Human Model Research Articles

Design of multi-row parallel-transmit coil arrays for enhanced SAR efficiency with deep brain electrodes at 3T: an electromagnetic simulation study.

Tissue heating near the implanted deep brain stimulation (DBS) during magnetic resonance imaging (MRI) poses a significant safety constraint. This study aimed to evaluate the performance of parallel transmit (pTx) head transmit radiofrequency (RF) coils in DBS patients, with a focus on excitation fidelity under specific absorption rate (SAR) control for brain imaging at 3T MRI. We employed electromagnetic simulations to assess different coil configurations, including multi-row pTx coils of 16-24 channels arranged in 1, 2, and 3 rows, and compared these to a circularly polarised and pTx birdcage coil using a realistic human model without and with DBS leads and electrodes. Two- and three-row pTx coils with overlapping loop elements exhibited similar performance, which was superior in excitation homogeneity and local SAR compared to the single-row coil and the birdcage coil both without and with DBS. These findings suggest that multi-row coils can enhance the safety and efficacy of MRI in patients with DBS devices, so potentially improving imaging performance in this expanding patient population. There was a minimal difference in performance between the 2 and 3-row coils, favouring the simpler, lower channel count design for practical implementation.

Computer Modeling of Bevacizumab Drug Distribution After Intravitreal Injection in Rabbit and Human Eyes.

Age-related macular degeneration (AMD) is a progressive eye disease that causes loss of central vision and has no cure. Wet AMD is the late neovascular form treated with vascular endothelial growth factor (VEGF) inhibitors. VEGF is the critical driver of wet AMD. One common off-label anti-VEGF drug used in AMD treatment is bevacizumab. Experimental efforts have been made to investigate the pharmacokinetic (PK) behavior of bevacizumab in vitreous and aqueous humor. Still, the quantitative effect of elimination routes and drug concentration in the macula are not well understood. In this work, we developed two spatial models representing rabbit and human vitreous to better understand the PK behavior of bevacizumab. This study explores different cases of drug elimination and the effects of injection location on drug concentration profiles. The models are validated by comparing them with experimental data. Our results suggest that anterior elimination is dominant for bevacizumab clearance from rabbit vitreous, whereas both anterior and posterior elimination have similar importance in drug clearance from the human vitreous. Furthermore, results indicate that drug injections closer to the posterior segment of the vitreous help maintain relevant drug concentrations for longer, improving bevacizumab duration of action in the vitreous. The rabbit and human models predict bevacizumab concentration in the vitreous and fovea, enhancing knowledge and understanding of wet AMD treatment.

Abstract B073: Leveraging micronuclei DNA from erythrocytes for early detection of hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is the most common form of liver cancer and accounts for ∼90% of cases globally. Detection of early-stage HCC is vital as it allows for potentially curative treatment, yet remains a significant challenge. Micronuclei (MN) are a hallmark of genomic instability. An increased frequency of MN is commonly observed in tumor cells as well as other somatic cells, including erythrocytes, in cancer patients. Here, we have established a technique (WO2021/228246 A1), for the isolation and analysis of micronuclei DNA (MN-DNA) in erythrocytes from peripheral blood. Significant changes in read densities at specific genomic locations within MN-DNA were identified in patients, termed as tumor- associated MN-DNA (taMN-DNA) features. This study evaluates the potential of these taMN- DNA features for early-stage HCC detection across human and murine model. Methods: To explore the potential of MN-DNA in cancer detection, we first collected 1-2 mL of whole blood from HDs (N = 53) and HCC patients (N = 53). MN-DNA was isolated and purified from erythrocytes, followed by whole-genome sequencing. Participants were randomly divided into training, test cohorts in an 8:2 ratio. We applied machine learning algorithms to leverage these features for cancer detection in the human model. Additionally, to investigate the formation of taMN-DNA features, we employed a well-established mouse model that mimics HCC development. Mice were injected with the genotoxic agent diethylnitrosamine (DEN) 2 weeks after birth, and developed malignant macroscopic liver nodules at approximately 40 weeks of age. Results: In the human cohort, we enrolled 106 participants, with more than half of the HCC cases were diagnosed at early-stage (stage 0-II). The cancer detection model utilizing taMN- DNA features achieved an overall accuracy of 86.3%, with a sensitivity of 81.8% and specificity of 90.9%. For early-stage, the model demonstrated sensitivities of 54.5% at a specificity of 90.9%. In the DEN-induced HCC mouse model, unsupervised hierarchical clustering based on these selected taMN-DNA features clearly separated WT and tumor mice into two distinct groups. Notably, mice that were not tumorigenic at 36 weeks post-treatment in the DEN-treated group fell into the WT group when classified by the same taMN-DNA features, indicating that the formation of taMN-DNA signatures is associated with the presence of tumors specifically. Conclusions: This pilot study highlights the potential of MN-DNA as a promising tool for early HCC detection. Leveraging these taMN-DNA features can accurately distinguish early-stage HCC patients from HDs with high sensitivity. In both human and murine models, there is a significant relationship between these signatures and tumor presence, suggesting that taMN- DNA features might reflect the diseased state across mammalian species. Research sponsor: Timing Biotech. Citation Format: Hui Lin, Haobo Sun, Xingyun Yao, Xiaoxiao Fan, Fei Meng, Honghao Liang, Xiaofei Gao. Leveraging micronuclei DNA from erythrocytes for early detection of hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B073.

