Engineered T cells secreting anti-BCMA T cell engagers control multiple myeloma and promote immune memory in vivo.

Multiple myeloma is the second most common hematological malignancy in adults and remains an incurable disease. B cell maturation antigen (BCMA)-directed immunotherapy, including T cells bearing chimeric antigen receptors (CARs) and systemically injected bispecific T cell engagers (TCEs), has shown remarkable clinical activity, and several products have received market approval. However, despite promising results, most patients eventually become refractory and relapse, highlighting the need for alternative strategies. Engineered T cells secreting TCE antibodies (STAb) represent a promising strategy that combines the advantages of adoptive cell therapies and bispecific antibodies. Here, we undertook a comprehensive preclinical study comparing the therapeutic potential of T cells either expressing second-generation anti-BCMA CARs (CAR-T) or secreting BCMAxCD3 TCEs (STAb-T) in a T cell-limiting experimental setting mimicking the conditions found in patients with relapsed/refractory multiple myeloma. STAb-T cells recruited T cell activity at extremely low effector-to-target ratios and were resistant to inhibition mediated by soluble BCMA released from the cell surface, resulting in enhanced cytotoxic responses and prevention of immune escape of multiple myeloma cells in vitro. These advantages led to robust expansion and persistence of STAb-T cells in vivo, generating long-lived memory BCMA-specific responses that could control multiple myeloma progression in xenograft models, outperforming traditional CAR-T cells. These promising preclinical results encourage clinical testing of the BCMA-STAb-T cell approach in relapsed/refractory multiple myeloma.

Subcutaneous Administration of a Zwitterionic Chitosan-Based Hydrogel for Controlled Spatiotemporal Release of Monoclonal Antibodies.

Subcutaneous (SC) administration of monoclonal antibodies (mAbs) is a proven strategy for improving therapeutic outcomes and patient compliance. The current FDA-/EMA-approved enzymatic approach, utilizing recombinant human hyaluronidase (rHuPH20) to enhance mAbs SC delivery, involves degrading the extracellular matrix's hyaluronate to increase tissue permeability. However, this method lacks tunable release properties, requiring individual optimization for each mAb. Seeking alternatives, physical polysaccharide hydrogels emerge as promising candidates due to their tunable physicochemical and biodegradability features. Unfortunately, none have demonstrated simultaneous biocompatibility, biodegradability, and controlled release properties for large proteins (≥150 kDa) after SC delivery in clinical settings. Here, a novel two-component hydrogel comprising chitosan and chitosan@DOTAGA is introduced that can be seamlessly mixed with sterile mAbs formulations initially designed for intravenous (IV) administration, repurposing them as novel tunable SC formulations. Validated in mice and nonhuman primates (NHPs) with various mAbs, including trastuzumab and rituximab, the hydrogel exhibited biodegradability and biocompatibility features. Pharmacokinetic studies in both species demonstrated tunable controlled release, surpassing the capabilities of rHuPH20, with comparable parameters to the rHuPH20+mAbs formulation. These findings signify the potential for rapid translation to human applications, opening avenues for the clinical development of this novel SC biosimilar formulation.

Mechanical stress confers nuclear and functional changes in derived leukemia cells from persistent confined migration.

Nuclear deformability plays a critical role in cell migration. During this process, the remodeling of internal components of the nucleus has a direct impact on DNA damage and cell behavior; however, how persistent migration promotes nuclear changes leading to phenotypical and functional consequences remains poorly understood. Here, we described that the persistent migration through physical barriers was sufficient to promote permanent modifications in migratory-altered cells. We found that derived cells from confined migration showed changes in lamin B1 localization, cell morphology and transcription. Further analysis confirmed that migratory-altered cells showed functional differences in DNA repair, cell response to chemotherapy and cell migration in vivo homing experiments. Experimental modulation of actin polymerization affected the redistribution of lamin B1, and the basal levels of DNA damage in migratory-altered cells. Finally, since major nuclear changes were present in migratory-altered cells, we applied a multidisciplinary biochemical and biophysical approach to identify that confined conditions promoted a different biomechanical response of the nucleus in migratory-altered cells. Our observations suggest that mechanical compression during persistent cell migration has a role in stable nuclear and genomic alterations that might handle the genetic instability and cellular heterogeneity in aging diseases and cancer.

