded as medical waste during neonatal cardiac surgeries. Compared with fetal thymus tissue, the abundant quantity of neonatal thymus tissue offers the opportunity to prepare over 1,000 NeoThy mice from an individual thymus donor. Here we describe a protocol for processing of the neonatal tissues (thymus and UCB) and hematopoietic stem and progenitor cell separation, human leukocyte antigen typing and matching of allogenic thymus and UCB tissues, creation of NeoThy mice, assessment of human immune cell reconstitution and all experimental steps from planning and design to data analysis. This entire protocol takes a total of ~19 h to complete, with steps broken up into multiple sessions of 4 h or less that can be paused and completed over multiple days. The protocol can be completed, after practice, by individuals with intermediate laboratory and animal handling skills, enabling researchers to make effective use of this promising in vivo model of human immune function., (© 2023. Springer Nature America, Inc.)

Biomedical devices comprise a major component of modern medicine, however immune-mediated fibrosis and rejection can limit their function over time. Here, we describe a humanized mouse model that recapitulates fibrosis following biomaterial implantation. Cellular and cytokine responses to multiple biomaterials were evaluated across different implant sites. Human innate immune macrophages were verified as essential to biomaterial rejection in this model and were capable of cross-talk with mouse fibroblasts for collagen matrix deposition. Cytokine and cytokine receptor array analysis confirmed core signaling in the fibrotic cascade. Foreign body giant cell formation, often unobserved in mice, was also prominent. Last, high-resolution microscopy coupled with multiplexed antibody capture digital profiling analysis supplied spatial resolution of rejection responses. This model enables the study of human immune cell-mediated fibrosis and interactions with implanted biomaterials and devices.

Immuno-oncology (IO)-based therapies such as checkpoint inhibitors, bi-specific antibodies, and CAR-T-cell therapies have shown significant success in the treatment of several cancer indications. However, these therapies can result in the development of severe adverse events, including cytokine release syndrome (CRS). Currently, there is a paucity of in vivo models that can evaluate dose-response relationships for both tumor control and CRS-related safety issues. We tested an in vivo PBMC humanized mouse model to assess both treatment efficacy against specific tumors and the concurrent cytokine release profiles for individual human donors after treatment with a CD19xCD3 bispecific T-cell engager (BiTE). Using this model, we evaluated tumor burden, T-cell activation, and cytokine release in response to bispecific T-cell-engaging antibody in humanized mice generated with different PBMC donors. The results show that PBMC engrafted NOD-scid Il2rg null mice lacking expression of mouse MHC class I and II (NSG-MHC-DKO mice) and implanted with a tumor xenograft predict both efficacy for tumor control by CD19xCD3 BiTE and stimulated cytokine release. Moreover, our findings indicate that this PBMC-engrafted model captures variability among donors for tumor control and cytokine release following treatment. Tumor control and cytokine release were reproducible for the same PBMC donor in separate experiments. The PBMC humanized mouse model described here is a sensitive and reproducible platform that identifies specific patient/cancer/therapy combinations for treatment efficacy and development of complications., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Prostate cancers are largely unresponsive to immune checkpoint inhibitors (ICIs), and there is strong evidence that programmed death-ligand 1 (PD-L1) expression itself must be inhibited to activate antitumor immunity. Here, we report that neuropilin-2 (NRP2), which functions as a vascular endothelial growth factor (VEGF) receptor on tumor cells, is an attractive target to activate antitumor immunity in prostate cancer because VEGF-NRP2 signaling sustains PD-L1 expression. NRP2 depletion increased T cell activation in vitro. In a syngeneic model of prostate cancer that is resistant to ICI, inhibition of the binding of VEGF to NRP2 using a mouse-specific anti-NRP2 monoclonal antibody (mAb) resulted in necrosis and tumor regression compared with both an anti-PD-L1 mAb and control immunoglobulin G. This therapy also decreased tumor PD-L1 expression and increased immune cell infiltration. We observed that the NRP2 , VEGFA , and VEGFC genes are amplified in metastatic castration-resistant and neuroendocrine prostate cancer. We also found that individuals with NRP2 High PD-L1 High metastatic tumors had lower androgen receptor expression and higher neuroendocrine prostate cancer scores than other individuals with prostate cancer. In organoids derived from patients with neuroendocrine prostate cancer, therapeutic inhibition of VEGF binding to NRP2 using a high-affinity humanized mAb suitable for clinical use also diminished PD-L1 expression and caused a substantial increase in immune-mediated tumor cell killing, consistent with the animal studies. These findings provide justification for the initiation of clinical trials using this function-blocking NRP2 mAb in prostate cancer, especially for patients with aggressive disease.

