Classification and counting of cells in brightfield microscopy images: an application of convolutional neural networks.

Microscopy is integral to medical research, facilitating the exploration of various biological questions, notably cell quantification. However, this process's time-consuming and error-prone nature, attributed to human intervention or automated methods usually applied to fluorescent images, presents challenges. In response, machine learning algorithms have been integrated into microscopy, automating tasks and constructing predictive models from vast datasets. These models adeptly learn representations for object detection, image segmentation, and target classification. An advantageous strategy involves utilizing unstained images, preserving cell integrity and enabling morphology-based classification-something hindered when fluorescent markers are used. The aim is to introduce a model proficient in classifying distinct cell lineages in digital contrast microscopy images. Additionally, the goal is to create a predictive model identifying lineage and determining optimal quantification of cell numbers. Employing a CNN machine learning algorithm, a classification model predicting cellular lineage achieved a remarkable accuracy of 93%, with ROC curve results nearing 1.0, showcasing robust performance. However, some lineages, namely SH-SY5Y (78%), HUH7_mayv (85%), and A549 (88%), exhibited slightly lower accuracies. These outcomes not only underscore the model's quality but also emphasize CNNs' potential in addressing the inherent complexities of microscopic images.

Extracellular vesicles in neuroblastoma: role in progression, resistance to therapy and diagnostics.

Neuroblastoma (NB) is the most common extracranial solid pediatric cancer, and is one of the leading causes of cancer-related deaths in children. Despite the current multi-modal treatment regimens, majority of patients with advanced-stage NBs develop therapeutic resistance and relapse, leading to poor disease outcomes. There is a large body of knowledge on pathophysiological role of small extracellular vesicles (EVs) in progression and metastasis of multiple cancer types, however, the importance of EVs in NB was until recently not well understood. Studies emerging in the last few years have demonstrated the involvement of EVs in various aspects of NB pathogenesis. In this review we summarize these recent findings and advances on the role EVs play in NB progression, such as tumor growth, metastasis and therapeutic resistance, that could be helpful for future investigations in NB EV research. We also discuss different strategies for therapeutic targeting of NB-EVs as well as utilization of NB-EVs as potential biomarkers.

Resiquimod Induces C-C Motif Chemokine Ligand 2 Via Nuclear Factor-Kappa B in SH-SY5Y Human Neuroblastoma Cells.

Toll-like receptor (TLR) 7 plays an important role in recognizing virus-derived nucleic acids. TLR7 signaling in astrocytes and microglia is critical for activating immune responses against neurotrophic viruses. Neurons express TLR7, similar to glial cells; however, the role of neuronal TLR7 has not yet been fully elucidated. This study sought to determine whether resiquimod, the TLR7/8 agonist, induces the expression of inflammatory chemokines in SH-SY5Y human neuroblastoma cells. Immunofluorescence microscopy revealed that TLR7 was constitutively expressed in SH-SY5Y cells. Stimulation with resiquimod induced C-C motif chemokine ligand 2 (CCL2) expression, accompanied by the activation of nuclear factor-kappa B (NF-κB) in SH-SY5Y cells. Resiquimod increased mRNA levels of C-X-C motif chemokine ligand 8 (CXCL8) and CXCL10, while the increase was slight at the protein level. Knockdown of NF-κB p65 eliminated resiquimod-induced CCL2 production. This study provides novel evidence that resiquimod has promising therapeutic potential against central nervous system viral infections through its immunostimulatory effects on neurons.

Ergosterol promotes neurite outgrowth, inhibits amyloid-beta synthesis, and extends longevity: In vitro neuroblastoma and in vivo Caenorhabditis elegans evidence.