HPB O04 - Human Splenic Ex-Vivo Perfusion Model: A Platform for Translational Basic Science Research

Abstract Background The function of the spleen has always been shrouded in mystery and since the Renaissance leading figures have attempted to understand its function. Vesalius questioned Galen and Marcello Malpighi (1628-1694) gave the first accurate description of the histology and the physiology. The spleen still poses unique challenges as invasive approaches may produce intraperitoneal bleeding, differences between human and animal spleens complicates comparative studies, and post-mortem examinations reveal only late-stage disease manifestations and functional studies are impossible. This study describes a human splenic ex-vivo perfusion model for translational research, using microbiological studies as markers of splenic physiological function. Method Patients with left-sided pancreatic cancers scheduled for distal pancreatic and splenic resection were identified from the Hepato-Pancreatic and Biliary MDT at the University Hospitals of Leicester NHS Trust. Patients were informed, consented, and the surgical approach remained standard, with splenic artery ligation deferred until specimen extraction. The artery was cannulated, and residual blood clots were cleared with Custodiol® HTK solution. The perfusion technique employs an extracorporeal circuit incorporateing anoxygenator and a heated water chamber maintaining a 37°C perfusate. The perfused spleen was infected with Streptococcus Pneumoniae strains, with regular biopsies and perfusate gas analysis performed. Results Since February 2023, nine spleens have been successfully perfused, with an average weight of 169.5 grams, cold ischemic time of 116 minutes, and warm ischemic time of 12.2 minutes. Pressure was maintained at 80 mmHg and hourly perfusate gas analysis demonstrated a mean pH of 7.1 and a gradual increase in lactate. Histopathological examination revealed clear distinctions between the lymphoid tissue and splenic red pulp, with intact central arterioles and healthy cell morphology at time 0 and after 6 hours of perfusion. Additionally, the perfused spleens cleared S. pneumoniae strains to below-detection levels. Conclusion Our work demonstrates that a stable human splenic ex-vivoperfusion model which maintains structural and physiological integrity can be achieved from tissue that would otherwise be discarded. The continued physiological function is confirmed by the clearance of S. pneumoniae. While currently employed for microbiological studies, this model has the potential to facilitate a wide range of different translational science studieswhile reducing the need for animal experimentation. The perfusion times employed for the present study were sufficient to demonstrate bacterial clearance which was the aim but there is capacity in the system to significantly prolong the duration.

Can human brain connectivity explain verbal working memory?

ABSTRACT The ability of humans to store spoken words in verbal working memory and build extensive vocabularies is believed to stem from evolutionary changes in cortical connectivity across primate species. However, the underlying neurobiological mechanisms remain unclear. Why can humans acquire vast vocabularies, while non-human primates cannot? This study addresses this question using brain-constrained neural networks that realize between-species differences in cortical connectivity. It investigates how these structural differences support the formation of neural representations for spoken words and the emergence of verbal working memory, crucial for human vocabulary building. We develop comparative models of frontotemporal and occipital cortices, reflecting human and non-human primate neuroanatomy. Using meanfield and spiking neural networks, we simulate auditory word recognition and examine verbal working memory function. The “human models”, characterized by denser inter-area connectivity in core language areas, produced larger cell assemblies than the “monkey models”, with specific topographies reflecting semantic properties of the represented words. Crucially, longer-lasting reverberant neural activity was observed in human versus monkey architectures, compatible with robust verbal working memory, a necessary condition for vocabulary building. Our findings offer insights into the structural basis of human-specific symbol learning and verbal working memory, shedding light on humans’ unique capacity for large vocabulary acquisition.