Activation kinetics of regulatory molecules during immunological synapse in T cells.

T cell activation through TCR stimulation leads to the formation of the immunological synapse (IS), a specialized adhesion organized between T lymphocytes and antigen presenting cells (APCs) in which a dynamic interaction among signaling molecules, the cytoskeleton and intracellular organelles achieves proper antigen-mediated stimulation and effector function. The kinetics of molecular reactions at the IS is essential to determine the quality of the response to the antigen stimulation. Herein, we describe methods based on biochemistry, flow cytometry and imaging in live and fixed cells to study the activation state and dynamics of regulatory molecules at the IS in the Jurkat T cell line CH7C17 and primary human and mouse CD4+ T lymphocytes stimulated by antigen presented by Raji and HOM2 B cell lines and human and mouse dendritic cells.

T cell activation and effector function in the human Jurkat T cell model.

In order to understand T cell function, it is necessary to completely decipher the molecular dynamics underlying T cell activation and effector function. In vitro easy-to-handle cellular models are valuable tools to study intracellular molecular mechanisms in live cells. The CD4 T cell line Jurkat (JK) has been widely employed to investigate intracellular signaling leading to T cell activation in response to T cell receptor (TCR) triggering. Here, we describe diverse, complementary protocols to evaluate the TCR- and costimulation-mediated T cell activation, as well as the immunological synapse assembly and cytokine production occurring as a consequence of successful early activation events. This in vitro model is extremely useful to address molecular mechanisms operating during T cell activation and effector function acting in diverse pathophysiological scenarios.

Smartphone accelerometry for quantifying core stability and developing exercise training progressions in people with multiple sclerosis.

BACKGROUND: Core stability exercise programs have become popular in recent years for preserving balance and functional independence in people with multiple sclerosis (PwMS); however, their real impact is not well-known as the main intervention target (i.e., core stability) theoretically responsible for balance or functional improvements is not measured. The objective of this study was to test the reliability of accelerometers integrated into smartphones for quantifying core stability and developing exercise progressions in PwMS. METHODS: Twenty participants with MS [age: 47.5±8.0 years; height: 1.62±0.07 m; mass: 63.4±10.9 kg; EDSS: 3.0 (1.5-6)] participated voluntarily in this study. CS was assessed in different variations of the front, side, and back bridges and bird-dog exercises by measuring the mean lumbopelvic acceleration in two testing sessions, separated by one week. Relative and absolute reliability of lumbopelvic acceleration of those exercise variations performed by more than 60% of the participants was analyzed by the intraclass correlation coefficient (ICC3,1), and the standard error of measurement (SEM) and the minimal detectable change (MDC), respectively. Repeated measures ANOVAs were performed to detect a potential learning effect between test-retest assessments. Statistical significance was set at p < 0.05. RESULTS: Reliability analyses revealed that good to excellent relative and absolute scores (0.850.05). CONCLUSION: Smartphone accelerometry seems a low cost, portable and easy-to-use tool to objectively and reliably track core stability changes in PwMS through. However, in spite of the popularity of bridging and bird-dog exercises, only the short and long bridges and the three-point bird-dog positions proved feasible for most participants. Overall, this study provides useful information to evaluate and guide the prescription of core stability exercise programs in PwMS with mild-to-moderate impairment.

A Thematic Survey on the Reporting Quality of Randomized Controlled Trials in Rehabilitation: The Case of Multiple Sclerosis.