AIMS: Alzheimer's disease (AD), the most common neurodegenerative disorder associated with aging, is characterized by amyloid-ß (Aß) plaques in the hippocampus. Ergosterol, a mushroom sterol, exhibits neuroprotective activities; however, the underlying mechanisms of ergosterol in promoting neurite outgrowth and preventing Aß-associated aging have never been investigated. We aim to determine the beneficial activities of ergosterol in neuronal cells and Caenorhabditis elegans (C. elegans). MATERIALS AND METHODS: The neuritogenesis and molecular mechanisms of ergosterol were investigated in wild-type and Aß precursor protein (APP)-overexpressing Neuro2a cells. The anti-amyloidosis properties of ergosterol were determined by evaluating in vitro Aß production and the potential inhibition of Aß-producing enzymes. Additionally, AD-associated transgenic C. elegans was utilized to investigate the in vivo attenuating effects of ergosterol. KEY FINDINGS: Ergosterol promoted neurite outgrowth in Neuro2a cells through the upregulation of the transmembrane protein Teneurin-4 (Ten-4) mRNA and protein expressions, phosphorylation of the extracellular signal-regulated kinases (ERKs), activity of cAMP response element (CRE), and growth-associated protein-43 (GAP-43). Furthermore, ergosterol enhanced neurite outgrowth in transgenic Neuro2A cells overexpressing either the wild-type APP (Neuro2a-APPwt) or the Swedish mutant APP (Neuro2a-APPswe) through the Ten-4/ERK/CREB/GAP-43 signaling pathway. Interestingly, ergosterol inhibited Aß synthesis in Neuro2a-APPwt cells. In silico analysis indicated that ergosterol can interact with the catalytic sites of ß- and γ-secretases. In Aß-overexpressing C. elegans, ergosterol decreased Aß accumulation, increased chemotaxis behavior, and prolonged lifespan. SIGNIFICANCE: Ergosterol is a potential candidate compound that might benefit AD patients by promoting neurite outgrowth, inhibiting Aß synthesis, and enhancing longevity.

Pleiotropic Nanostructures Built from l-Histidine Show Biologically Relevant Multicatalytic Activities.

The essential amino acid histidine plays a central role in the manifestation of several metabolic processes, including protein synthesis, enzyme-catalysis, and key biomolecular interactions. However, excess accumulation of histidine causes histidinemia, which shows brain-related medical complications, and the molecular mechanism of such histidine-linked complications is largely unknown. Here, we show that histidine undergoes a self-assembly process, leading to the formation of amyloid-like cytotoxic and catalytically active nanofibers. The kinetics of histidine self-assembly was favored in the presence of Mg(II) and Co(II) ions. Molecular dynamics data showed that preferential noncovalent interactions dominated by H-bonds between histidine molecules facilitate the formation of histidine nanofibers. The histidine nanofibers induced amyloid cross-seeding reactions in several proteins and peptides including pathogenic Aß1-42 and brain extract components. Further, the histidine nanofibers exhibited oxidase activity and enhanced the oxidation of neurotransmitters. Cell-based studies confirmed the cellular internalization of histidine nanofibers in SH-SY5Y cells and subsequent cytotoxic effects through necrosis and apoptosis-mediated cell death. Since several complications including behavioral abnormality, developmental delay, and neurological disabilities are directly linked to abnormal accumulation of histidine, our findings provide a foundational understanding of the mechanism of histidine-related complications. Further, the ability of histidine nanofibers to catalyze amyloid seeding and oxidation reactions is equally important for both biological and materials science research.

Molecular docking analysis of chlorpyrifos at the human α7-nAChR and its potential relationship with neurocytoxicity in SH-SY5Y cells.

The organophosphate insecticide chlorpyrifos (CPF), an acetylcholinesterase inhibitor, has raised serious concerns about human safety. Apart from inducing synaptic acetylcholine accumulation, CPF could also act at nicotinic acetylcholine receptors, like the α7-isoform (α7-nAChR), which could potentially be harmful to developing brains. Our aims were to use molecular docking to assess the binding interactions between CPF and α7-nAChR through, to test the neurocytotoxic and oxidative effects of very low concentrations of CPF on SH-SY5Y cells, and to hypothesize about the potential mediation of α7-nAChR. Docking analysis showed a significant binding affinity of CPH for the E fragment of the α7-nAChR (ΔGibbs: -5.63 to -6.85 Kcal/mol). According to the MTT- and Trypan Blue-based viability assays, commercial CPF showed concentration- and time-dependent neurotoxic effects at a concentration range (2.5-20 µM), ten-folds lower than those reported to have crucial effects for sheer CPF. A rise of the production of radical oxygen species (ROS) was seen at even lower concentrations (1-2.5 µM) of CPF after 24h. Notably, our docking analysis supports the antagonistic actions of CPF on α7-nAChR that were recently published. In conclusion, while α7-nAChR is responsible for neuronal survival and neurodevelopmental processes, its activity may also mediate the neurotoxicity of CPF.

Novel 6-alkyl-bridged 4-arylalkylpiperazin-1-yl derivatives of azepino[4,3-b]indol-1(2H)-one as potent BChE-selective inhibitors showing protective effects against neurodegenerative insults.