Increased exercise tolerance in humanized G6PD-deficient mice

AbstractGlucose-6-phosphate dehydrogenase (G6PD) deficiency affects 500 million people globally, affecting red blood cell (RBC) antioxidant pathways and increasing susceptibility to hemolysis under oxidative stress. Despite the systemic generation of reactive oxygen species during exercise, the effects of exercise on individuals with G6PD deficiency remain poorly understood This study used humanized mouse models expressing the G6PD Mediterranean variant (S188F, with 10% enzymatic activity) to investigate exercise performance and molecular outcomes. Surprisingly, despite decreased enzyme activity, G6PD-deficient mice have faster critical speed than mice expressing human canonical G6PD. After exercise, deficient mice did not exhibit differences in RBC morphology or hemolysis, but had improved cardiac function, including cardiac output, stroke volume, sarcomere length, and mitochondrial content. Proteomics analyses of cardiac and skeletal muscles (gastrocnemius, soleus) from G6PD-deficient compared with sufficient mice revealed improvements in mitochondrial function and increased protein turnover via ubiquitination, especially for mitochondrial and structural myofibrillar proteins. Mass spectrometry–based metabolomics revealed alterations in energy metabolism and fatty acid oxidation. These findings challenge the traditional assumptions regarding hemolytic risk during exercise in G6PD deficiency, suggesting a potential metabolic advantage in exercise performance for individuals carrying noncanonical G6PD variants.

Vision-Based Fall Detector for Elderly Based on Sliding Window Approach and Feature Engineering

A new vision-based fall detector is proposed that uses the tsfresh tool to generate features from the bounding box motion parameters of an object and performs classification in a sliding window mode. The efficiency of the generated features is demonstrated compared to the primary ones. Using the auto-sklearn library and a generalized dataset compiled from the UR Fall Detection and CAUCAFall datasets, the best human fall detection model is found. This model, based on a gradient boosting classifier, achieved 96% accuracy, which is not inferior to well-known detection algorithms, but uses only two primary motion parameters to generate secondary features. A PCA-based class separability study showed that for secondary features, 99% of the variance is captured by the first 4 principal components, while for primary features, the first 10 principal components contain only 80% of the data variance. Furthermore, the processing time for generating secondary features and making predictions was found to be relatively short, taking only a few seconds per sequence, highlighting the practical applicability of the proposed approach in real-time fall monitoring systems.

Research on Key Technologies of Image Steganography Based on Simultaneous Deception of Vision and Deep Learning Models

As machine learning continues to evolve, traditional image steganography techniques find themselves increasingly unable to meet the dual challenge of deceiving both the human visual system and machine learning models. In response, this paper introduces the Visually Robust Image Steganography (VRIS) model, specifically tailored for this dual deception task. The VRIS model elevates visual security through the use of specialized feature extraction and processing methodologies. It meticulously conducts feature-level fusion between secret images and cover images, ensuring a high level of visual similarity between them. To effectively mislead machine learning models, the VRIS model incorporates a sophisticated strategy utilizing random noise factors and discriminators. This involves adding controlled amounts of random Gaussian noise to the encrypted image, thereby enhancing the difficulty for machine learning frameworks to recognize it. Furthermore, the discriminator is trained to discern between the noise-infused encrypted image and the original cover image. Through adversarial training, the discriminator and VRIS model refine each other, successfully deceiving the machine learning systems. Additionally, the VRIS model presents an innovative method for extracting and reconstructing secret images. This approach safeguards secret information from unauthorized access while enabling legitimate users to non-destructively extract and reconstruct images by leveraging multi-scale features from the encrypted image, combined with advanced feature fusion and reconstruction techniques. Experimental results validate the effectiveness of the VRIS model, achieving high PSNR and SSIM scores on the LFW dataset and demonstrating significant deception capabilities against the ResNet50 model on the Mini-ImageNet dataset, with an impressive misclassification rate of 99.24%.