BACKGROUND AND PURPOSE: Optimal reporting is a critical element of scholarly communications. Several initiatives, such as the EQUATOR checklists, have raised authors' awareness about the importance of adequate research reports. On these premises, we aimed at appraising the reporting quality of published randomized controlled trials (RCTs) dealing with rehabilitation interventions. Given the breadth of such literature, we focused on rehabilitation for multiple sclerosis (MS), which was taken as a model of a challenging condition for all the rehabilitation professionals.A thematic methodological survey was performed to critically examine rehabilitative RCTs published in the last 2 decades in MS populations according to 3 main reporting themes: (1) basic methodological and statistical aspects; (2) reproducibility and responsiveness of measurements; and (3) clinical meaningfulness of the change. SUMMARY OF KEY POINTS: Of the initial 526 RCTs retrieved, 370 satisfied the inclusion criteria and were included in the analysis. The survey revealed several sources of weakness affecting all the predefined themes: among these, 25.7% of the studies complemented the P values with the confidence interval of the change; 46.8% reported the effect size of the observed differences; 40.0% conducted power analyses to establish the sample size; 4.3% performed retest procedures to determine the outcomes' reproducibility and responsiveness; and 5.9% appraised the observed differences against thresholds for clinically meaningful change, for example, the minimal important change. RECOMMENDATIONS FOR CLINICAL PRACTICE: The RCTs dealing with MS rehabilitation still suffer from incomplete reporting. Adherence to evidence-based checklists and attention to measurement issues and their impact on data interpretation can improve study design and reporting in order to truly advance the field of rehabilitation in people with MS.Video Abstract available for more insights from the authors (see the Video, Supplemental Digital Content 1 available at: http://links.lww.com/JNPT/A424 ).

Autonomous Vehicle Dataset with Real Multi-Driver Scenes and Biometric Data.

The development of autonomous vehicles is becoming increasingly popular and gathering real-world data is considered a valuable task. Many datasets have been published recently in the autonomous vehicle sector, with synthetic datasets gaining particular interest due to availability and cost. For a real implementation and correct evaluation of vehicles at higher levels of autonomy, it is also necessary to consider human interaction, which is precisely something that lacks in existing datasets. In this article the UPCT dataset is presented, a public dataset containing high quality, multimodal data obtained using state-of-the-art sensors and equipment installed onboard the UPCT's CICar autonomous vehicle. The dataset includes data from a variety of perception sensors including 3D LiDAR, cameras, IMU, GPS, encoders, as well as driver biometric data and driver behaviour questionnaires. In addition to the dataset, the software developed for data synchronisation and processing has been made available. The quality of the dataset was validated using an end-to-end neural network model with multiple inputs to obtain the speed and steering wheel angle and it obtained very promising results.

Effect of Trunk-Focused Exercises on Pain, Disability, Quality of Life, and Trunk Physical Fitness in Low Back Pain and How Potential Effect Modifiers Modulate Their Effects: A Systematic Review With Meta-analyses.

OBJECTIVE: To analyze the effect of trunkfocused exercise programs (TEPs) and moderator factors on chronic nonspecific low back pain (LBP). DESIGN: Systematic review with meta-analyses. LITERATURE SEARCH: We searched the PubMed, Scopus, Embase, SPORTDiscus, and CENTRAL databases from their inception to June 2022. STUDY SELECTION CRITERIA: We included randomized controlled trials comparing TEPs to control or general exercises. DATA SYNTHESIS: We used random-effects models to calculate the standardized mean difference (SMD) plus confidence interval (CI) and heterogeneity (I2) for pain, disability, quality of life, and trunk performance. The impact of moderator factors was analyzed through meta-regression. RESULTS: Forty randomized controlled trials (n = 2391) were included. TEPs showed positive effects for all outcomes versus control (SMD 0.90-2.46; 95% CI, -0.04 to 4.96; I2 61%-98%). There were small effects in favor of TEPs versus general exercises for pain (SMD = 0.20; 95% CI, 0.03-0.37; I2 = 13.4%) and disability (SMD = 0.20; 95% CI, 0.02-0.38; I2 = 0%). Trunk and/or hip range-of-motion improvements were associated with greater reductions in pain (P<.01; ß = 0.56; 95% CI, 0.25-0.87) and disability (P<.01; ß = 0.66; 95% CI, 0.27-1.05). Low body mass was associated with higher pain reduction (P = .03; ß = -0.17; 95% CI, -0.32 to -0.02). CONCLUSIONS: Trunk-focused exercise programs had positive effects on pain, disability, quality of life, and trunk performance compared to control groups, and on pain and disability compared to general exercises. Increasing trunk and/or hip range of motion was associated with greater pain and disability reduction, and lower body mass with higher pain reduction. J Orthop Sports Phys Ther 2023;53(2):64-93. Epub: 16 January 2023. doi:10.2519/jospt.2023.11091.