Agents that induce inflammation have been used since the 18th century for the treatment of cancer. The inflammation induced by agents such as Toll-like receptor agonists is thought to stimulate tumor-specific immunity in patients and augment control of tumor burden. While NOD-scid IL2rγnull mice lack murine adaptive immunity (T cells and B cells), these mice maintain a residual murine innate immune system that responds to Toll-like receptor agonists. Here we describe a novel NOD-scid IL2rγnull mouse lacking murine TLR4 that fails to respond to lipopolysaccharide. NSG-Tlr4null mice support human immune system engraftment and enable the study of human-specific responses to TLR4 agonists in the absence of the confounding effects of a murine response. Our data demonstrate that specific stimulation of TLR4 activates human innate immune systems and delays the growth kinetics of a human patient-derived xenograft melanoma tumor., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Hand orthoses are often provided to improve performance of activities of daily life (ADL). Yet, the manufacturing process of conventional custom-fabricated hand orthoses is a time-consuming and labour-intensive process. Even though three-dimensional (3D) printing of orthoses is a rapidly growing area that can facilitate the manufacturing process of hand orthoses, evidence on the effectiveness, costs and production time of 3D-printed orthoses in chronic hand conditions is scarce. This study aims to assess (1) the preliminary effectiveness of 3D-printed orthoses compared with conventionally custom-fabricated orthoses in persons with chronic hand conditions, (2) production time and costs of both orthoses and (3) experiences of the participants and orthotists with the manufacturing process of the 3D-printed orthosis., Methods and Analysis: In this prospective non-randomised interventional feasibility study, 20 adults with various chronic hand conditions using a conventional thumb, wrist or wrist-thumb orthosis will be provided with a 3D-printed corresponding type of orthosis. Assessments will be done 2 weeks prior to the intervention and at baseline for the conventional orthosis, and at 1 month and 4 months follow-up for the 3D-printed orthosis. The primary outcome is change from baseline in ADL performance (custom short form Dutch-Flemish Patient-Reported Outcomes Measurement Information System-Upper Extremity; ADL domain Michigan Hand Outcomes Questionnaire Dutch language version (MHQ-DLV)) at 4 months follow-up. Secondary outcomes include general hand function (MHQ-DLV), satisfaction with the orthosis (Dutch Client Satisfaction with Device; Dutch version of the Quebec User Evaluation of Satisfaction with Assistive Technology), usability (in-house questionnaire) and quality of life (EuroQoL 5-Dimension 5-Level). Costs and production time of the conventional and 3D-printed orthoses will be prospectively recorded. Experiences regarding the manufacturing process will be obtained from participants and orthotists (in-house questionnaire)., Ethics and Dissemination: The Medical Ethics Committee of the Amsterdam UMC, Academic Medical Centre, has waived the requirement for ethical review of this study. Results will be disseminated through peer-reviewed journals, scientific conferences, and media aimed at a broad audience including patients., Trial Registration Number: NCT05320211., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Genetic susceptibility to type 1 diabetes is associated with homozygous expression of major histocompatibility complex class II alleles that carry specific beta chain polymorphisms. Why heterozygous expression of these major histocompatibility complex class II alleles does not confer a similar predisposition is unresolved. Using a nonobese diabetic mouse model, here we show that heterozygous expression of the type 1 diabetes-protective allele I-A g7 β56P/57D induces negative selection to the I-A g7 -restricted T cell repertoire, including beta-islet-specific CD4 + T cells. Surprisingly, negative selection occurs despite I-A g7 β56P/57D having a reduced ability to present beta-islet antigens to CD4 + T cells. Peripheral manifestations of non-cognate negative selection include a near complete loss of beta-islet-specific CXCR6 + CD4 + T cells, an inability to cross-prime islet-specific glucose-6-phosphatase catalytic subunit-related protein and insulin-specific CD8 + T