A modified human shoulder model considering the migration of the humeral head from the scapular glenoid fossa for the design of shoulder exoskeletons

The wearable upper limb exoskeletons designed using the traditional shoulder model have a serious issue related to joint axis misalignment with the human shoulder. The misalignment is primarily due to the limited degrees of freedom model assumed for the shoulder. The glenohumeral joint in the conventional upper limb model is considered a single spherical joint, which, when considering the human anatomy, neglects the humeral head translation from the glenoid fossa of the scapula. This shoulder model cannot be used for designing the wearable upper limb exoskeletons, which can damage the shoulder joint due to interactive forces generated from the human-exoskeleton joint misalignment. This paper presents a modified, anatomically similar, human upper limb model where the single spherical joint of the glenohumeral joint is modified into two spherical joints connected by a prismatic joint. The kinematic and dynamic model of the proposed shoulder model is explained in detail. The elbow reachable space for this model has been calculated and compared with that of the existing models, and it is found to be closer to the actual workspace. The proposed model is simulated with a conventional exoskeleton attachment to show the effect of misalignment during humeral head translation and the benefits of using the proposed model in the design of the shoulder exoskeleton. An exoskeleton test bench that uses the modified shoulder model is also presented.

Abstract 4122301: The Role of Hepatic CDS2 in Phosphatidylinositol Homeostasis and Lipid Metabolism

Background: Recent studies in both human genetics and mouse models have highlighted the significance of phosphatidylinositol (PI) in the progression of fatty liver disease (FLD). Despite this, the precise mechanisms underlying PI's role remain elusive. Aims: This study aimed to elucidate the involvement of PI in FLD by disrupting hepatic PI biosynthesis through modulation of CDP-diacylglycerol synthase 2 (CDS2) expression, the primary enzyme in PI biosynthesis (A). Methods: We generated liver-specific knockouts of CDS2 and SCAP (SREBP cleavage-activating protein) using adeno-associated virus (AAV) expressing sgRNA against each gene. Phospholipid species were analyzed and quantified by LC/MS. Results: CDS2 liver-specific knockout mice (B, C) exhibited a pronounced development of fatty liver, characterized by elevated triglyceride and cholesterol levels (C). Notably, liver PI levels were significantly reduced in CDS2 knockout mice (E), accompanied by dysregulated activation of sterol regulatory element-binding protein (SREBP) even during fasting conditions (F), leading to increased de novo lipogenesis. Intraperitoneal administration of exogenous PI effectively normalized SREBP cleavage (G), highlighting PI's critical role in SREBP regulation. Furthermore, simultaneous knockout of SCAP with CDS2 restored the normal liver phenotype, implicating SREBP activation as a central mechanism underlying fatty liver development in CDS2 knockout mice (H, I). Conclusion: Deletion of CDS2 in the liver reduced PI levels, subsequently inducing significant fatty liver due to dysregulated SREBP activation. This study underscores the importance of CDS2 in maintaining PI homeostasis, which in turn regulates SREBP activity and lipid metabolism. Our findings provide valuable insights into the pathogenesis of metabolic disorders and offer potential therapeutic strategies for targeting FLD.

Effects of iron overload in human joint tissue explant cultures and animal models.