Trunk muscle activation in prone plank exercises with different body tilts.

BACKGROUND: Body tilt changes could affect the intensity/difficulty of core stability exercises, but there is still a lack of knowledge about its impact. OBJECTIVE: To analyse the trunk muscles activation during prone plank exercises at different body tilts. METHODS: Twenty-four young adults who performed recreational gymnastic activities participated in this study. Electromyography activity of the rectus abdominis (RA), external oblique (EO), internal oblique (IO) and erector spinae (ES) was recorded during the performance of six variations of the prone plank exercise (planking with feet supported on the ground [conventional horizontal position] and planking with feet supported on wall bars at five different heights increasing the angle tilt) and an inverted position exercise. RESULTS: The RA, EO and IO activation in all prone plank variations were higher than those observed in the conventional prone plank. In addition, the coefficient of variation of the muscle activation increased with the declination angle, reaching the highest values in the inverted position for the RA and ES muscles. CONCLUSION: The results seem to indicate that the body tilt variation could be used as an easy and inexpensive strategy for modulating the neuromuscular demands and the motor control challenge during planking exercises.

Effects of playing 1 vs 3 matches in a one-week period on physical performance in young soccer players.

The aim of this study was to analyse the effects of playing 1 vs 3 matches in a one-week period on physical performance in young soccer players. Twelve youth soccer players completed a battery of physical tests (countermovement jump [CMJ], 25 m sprint, 5-0-5 agility test, ankle dorsiflexion range of motion [AD ROM]) 72 h after a match. These tests were performed on two different occasions: during a week with 1 competitive match, and during a week in which 3 matches were played. Three matches in a week caused from most likely to very likely impairments in CMJ (ES = 0.81), the 5-0-5 agility test (ES = 1.03), and in AD ROM (ES = 0.46-0.63) compared with the 1 match in a week. For the 25 m sprint test, performance impairments were found in the split times for 10-15 m (ES = 0.72), 15-20 m (ES = 0.52) and 20-25 m (ES = 0.90) compared with 1 match in a week. Jumping, sprinting, change of direction (COD) performance and AD ROM are significantly affected during congested calendars in young soccer players. The monitoring of these variables is a useful tool to assess players' recovery and may help in determining players' readiness for the next matches.

A novel ground truth multispectral image dataset with weight, anthocyanins, and Brix index measures of grape berries tested for its utility in machine learning pipelines.

BACKGROUND: The combination of computer vision devices such as multispectral cameras coupled with artificial intelligence has provided a major leap forward in image-based analysis of biological processes. Supervised artificial intelligence algorithms require large ground truth image datasets for model training, which allows to validate or refute research hypotheses and to carry out comparisons between models. However, public datasets of images are scarce and ground truth images are surprisingly few considering the numbers required for training algorithms. RESULTS: We created a dataset of 1,283 multidimensional arrays, using berries from five different grape varieties. Each array has 37 images of wavelengths between 488.38 and 952.76 nm obtained from single berries. Coupled to each multispectral image, we added a dataset with measurements including, weight, anthocyanin content, and Brix index for each independent grape. Thus, the images have paired measures, creating a ground truth dataset. We tested the dataset with 2 neural network algorithms: multilayer perceptron (MLP) and 3-dimensional convolutional neural network (3D-CNN). A perfect (100% accuracy) classification model was fit with either the MLP or 3D-CNN algorithms. CONCLUSIONS: This is the first public dataset of grape ground truth multispectral images. Associated with each multispectral image, there are measures of the weight, anthocyanins, and Brix index. The dataset should be useful to develop deep learning algorithms for classification, dimensionality reduction, regression, and prediction analysis.

Altered CXCR4 dynamics at the cell membrane impairs directed cell migration in WHIM syndrome patients.