cells and disease arrest at the insulitis stage. These data reveal that negative selection on non-cognate self-antigens in the thymus can promote T cell tolerance and protection from autoimmunity., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Endemic Burkitt lymphoma (BL) is a childhood cancer in sub-Saharan Africa characterized by Epstein-Barr virus and malaria-associated aberrant B-cell activation and MYC chromosomal translocation. Survival rates hover at 50% after conventional chemotherapies; therefore, clinically relevant models are necessary to test additional therapies. Hence, we established five patient-derived BL tumor cell lines and corresponding NSG-BL avatar mouse models. Transcriptomics confirmed that our BL lines maintained fidelity from patient tumors to NSG-BL tumors. However, we found significant variation in tumor growth and survival among NSG-BL avatars and in Epstein-Barr virus protein expression patterns. We tested rituximab responsiveness and found one NSG-BL model exhibiting direct sensitivity, characterized by apoptotic gene expression counterbalanced by unfolded protein response and mTOR pro-survival pathways. In rituximab-unresponsive tumors, we observed an IFN-α signature confirmed by the expression of IRF7 and ISG15. Our results demonstrate significant inter-patient tumor variation and heterogeneity, and that contemporary patient-derived BL cell lines and NSG-BL avatars are feasible tools to guide new therapeutic strategies and improve outcomes for these children., (© 2023 Saikumar Lakshmi et al.)

Immunotherapy has emerged as a promising treatment paradigm for many malignancies and is transforming the drug development landscape. Although immunotherapeutic agents have demonstrated clinical efficacy, they are associated with variable clinical responses, and substantial gaps remain in our understanding of their mechanisms of action and specific biomarkers of response. Currently, the number of preclinical models that faithfully recapitulate interactions between the human immune system and tumours and enable evaluation of human-specific immunotherapies in vivo is limited. Humanized mice, a term that refers to immunodeficient mice co-engrafted with human tumours and immune components, provide several advantages for immuno-oncology research. In this Review, we discuss the benefits and challenges of the currently available humanized mice, including specific interactions between engrafted human tumours and immune components, the development and survival of human innate immune populations in these mice, and approaches to study mice engrafted with matched patient tumours and immune cells. We highlight the latest advances in the generation of humanized mouse models, with the aim of providing a guide for their application to immuno-oncology studies with potential for clinical translation., (© 2023. Springer Nature Limited.)

Background: Aerobic exercise aims to improve aerobic capacity., Objective: To summarize the evidence on the efficacy of aerobic exercise on aerobic capacity in slowly progressive neuromuscular diseases (NMDs)., Methods: We searched the electronic databases MEDLINE, EMBASE, SPORTDiscus and Web of Science Conference Proceedings Index for articles published up to June 17, 2021, selecting randomized controlled trials that included adults with slowly progressive NMDs and compared aerobic exercise to no aerobic exercise. The primary outcome was peak oxygen uptake (VO 2 peak) directly post-intervention. Secondary outcomes included other peak test parameters, submaximal test parameters, long-term outcomes ≥8 weeks post-intervention, adherence and adverse events. Meta-analyses were performed for the primary outcome and for secondary outcomes when reported in more than 2 studies. Risk of bias was assessed with the Cochrane Risk of Bias tool and quality of evidence according to GRADE., Results: Nine studies were included (195 participants with 8 different NMDs). Eight studies were rated at high risk of bias and 1 study was rated at some concerns. Duration of exercise programs ranged from 6 to 26 weeks, with 3 weekly training sessions of 20 to 40 min, based on maximal capacity. Meta-analyses revealed short-term moderate beneficial effects of aerobic exercise on VO 2 peak (standardized mean difference [SMD] 0.55, 95% CI 0.23; 0.86) and peak workload (SMD 0.61, 95% CI 0.24; 0.99). Long-term effects were not assessed. Most training