Osteoarthritis (OA) is a prevalent degenerative joint disease affecting over 530 million individuals worldwide. Recent studies suggest a potential link between iron overload, a condition characterised by the excessive accumulation of iron in the body, and the onset of OA. Iron is essential for various biological processes, and any disruption in its homeostasis can trigger significant health effects, including OA. This study aimed to elucidate the effects of excess iron on joint tissue and the underlying mechanisms associated with excess iron and OA development. Human articular cartilage (n = 6) and synovium (n = 4) were collected from patients who underwent total knee arthroplasty. Cartilage and synovium explants were incubated with a gradually increasing concentration of ferric ammonium citrate for 3days respectively. The effects of iron homeostasis in tissue explants were analysed using a Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS). To further study the effects of iron excess on OA initiation and development, male 3-week-old Hfe-/- and 5-week-old Tfr2-/- mice, animal models of hereditary haemochromatosis were established. Littermate wild-type mice were fed a high-iron diet to induce dietary overload. All animals were sacrificed at 8weeks of age, and knee joints were harvested for histological analysis. The LA-ICP-MS analysis unveiled changes in the elemental composition related to iron metabolism, which included alterations in FTH1, FPN1, and HAMP within iron(III)-treated cartilage explants. While chondrocyte viability remained stable under different iron concentrations, ex vivo treatment with a high concentration of Fe3+ increased the catabolic gene expression of MMP13. Similar alterations were observed in the synovium, with added increases in GAG content and inflammation markers. In vivo studies further supported the role of iron overload in OA development as evidenced by spontaneous OA symptoms, proteoglycan loss, increased Mankin scores, synovial thickening, and enhanced immunohistochemical expression of MMP13, ADAMTS5, and P21 in Hfe-/-, Tfr2-/-, and diet-induced iron overload mouse models. Our findings elucidate the specific pathways through which excess iron accelerates OA progression and highlights potential targets for therapeutic intervention aimed at modulating iron levels to mitigate OA symptoms. KEY MESSAGES: Iron overload alters joint iron metabolism, increasing OA markers in cartilage and synovium. High iron levels in mice accelerate OA, highlighting genetic and dietary impacts. Excess iron prompts chondrocyte iron storage response, signalling potential OA pathways. Iron dysregulation linked to increased cartilage degradation and synovial inflammation. Our findings support targeted therapies for OA based on iron modulation strategies.

Abstract 4140108: A CRISPR-Activation CROP-seq Screen Identifies HMGN1 as a Dosage-Sensitive Regulator of Heart Defects in Down Syndrome

Introduction/Background: Congenital heart defects (CHD) are the most common form of developmental abnormalities, occurring in ~1% of live births, and can arise due to altered dosage of genes essential for cardiogenesis. Aneuploidy accounts for nearly 15% of CHD and the most frequent form involves trisomy of chromosome 21 (Ch21), resulting in Down Syndrome (DS). CHD is present in ~50% of DS cases, with a 1000-fold enrichment of atrioventricular canal (AVC) defects that disrupt the junction of the atria and ventricles. Hypothesis: We hypothesize that the presence of a third copy of one or more genes on Ch21 underlies AVC and other cardiac defects; however, the dosage-sensitive CHD gene(s) on Ch21 causing this are not fully defined. Methods: We used human pluripotent stem cells derived from an individual mosaic for DS, allowing us to compare Ch21 trisomy cells with isogenic control cells disomic for Ch21. We performed single cell RNA-sequencing (scRNA-seq) to define the transcriptional dysregulation associated with DS in atrioventricular canal myocardial cells (AVCM). We then introduced a CRISPR-activation transgene to disomic cells and delivered small guide RNAs (sgRNAs) against 66 candidate Ch21 CHD genes. We developed a new machine learning algorithm to assign a trisomic score to CRISPR perturbed (CROP-seq) cells. Finally, we reduced the copy number of our top candidate gene in a mouse model of Down Syndrome and used microCT to assess incidence of cardiac septal defects. Results: Using human pluripotent stem cell and mouse models of DS, we identify HMGN1, a nucleosome-binding epigenetic factor on Ch21, as a dosage-sensitive regulator of cardiac defects in DS. Single cell transcriptomics revealed that human AVC cardiomyocytes shifted to a more ventricular myocardial state in trisomy 21 hiPS-derived cells. CRISPR-activation in isogenic disomic cells of 66 candidate Ch21 genes expressed in heart development, followed by single cell RNA-sequencing (CROP-seq) combined with machine learning predictions indicated that increased expression levels of HMGN1 altered the transcriptional state of atrioventricular cardiomyocytes similar to trisomy 21. Finally, reduction of Hmgn1 from three to two alleles in a mouse model of DS attenuated the incidence of cardiac septal defects. Conclusion: Our results identify HMGN1 as a dosage sensitive regulator of heart defects in DS and represent a generalizable approach for identifying candidate genes within areas of aneuploidy.

In Vitro Cholesterol Uptake by the Microflora of Selected Kefir Starter Cultures.