Due to the putative role of butyrylcholinesterase (BChE) in regulation of acetylcholine levels and functions in the late stages of the Alzheimer's disease (AD), the potential of selective inhibitors (BChEIs) has been envisaged as an alternative to administration of acetylcholinesterase inhibitors (AChEIs). Starting from our recent findings, herein the synthesis and in vitro evaluation of cholinesterase (ChE) inhibition of a novel series of some twenty 3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one derivatives, bearing at the indole nitrogen diverse alkyl-bridged 4-arylalkylpiperazin-1-yl chains, are reported. The length of the spacers, as well as the type of arylalkyl group affected the enzyme inhibition potency and BChE/AChE selectivity. Two compounds, namely 14c (IC50 = 163 nM) and 14d (IC50 = 65 nM), bearing at the nitrogen atom in position 6 a n-pentyl- or n-heptyl-bridged 4-phenethylpiperazin-1-yl chains, respectively, proved to be highly potent mixed-type inhibitors of both equine and human BChE isoforms, showing more than two order magnitude of selectivity over AChE. The study of binding kinetics through surface plasmon resonance (SPR) highlighted differences in their BChE residence times (8 and 47 s for 14c and 14d, respectively). Moreover, 14c and 14d proved to hit other mechanisms known to trigger neurodegeneration underlying AD and other CNS disorders. Unlike 14c, compound 14d proved also capable of inhibiting by more than 60% the in vitro self-induced aggregation of neurotoxic amyloid-ß (Aß) peptide at 100 µM concentration. On the other hand, 14c was slightly better than 14d in counteracting, at 1 and 10 µM concentration, glutamate excitotoxicity, due to over-excitation of NMDA receptors, and hydrogen peroxide-induced oxidative stress assessed in neuroblastoma cell line SH-SY5Y. This paper is dedicated to Prof. Marcello Ferappi, former dean of the Faculty of Pharmacy of the University of Bari, in the occasion of his 90th birthday.

Mdivi-1: Effective but complex mitochondrial fission inhibitor.

Mdivi-1, Mitochondrial DIVIsion inhibitor 1, has been widely employed in research under the assumption that it exclusively influences mitochondrial fusion, but effects other than mitochondrial dynamics have been underinvestigated. This paper provides transcriptome and DNA methylome-wide analysis for Mdivi-1 treated SH-SY5Y human neuroblastoma cells using RNA sequencing (RNA-seq) and methyl capture sequencing (MC-seq) methods. Gene ontology analysis of RNA sequences revealed that p53 transcriptional gene network and DNA replication initiation-related genes were significantly up and down-regulated, respectively, showing the correlation with the arrest cell cycle in the G1 phase. MC-seq, a powerful sequencing method for capturing DNA methylation status in CpG sites, revealed that although Mdivi-1 does not induce dramatic DNA methylation change, the subtle alterations were concentrated within the CpG island. Integrative analysis of both sequencing data disclosed that the p53 transcriptional network was activated while the Parkinson's disease pathway was halted. Next, we investigated several changes in mitochondria in response to Mdivi-1. Copy number and transcription of mitochondrial DNA were suppressed. ROS levels increased, and elevated ROS triggered mitochondrial retrograde signaling rather than inducing direct DNA damage. In this study, we could better understand the molecular network of Mdivi-1 by analyzing DNA methylation and mRNA transcription in the nucleus and further investigating various changes in mitochondria, providing inspiration for studying nuclear-mitochondrial communications.

[Different concentrations of adapalene induce differentiation and apoptosis of SH-SY5Y cells]. / 不同浓度阿达帕林诱导SH-SY5Y细胞的分化及凋亡.

OBJECTIVES: To investigate the effects of different concentrations of adapalene on the morphology and functions of neuroblastoma cell line SH-SY5Y, as well as its role in inducing cell differentiation and apoptosis. METHODS: SH-SY5Y cells were divided into control group, low concentration (0.1 µM and 1 µM) adapalene groups, and high concentration (10 µM) adapalene group. Time-lapse microscopy was used to observe the morphological changes of SH-SY5Y cells. Immunofluorescence staining was performed to detect the expression of neuronal specific marker ßIII-tubulin and mature neuronal marker neurofilament heavy polypeptide (NFH). Multi-electrode array was used to record the electrophysiological features of SH-SY5Y cells. Cell apoptosis was evaluated using a cell apoptosis detection kit. RESULTS: Low concentrations of adapalene promoted the formation of neurite outgrowth in SH-SY5Y cells, with the neurites interconnected to form a network. Spontaneous discharge activity was observed in SH-SY5Y cells treated with low concentrations of adapalene. Compared to the control group, the expression of ßIII-tubulin and NFH increased in the 1 µM adapalene group, while the level of cell apoptosis increased in the high concentration adapalene group (P<0.05). CONCLUSIONS: Low concentrations of adapalene can induce differentiation of SH-SY5Y cells into mature functional neurons, while high concentrations of adapalene can induce apoptosis in SH-SY5Y cells.