sessions (83-97%) were completed, but time spent in target intensity zones was not reported. Included studies lacked detailed adverse event reporting., Conclusions: There is low-quality evidence that aerobic exercise is safe and leads to moderate improvement of aerobic capacity directly post-intervention in slowly progressive NMDs, but the long-term efficacy remains unclear. Detailed information about the time spent in target intensity zones and adverse events is lacking., Prospero: CRD42020200083., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2022 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Background: Lower limb orthoses intend to improve walking in adults with neuromuscular disorders (NMD). Yet, reported group effects of lower limb orthoses on treatment outcomes have generally been small and heterogeneous. We propose that guideline-based orthotic care within a multidisciplinary expert setting may improve treatment outcomes., Aim: To examine the effectiveness of specialist care orthoses compared to usual care orthoses on personal goal attainment and walking ability., Design: Cohort study., Population: Adults with NMD who experienced walking problems due to calf and/or quadriceps muscle weakness and were provided with a specialist care lower limb orthosis between October 2011 and January 2021., Methods: Three months after provision, the specialist care orthosis was compared to the usual care orthosis worn at baseline in terms of personal goal attainment (Goal Attainment Scaling (GAS)), comfortable walking speed (m/s), net energy cost (J/kg/m) (both assessed during a 6-minute walk test), perceived walking ability and satisfaction., Results: Sixty-four adults with NMD were eligible for analysis. The specialist care orthoses comprised 19 dorsiflexion-restricting ankle-foot orthoses (AFOs), 22 stance-control knee-ankle-foot orthoses (KAFOs) and 23 locked KAFOs. Overall, 61% of subjects showed a clinically relevant improvement in GAS score. Perceived safety, stability, intensity, fear of falling and satisfaction while walking all improved (p≤0.002), and subjects were satisfied with their specialist care orthosis and the services provided. Although no effects on walking speed or net energy cost were found in combined orthosis groups, specialist care AFOs significantly reduced net energy cost (by 9.5%) compared to usual care orthoses (from mean (SD) 3.81 (0.97) to 3.45 (0.80) J/kg/m, p = 0.004)., Conclusion: Guideline-based orthotic care within a multidisciplinary expertise setting could improve treatment outcomes in adults with NMD compared to usual orthotic care by improvements in goal attainment and walking ability. A randomized controlled trial is now warranted to confirm these results., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 van Duijnhoven et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: To investigate whether 6-min walking is fatiguing for polio survivors, and how fatigue influences their normal and adaptive walking., Design: Cross-sectional study., Patients: Polio survivors (n = 23) with ≥ 1 fall and/or fear of falling reported in the previous year and healthy individuals (n = 11)., Methods: Participants performed 1 normal-walk test and 2 walking-adaptability tests (target stepping and narrow-beam walking) on an instrumented treadmill at fixed self-selected speed, each test lasting 6 min. Leg-muscle fatigue (leg-muscle activation, measured with surface electromyography), cardiorespiratory fatigue (heart rate, rate of perceived exertion), gait and walking-adaptability performance were assessed. The study compared: (i) the first and last minute per test, (ii) normal and adaptive walking, and (iii) groups., Results: Leg-muscle activation did not change during normal walking (p > 0.546), but declined over time during adaptive walking, especially in polio survivors (p < 0.030). Cardiorespiratory fatigue increased during all tests (p < 0.001), especially in polio survivors (p < 0.01), and was higher during adaptive than normal walking (p < 0.007). Target-stepping performance declined in both groups (p = 0.007), while narrow-beam walking improved in healthy individuals (p < 0.001) and declined in polio survivors (p < 0.001)., Conclusion: Cardiorespiratory fatigue might further degrade walking adaptability, especially among polio survivors during narrow-beam walking. This might increase the risk of falls among polio survivors.