Kefir, a fermented milk beverage, is recognized for its potential health benefits, including its cholesterol-lowering properties. This study demonstrated that selected kefir starter cultures, including Lactococcus strains and yeasts, significantly reduce cholesterol-binding capacity under simulated gastrointestinal conditions, underscoring the challenges of probiotic delivery. We compared the performance of these cultures under laboratory conditions (growth broths) and simulated digestive juice models. Lactococcus strains showed significant differences in cholesterol binding between the two environments, highlighting the limitations of relying solely on laboratory testing. Yeast cultures also exhibited greater cholesterol binding in their native broths, but their survival was limited in digestive models. Our findings suggest that effective probiotic formulations should prioritize strains with high cholesterol-binding capacity and robust survival rates throughout the digestive tract. This study provides valuable insights for future research on the mechanisms behind these functionalities and the potential of kefir yeast strains for use in human digestive models. Our results can be used to inform the development of improved probiotic formulations for cholesterol management.

A new exvivo human skin model for the topographic and biological analysis of cosmetic formulas.

Existing methods to evaluate skin care products suffer limitations. This is the case for exvivo skin explants, a first-choice 3D model. While essential to analyse mid- to long-term biological effects, this classical model hinders assessing microrelief variations. To circumvent these limitations, we developed an exvivo PERFused EXplant setup (Perfex) that maintains the outer skin surface in the open air, closely mirroring physiological conditions. A custom-designed reservoir enables perfusing the dermal side of explants with buffered, temperature-controlled medium, while the epidermis is subjected to "normal" conditions. Skin tension and characteristics of the stratum corneum, microrelief, histology and immunohistology (collagen types I and III, elastin and fibrillin-1) were analysed and compared to those of explants maintained under conventional conditions or invivo skin. The effects of skin care formulas intended to induce short- and/or mid- to long-term effects were also assessed. Skin explants maintained with the Perfex setup exhibit characteristics (firmness, elasticity, hydration and barrier function) closer to those of invivo skin than with conventional conditions. Moreover, Perfex-maintained explants present no alteration in histology after 7 days and slight variation in the expression of key protein markers. Microrelief characteristics also remain mostly stable over 7 days. Formula applications corroborate that skin tensor-containing products primarily induce short-term changes in the microrelief, while those with biologically active ingredients mainly lead to mid- to long-term effects on the histology and expression of molecular markers. Furthermore, maintaining skin explants with a physiologically relevant skin surface enabled analysing the relationship between microrelief and key markers, showing that fibrillin-1 is the protein most correlated with microrelief characteristics. The Perfex setup allows for similar preservation of skin explant histology and key protein expression as the conventional system, yet it maintains a skin surface close to that of invivo skin. Therefore, it is valuable to analyse both the short- and mid- to long-term impacts of skin care formulas and better comprehend their effects. The Perfex system also offers a new tool for investigating fundamental questions, such as the link that can exist between dermal proteins and skin surface properties.

Structure–activity relationship studies of Imidazo[1′,2′:1,6]pyrido[2,3-d]pyrimidine derivatives to develop selective FGFR inhibitors as anticancer agents for FGF19-overexpressed hepatocellular carcinoma

The aberrant activation of fibroblast growth factor (FGF) and FGF receptor (FGFR)-mediated signaling pathways are associated with cancer development, including hepatocellular carcinoma (HCC). A novel series of imidazo[1′,2′:1,6]pyrido[2,3-d]pyrimidine, containing an acrylamide covalent warhead, were synthesized as selective FGFR 1–4 inhibitors. Compound 7n was identified as the most potent inhibitor against FGFR1, 2, and 4, with IC50 values of 8/4 nM (FGFR1/2) and 3.8 nM (FGFR4), and the covalent docking analyses suggested that 7n form a covalent adduct with cysteine residue on the hinge or p-loop of FGFR. Compound 7n exhibited a favorable pharmacokinetic profile and significant in vivo antitumor efficacy in human liver cancer xenograft mouse models (xenograft, FGF/FGFR-dependent HCC cells).

Enhancement of human native skin fibroblast proliferation in natural salso-bromo-iodic mineral water added to in vitro culture.