Chemokine receptor nanoscale organization at the cell membrane is orchestrated by the actin cytoskeleton and influences cell responses. Using single-particle tracking analysis we show that CXCR4R334X, a truncated mutant chemokine receptor linked to WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis), fails to nanoclusterize after CXCL12 stimulation, and alters the lateral mobility and spatial organization of CXCR4 when coexpressed. These findings correlate with multiple phalloidin-positive protrusions in cells expressing CXCR4R334X, and their inability to correctly sense chemokine gradients. The underlying mechanisms involve inappropriate actin cytoskeleton remodeling due to the inadequate ß-arrestin1 activation by CXCR4R334X, which disrupts the equilibrium between activated and deactivated cofilin. Overall, we provide insights into the molecular mechanisms governing CXCR4 nanoclustering, signaling and cell function, and highlight the essential scaffold role of ß-arrestin1 to support CXCL12-mediated actin reorganization and receptor clustering. These defects associated with CXCR4R334X expression might contribute to the severe immunological symptoms associated with WHIM syndrome.

Overcoming CAR-Mediated CD19 Downmodulation and Leukemia Relapse with T Lymphocytes Secreting Anti-CD19 T-cell Engagers.

Chimeric antigen receptor (CAR)-modified T cells have revolutionized the treatment of CD19-positive hematologic malignancies. Although anti-CD19 CAR-engineered autologous T cells can induce remission in patients with B-cell acute lymphoblastic leukemia, a large subset relapse, most of them with CD19-positive disease. Therefore, new therapeutic strategies are clearly needed. Here, we report a comprehensive study comparing engineered T cells either expressing a second-generation anti-CD19 CAR (CAR-T19) or secreting a CD19/CD3-targeting bispecific T-cell engager antibody (STAb-T19). We found that STAb-T19 cells are more effective than CAR-T19 cells at inducing cytotoxicity, avoiding leukemia escape in vitro, and preventing relapse in vivo. We observed that leukemia escape in vitro is associated with rapid and drastic CAR-induced internalization of CD19 that is coupled with lysosome-mediated degradation, leading to the emergence of transiently CD19-negative leukemic cells that evade the immune response of engineered CAR-T19 cells. In contrast, engineered STAb-T19 cells induce the formation of canonical immunologic synapses and prevent the CD19 downmodulation observed in anti-CD19 CAR-mediated interactions. Although both strategies show similar efficacy in short-term mouse models, there is a significant difference in a long-term patient-derived xenograft mouse model, where STAb-T19 cells efficiently eradicated leukemia cells, but leukemia relapsed after CAR-T19 therapy. Our findings suggest that the absence of CD19 downmodulation in the STAb-T19 strategy, coupled with the continued antibody secretion, allows an efficient recruitment of the endogenous T-cell pool, resulting in fast and effective elimination of cancer cells that may prevent CD19-positive relapses frequently associated with CAR-T19 therapies.

Synapse topology and downmodulation events determine the functional outcome of anti-CD19 T cell-redirecting strategies.

Cancer immunotherapy strategies based on the endogenous secretion of T cell-redirecting bispecific antibodies by engineered T lymphocytes (STAb-T) are emerging as alternative or complementary approaches to those based on chimeric antigen receptors (CAR-T). The antitumor efficacy of bispecific anti-CD19 × anti-CD3 (CD19×CD3) T cell engager (BiTE)-secreting STAb-T cells has been demonstrated in several mouse models of B-cell acute leukemia. Here, we have investigated the spatial topology and downstream signaling of the artificial immunological synapses (IS) that are formed by CAR-T or STAb-T cells. Upon interaction with CD19-positive target cells, STAb-T cells form IS with structure and signal transduction, which more closely resemble those of physiological cognate IS, compared to IS formed by CAR-T cells expressing a second-generation CAR bearing the same CD19-single-chain variable fragment. Importantly, while CD3 is maintained at detectable levels on the surface of STAb-T cells, indicating sustained activation mediated by the secreted BiTE, the anti-CD19 CAR was rapidly downmodulated, which correlated with a more transient downstream signaling. Furthermore, CAR-T cells, but not STAb-T cells, provoke an acute loss of CD19 in target cells. Such differences might represent advantages of the STAb-T strategy over the CAR-T approach and should be carefully considered in order to develop more effective and safer treatments for hematological malignancies.