Von Willebrand disease (VWD) is a bleeding disorder with different levels of severity. VWD-associated mutations are located in the von Willebrand factor (VWF) gene, coding for the large multidomain plasma protein VWF with essential roles in hemostasis and thrombosis. On the one hand, a variety of mutations in the C-domains of VWF are associated with increased bleeding upon vascular injury. On the other hand, VWF gain-of-function (GOF) mutations in the C4 domain have recently been identified, which induce an increased risk of myocardial infarction. Mechanistic insights into how these mutations affect the molecular behavior of VWF are scarce and holistic approaches are challenging due to the multidomain and multimeric character of this large protein. Here, we determine the structure and dynamics of the C6 domain and the single nucleotide polymorphism (SNP) variant G2705R in C6 by combining nuclear magnetic resonance spectroscopy, molecular dynamics simulations and aggregometry. Our findings indicate that this mutation mostly destabilizes VWF by leading to a more pronounced hinging between both subdomains of C6. Hemostatic parameters of variant G2705R are close to normal under static conditions, but the missense mutation results in a gain-of-function under flow conditions, due to decreased VWF stem stability. Together with the fact that two C4 variants also exhibit GOF characteristics, our data underline the importance of the VWF stem region in VWF's hemostatic activity and the risk of mutation-associated prothrombotic properties in VWF C-domain variants due to altered stem dynamics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mast cells (MC) are key drivers of allergic and inflammatory diseases. Sialic acid-binding immunoglobulin-like lectin (Siglec)-6 is an immunoregulatory receptor found on MCs. While it is recognized that engaging Siglecs with antibodies mediates inhibition across immune cells, the mechanisms that govern this agonism are not understood. Here we generated Siglec-6 mAb clones (AK01 to AK18) to better understand Siglec-6-mediated agonism. Siglec-6 mAbs displayed epitope-dependent receptor internalization and inhibitory activity. We identified a Siglec-6 mAb (AK04) that required Fc-mediated interaction for receptor internalization and induced inhibition and antibody-dependent cellular phagocytosis against MCs. AK04-mediated MC inhibition required Siglec-6 immunoreceptor tyrosine-based inhibitory motif (ITIM) and ITIM-like domains and was associated with receptor cluster formation containing inhibitory phosphatases. Treatment of humanized mice with AK04 inhibited systemic anaphylaxis with a single dose and reduced MCs with chronic dosing. Our findings suggest Siglec-6 activity is epitope dependent and highlight an agonistic Siglec-6 mAb as a potential therapeutic approach in allergic disease., (© 2022. The Author(s).)

Objective: To explore factors associated with walking adaptability and associations between walking adaptability and falling in polio survivors., Design: Cross-sectional study., Setting: Outpatient expert polio clinic., Participants: Polio survivors (N=46) who fell in the previous year and/or reported fear of falling., Interventions: Not applicable., Main Outcome Measures: Walking adaptability was assessed on an interactive treadmill and operationalized as variable target-stepping and reactive obstacle avoidance performance. Further, we collected walking speed and assessed leg muscle strength, balance performance (Berg Balance Scale and Timed-Up-and-Go Test), balance confidence (Activities-specific Balance Confidence scale), ambulation level, orthosis use, fear of falling, and number of falls in the previous year., Results: With walking speed included as a covariate, muscle weakness of the most affected leg and balance confidence explained 54% of the variance in variable target-stepping performance. For reactive obstacle avoidance performance, muscle weakness of the most affected leg and knee extensor strength of the least affected leg explained 32% of the variance. Only target-stepping performance was significantly related to the number of falls reported in the previous year (R 2 =0.277, P<.001) and mediated the relation between leg muscle weakness and balance confidence with falling., Conclusion: Our exploratory study suggests that leg muscle weakness and reduced balance confidence limit walking adaptability in polio survivors. Because poorer target stepping rather than obstacle avoidance performance was associated with falling, our results indicate that a limited ability to ensure safe foot placement may be a fall risk factor in this group. These findings should be confirmed in a larger sample., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is critically involved in hemostasis. Biosynthesis of long VWF concatemers in the endoplasmic reticulum and the trans-Golgi is still not fully understood. We use the single-molecule force spectroscopy technique magnetic tweezers to analyze a previously hypothesized conformational change in the D'D3 domain crucial for VWF multimerization. We find that the interface formed by submodules C8-3, TIL3, and E3 wrapping around VWD3 can open and expose 2 buried cysteines, Cys1099 and Cys1142, that are vital for multimerization. By characterizing the conformational change at varying levels of force, we can quantify the kinetics of the transition and stability of the interface. We find a pronounced destabilization of the interface on lowering the pH from 7.4 to 6.2 and 5.5. This is consistent with initiation of the conformational change that enables VWF multimerization at the D'D3 domain by a decrease in pH in the trans-Golgi network and Weibel-Palade bodies. Furthermore, we find a stabilization of the interface in the presence of coagulation factor VIII, providing evidence for a previously hypothesized binding site in submodule C8-3. Our findings highlight the critical role of the D'D3 domain in VWF biosynthesis and function, and we anticipate our methodology to be applicable to study other, similar conformational changes in VWF and beyond., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)