The favorable regenerative effects of some mineral waters on wound healing have long been empirically demonstrated. The aim of this experimental study is to investigate the effects of an Italian salso-bromo-iodic mineral water (Rivanazzano, Italy) on an in vitro human native fibroblast culture model to identify any potential regenerative actions. Human native fibroblasts were cultured under different experimental conditions: - Dulbecco's modified Eagle's medium (DMEM) reconstituted with distilled water (control); - DMEM reconstituted with filtered mineral water collected from the spring; - DMEM reconstituted with filtered mineral water collected at the balneotherapy facility; - DMEM reconstituted with filtered, heated mineral water collected at the balneotherapy facility; - DMEM partially replaced with filtered mineral water collected from the spring at different concentrations (10%, 20%, 30%, 40%, 50%); - DMEM partially replaced with filtered, heated mineral water collected at the balneotherapy facility at different concentrations (10%, 20%, 30%, 40%, 50%); - DMEM partially replaced with filtered mineral water collected at the balneotherapy facility at different concentrations (10%, 20%, 30%, 40%, 50%). Cell proliferation and viability were evaluated using spectrophotometric analysis following staining with the XTT Microculture Tetrazolium Assay. Statistical analyses were performed for each experimental condition at 24, 48 and 72 h. The best outcomes were observed in fibroblasts cultured with DMEM partially replaced with filtered mineral water collected from the spring, within the range of 20-50%. Our research results showed that Rivanazzano salso-bromo-iodic mineral water has a stimulating effect on in vitro human native fibroblast cultures. This activity was most pronounced with water collected from the spring, and it decreased with water collected at the balneotherapy facilities. These findings could form the basis for clinical applications in wound healing and balneotherapy.

Abstract 4140191: ChREBP Drives Brown Adipocyte Differentiation and Lipid Utilization for Excess Energy Expenditure

Introduction: Although several anti-obesity drugs have become recently available, they reduce patients’ appetite, which might worsen cardiovascular disease (CVD) prognosis by malnutrition/cachexia. Brown adipose tissue (BAT) oxidizes glucose and lipids for thermogenesis, a process that requires excess energy expenditure provided by uncoupling protein 1 (UCP1) + cells. Recently, critical roles of BAT in cardio-protection have been discovered. Taken together, boosting BAT activity can be a novel anti-obesity treatment suitable for CVD patients. In this study, we aimed to establish anti-obesity strategy by manipulation of the glucose-sensing lipid-storage transcription factor, ChREBP, with unexpected function of boosting lipid utilization in BAT. Methods and Results: Through mouse/human single-cell/nucleus RNA sequencing analyses, we identified ChREBP as specifically expressed in highly thermogenic UCP1 + brown adipocytes ( Fig.A ). To investigate the role of ChREBP in BAT formation, we generated human brown adipocyte-like model from induced pluripotent stem cells (iPSC-BAT, Fig.B ). The iPSC-BAT exhibited high UCP1 and ChREBP expression, multi-locular lipid droplets, and vigorous oxygen consumption. Knocking down ChREBP strongly blocked iPSC-BAT differentiation ( Fig.C ) and impaired lipid utilization ( Fig.D ). Consistently, in vitro deletion of ChREBP in mouse BAT progenitors impeded differentiation, lipid droplet accumulation, and oxygen consumption ( Fig.E-F ). In vivo, BAT-specific deletion of all ChREBP isoforms reduced lipid accumulation and BAT characteristics. Conversely, using a new mouse model for Cre-mediated ChREBPβ overexpression, we showed that ChREBPβ overexpression in BAT (Chβ BATOX) increased BAT cellularity and activated lipogenic machineries, with paradoxical decreased lipid content, suggesting excessively activated lipid consumption in BAT ( Fig.G ). More importantly, Chβ BATOX maintained UCP1 expression and reduced fat volume ( Fig.H ) even after strongly inhibiting BAT activity. Conclusion: In summary, ChREBP is essential for BAT development and thermogenic capacity by affecting lipid utilization. Our results suggest that ChREBPβ activation may be a novel anti-obesity avenue for CVD patients.

Identification of a Resistance Exercise-Specific Signaling Pathway that Drives Skeletal Muscle Growth.

A human model of unilateral endurance versus resistance exercise, in conjunction with deep phosphoproteomic analyses, was used to identify exercise mode-specific phosphorylation events. Among the outcomes, a resistance exercise-specific cluster of events was identified, and a multitude of bioinformatic- and literature-based predictions suggested that this was mediated by prolonged activation of a pathway involving MKK3b/6, p38, MK2, and mTORC1. Follow-up studies in humans and mice provide consistent support for the predictions and also revealed that resistance exercise-induced signaling through MKK3b and the induction of protein synthesis are highly correlated events (R = 0.87). Moreover, genetic activation of MKK3b/6 in skeletal muscles was sufficient to induce signaling through the members of the resistance exercise-specific pathway, as well as an increase in protein synthesis and fiber size. Thus, we propose that we have identified some of the core components of a signaling pathway that drives the growth-promoting effects of resistance exercise.