Effect of laser photocoagulation, antiangiogenic therapy or a combined treatment on refractive outcomes of newborns with ROP.

PURPOSE: To determine the refractive outcome after treatment with laser photocoagulation, intravitreal antiangiogenic injection or both, for the treatment of type 1 ROP and/or aggressive posterior ROP using defocus equivalent terms. These results will be compared with the spherical equivalent (SE) notation in an initial refraction (during the first 3 years after treatment) and in a final evaluation (three years after treatment). METHODS: Clinical charts review of preterm patients who developed type 1 ROP and/or aggressive posterior ROP who were treated with laser photocoagulation, intravitreal antiangiogenic injection, or both, between January 2007 and December 2014 at Fundación Oftalmológica de Santander were reviewed. Refractive error was evaluated at two different time points in three different treatment groups. RESULTS: Seventy-seven eyes (56 patients) with ROP diagnosis treated with laser, antiangiogenics or in a combined scheme were included. Median gestational age was 29 weeks (IQR = 3), median birth weight was 1100 (IQR = 335) and mean corrected age at the time of treatment was 37.3 weeks (SD 2.2). Refractive outcomes in SE terms for the first refraction had a median of -0.50 diopters and in defocus terms, 4.00 diopters (D). For the second refraction, -3.00 diopters and 4.00 diopters (D), respectively. Pearson correlation test result for the first measurement was 93% (p = 0.000) and for the second evaluation was 99% (p = 0.000). CONCLUSIONS: Low birth weight had a statistically significant association to the increase of the refractive outcome. The high correlation between SE and DE for refractive errors allows us to confirm that DE is an appropriate metric to report refractive outcomes in this group of patients due to the presence of combined refractive ametropias.

CRISPR/Cas9-mediated genome editing assists protein dynamics studies in live cells.

Spatial and temporal regulation of molecular reactions dictates cell fate. Thus, studying molecular dynamics is essential to understand how cells decide what to do and the fundamental perturbations causing disease. Classically, molecular dynamics has been studied by protocols based in the overexpression of fluorescent fusion proteins. However, overexpression is associated to altered stoichiometry, molecular dynamics and subcellular distribution. We here discuss the necessity to study molecular dynamics of fluorescent fusion proteins expressed under physiological mechanisms in the cell, pointing to CRISPR/Cas9-mediated genome editing as the ideal means to do so. Current genome editing protocols enable us to study molecular dynamics while avoiding drawbacks associated to overexpression.

Trispecific T-cell engagers for dual tumor-targeting of colorectal cancer.

Retargeting of T lymphocytes toward cancer cells by bispecific antibodies has demonstrated its therapeutic potential, with one such antibody approved for the treatment of acute lymphoblastic leukemia (blinatumomab) and several other in clinical trials. However, improvement of their efficacy and selectivity for solid tumors is still required. Here, we describe a novel tandem T-cell recruiting trispecific antibody for the treatment of colorectal cancer (CRC). This construct, termed trispecific T-cell engager (TriTE), consists of a CD3-specific single-chain Fv (scFv) flanked by anti-epidermal growth factor receptor (EGFR) and anti-epithelial cell adhesion molecule (EpCAM) single-domain VHH antibodies. The TriTE was well expressed in mammalian and yeast cells, bound the cognate antigens of the three parental antibodies, and enabled the specific cytolysis of EGFR- and/or EpCAM-expressing cancer cells, without inducing T cell activation and cytoxicity against double-negative (EGFR-EpCAM-) cancer cells. Bivalent bispecific targeting of double-positive HCT116 cells by TriTE improved in vitro potency up to 100-fold compared to single-positive cells and significantly prolonged survival in vivo. In addition, it was less efficient at killing single-positive target cells than the corresponding bispecific controls, leading to potentially enhanced tumor specificity. Moreover, dual targeting of two tumor-associated antigens may contribute toward preventing the tumor escape by antigen loss caused by selective pressures from conventional single-targeting T-cell engagers, and may help to overcome antigenic heterogeneity.