TLR4 TIR domain and nucleolin GAR domain synergistically mediate RSV infection and induce neuronal inflammatory damage in SH-SY5Y cells.

Previous research results of our group showed that Toll-like receptor 4 (TLR4) and nucleolin synergistically mediate respiratory syncytial virus (RSV) infection in human central neuron cells, but the specific mechanism remains unclear. Here we designed and synthesized lentiviruses with TIR (674-815 aa), TLR4 (del 674-815 aa), GAR (645-707 aa), and NCL (del 645-707 aa) domains, and obtained stable overexpression cell lines by drug screening, and subsequently infected RSV at different time points. Laser confocal microscopy and coimmunoprecipitation were used for the observation of co-localization and interaction of TIR/GAR domains. Western blot analysis was used for the detection of p-NF-κB and LC3 protein expression. Real-time PCR was used for the detection of TLR4/NCL mRNA expression. ELISA assay was used to measure IL-6, IL-1ß, and TNF-α concentrations and flow cytometric analysis was used for the study of apoptosis. Our results suggest that overexpression of TIR and GAR domains can exacerbate apoptosis and autophagy, and that TIR and GAR domains can synergistically mediate RSV infection and activate the NF-κB signaling pathway, which regulates the secretion of downstream inflammatory factors, such as IL-6, IL-1ß, and TNF-α, and ultimately leads to neuronal inflammatory injury.

Single cell analysis reveals the roles and regulatory mechanisms of type-I interferons in Parkinson's disease.

The pathogenesis of Parkinson's disease (PD) is strongly associated with neuroinflammation, and type I interferons (IFN-I) play a crucial role in regulating immune and inflammatory responses. However, the specific features of IFN in different cell types and the underlying mechanisms of PD have yet to be fully described. In this study, we analyzed the GSE157783 dataset, which includes 39,024 single-cell RNA sequencing results for five PD patients and six healthy controls from the Gene Expression Omnibus database. After cell type annotation, we intersected differentially expressed genes in each cell subcluster with genes collected in The Interferome database to generate an IFN-I-stimulated gene set (ISGs). Based on this gene set, we used the R package AUCell to score each cell, representing the IFN-I activity. Additionally, we performed monocle trajectory analysis, and single-cell regulatory network inference and clustering (SCENIC) to uncover the underlying mechanisms. In silico gene perturbation and subsequent experiments confirm NFATc2 regulation of type I interferon response and neuroinflammation. Our analysis revealed that microglia, endothelial cells, and pericytes exhibited the highest activity of IFN-I. Furthermore, single-cell trajectory detection demonstrated that microglia in the midbrain of PD patients were in a pro-inflammatory activation state, which was validated in the 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model as well. We identified transcription factors NFATc2, which was significantly up-regulated and involved in the expression of ISGs and activation of microglia in PD. In the 1-Methyl-4-phenylpyridinium (MPP+)-induced BV2 cell model, the suppression of NFATc2 resulted in a reduction in IFN-ß levels, impeding the phosphorylation of STAT1, and attenuating the activation of the NF-κB pathway. Furthermore, the downregulation of NFATc2 mitigated the detrimental effects on SH-SY5Y cells co-cultured in conditioned medium. Our study highlights the critical role of microglia in type I interferon responses in PD. Additionally, we identified transcription factors NFATc2 as key regulators of aberrant type I interferon responses and microglial pro-inflammatory activation in PD. These findings provide new insights into the pathogenesis of PD and may have implications for the development of novel therapeutic strategies.

Monocarboxylate Transporters 1 and 2 Are Responsible for L-Lactate Uptake in Differentiated Human Neuroblastoma SH-SY5Y Cells.