IMMU-21. OVEREXPRESSING CAR T-CELL GLUCOSE TRANSPORTER TO ENHANCE ANTI-TUMOR ACTIVITY IN GLIOBLASTOMA

Abstract INTRODUCTION Despite aggressive interventions, Glioblastoma (GBM) remains as a challenge for neuro-oncologists. Standard treatments are often followed by disease recurrence. Adoptive cell therapies have emerged as a viable therapeutic for brain malignancies. CAR-T cell therapy has generated substantial interest because of its efficacy and specificity in treating various cancers. Despite these efficacies, studies reveal limitations of CAR-T therapy due to the altered metabolic environment in GBM. GBM tumors shift towards aerobic glycolysis, regardless of oxygen availability leading to massive glucose consumption. This results in impeding T-cell glycolysis and activation. OBJECTIVE The GBM microenvironment provides local restraints via metabolic competition suppressing antitumor immunity, specifically inhibiting infiltration and tumoricidal functions of host and adoptively transferred tumor-reactive T-cells. In our study, we manipulated the metabolic fitness of the therapeutic T-cells to enhance their tumoricidal activity, by overexpressing glucose transporter GLUT1 and GLUT3. We show that this manipulation can enhance their anti-tumor activity. METHODS The T-cell glucose metabolic pathway was modulated via glucose transporter overexpression. The functionality of metabolically modified T-cells was investigated in murine and human models. RESULTS We demonstrated that tumor cells impose glucose restriction when competing for glucose with T-cells leading to T-cell hypo responsiveness. Overexpression of glucose transporters such as Glut1 and Glut3 increased T-cell glucose utilization and provide survival advantage leading to enhanced T-cell activation in glucose-restricted conditions. The metabolic modifications regulate T-cell efficacy by enhancing intra-tumoral trafficking. We tested this approach in the context of CD70CAR T cell therapy. It was established that glucose transporter overexpression in CD70 CAR T-cells not only improves intracranial tumor invasion but also elicit functional anti-tumor cell response thus improving survival. CONCLUSIONS This study integrates fundamental concepts of tumor and immune metabolism in the design of immunotherapy and confirms that immunometabolism represents a viable target for new cancer therapy to treat brain tumors.

MiR-24-3p secreted as extracellular vesicle cargo by cardiomyocytes inhibits fibrosis in human cardiac microtissues.

Cardiac fibrosis in response to injury leads to myocardial stiffness and heart failure. At the cellular level, fibrosis is triggered by the conversion of cardiac fibroblasts (CF) into extracellular matrix-producing myofibroblasts. miR-24-3p regulates this process in animal models. Here, we investigated whether miR-24-3p plays similar roles in human models. Gain- and loss-of-function experiments were performed using human induced pluripotent stem cell-derived cardiomyocytes (hCM) and primary hCF under normoxic or ischaemia-simulating conditions. hCM-derived extracellular vesicles (EVs) were added to hCF. Similar experiments were performed using three-dimensional human cardiac microtissues and ex vivo-cultured human cardiac slices.hCF transfection with miR-24-3p mimic prevented TGFβ1-mediated induction of FURIN, CCND1 and SMAD4-miR-24-3p target genes participating in TGFβ1-dependent fibrinogenesis -, regulating hCF-to-myofibroblast conversion. hCM secreted miR-24-3p as EV cargo. hCM-derived EVs modulated hCF activation. Ischaemia-simulating conditions induced miR-24-3p depletion in hCM-EVs and microtissues. Similarly, hypoxia downregulated miR-24-3p in cardiac slices. Analyses of clinical samples revealed decreased miR-24-3p levels in circulating EVs in acute myocardial infarction (AMI) patients, compared with healthy subjects. Post-mortem RNAScope analysis showed miR-24-3p downregulation in myocardium from AMI patients, compared with patients who died from noncardiac diseases. Berberin, a plant-derived agent with miR-24-3p-stimulatory activity, increased miR-24-3p contents in hCM-EVs, downregulated FURIN, CCND1 and SMAD4, and inhibited fibrosis in cardiac microtissues. These findings suggest that hCM may control hCF activation through miR-24-3p secreted as EV cargo. Ischaemia impairs this mechanism, favouring fibrosis.