L-Lactate transport via monocarboxylate transporters (MCTs) in the central nervous system, represented by the astrocyte-neuron lactate shuttle (ANLS), is crucial for the maintenance of brain functions, including memory formation. Previously, we have reported that MCT1 contributes to L-lactate transport in normal human astrocytes. Therefore, in this study, we aimed to identify transporters that contribute to L-lactate transport in human neurons. SH-SY5Y cells, which are used as a model for human neurons, were differentiated using all-trans-retinoic acid. L-Lactate uptake was measured using radiolabeled L-lactate, and the expression of MCT proteins was confirmed Western blotting. L-Lactate transport was pH-dependent and saturated at high concentrations. Kinetic analysis suggested that L-lactate uptake was biphasic. Furthermore, MCT1, 2 selective inhibitors inhibited L-lactate transport. In addition, the expression of MCT1 and 2 proteins, but not MCT4, was confirmed. In this study, we demonstrated that MCT1 and 2 are major contributors to L-lactate transport in differentiated human neuroblastoma SH-SY5Y cells from the viewpoint of kinetic analysis. These results lead to a better understanding of ANLS in humans, and further exploration of the factors that can promote MCT1 and 2 functions is required.

A Rare Case of Abnormal Diffuse Brain Uptake on an 123I MIBG Scan in a Patient With High-Risk Neuroblastoma.

ABSTRACT: 123I-meta-iodobenzylguanidine (123I-MIBG) is extensively used for initial staging and response evaluation in children with neuroblastoma. Physiological uptake of 123I-MIBG occurs in the salivary glands, liver, adrenal gland, myocardium, bowel, and thyroid gland. 123I-MIBG cannot cross an intact blood-brain barrier. We present the rare case of a 3-year-old boy with neuroblastoma and meningeal metastases who underwent an 123I-MIBG scan for disease restaging that showed abnormal brain uptake. Abnormal MIBG uptake in the brain can occur if there is disruption of the blood-brain barrier either secondary to metastases or after damage to blood-brain barrier.

Clinical Presentation, Management, and Diagnostic Performance of 2021 Criteria for Paraneoplastic Neurologic Syndromes in Childhood.

BACKGROUND AND OBJECTIVES: Paraneoplastic neurologic syndromes (PNSs) are remote neurologic immune-related effects of tumors. The clinical characteristics of pediatric PNSs remain unclear. We retrospectively examined the clinical characteristics of cases of pediatric PNSs and assessed the performance of the 2021 diagnostic criteria in children. METHODS: Patients hospitalized in the Beijing Children's Hospital between June 2015 and June 2023 and fulfilling the description of definite by 2004 diagnostic criteria of PNSs were included. A retrospective analysis of clinical characteristics was conducted, and the 2021 diagnostic criteria were applied to rediagnostic stratification. RESULTS: Among the 42 patients included, the most common neurologic syndrome was opsoclonus-myoclonus syndrome (OMS) (62%), followed by rapidly progressive cerebellar syndrome (26%). Most tumors were neuroblastomas (88%), with few being ovarian teratomas (10%). Approximately 71% (30/42) of patients were classified as definite and 24% (10/42) as probable according to the 2021 criteria. All cases judged as probable exhibited rapidly progressive cerebellar ataxia with neuroblastoma. For OMS, chemotherapy was administered based on the tumor's risk stage, accompanied by regular infusion of IV gamma globulin and oral steroids following tumor diagnosis. Twenty-one patients underwent regular follow-ups over 4.92 (0.58-7.58) years. The initial hospitalization recorded a median score of 12 (7-14) on the Mitchell and Pike OMS rating scale, decreasing to 0 (0-5) at the final follow-up. In cases of rapidly progressive cerebellar syndrome, a similar therapeutic regimen was used. Nine patients underwent regular follow-ups over 4.42 (1.17-7.50) years. The mean modified Rankin scale score at first hospitalization was 4 (3-4), reducing to 1 (0-4) at the final follow-up. Only 17% (5/30) of patients across both groups exhibited poor response to this regimen. Among these 5 patients, 4 belonged to the low-risk group (without chemotherapy). DISCUSSION: OMS followed by rapidly progressive cerebellar ataxia are the most common forms of PNSs in children and are associated with neuroblastoma. An aggressive approach with multiple immunotherapies may improve the prognosis of neuroblastoma-associated PNSs. The 2021 criteria perform well in pediatric PNSs. However, we propose upgrading the classification of antibody-negative rapidly progressive cerebellar ataxia with neuroblastoma to definite diagnosis. This adjustment aims to further improve the diagnostic efficacy of this diagnostic criterion in childhood.

Astragaloside IV combined with ligustrazine ameliorates abnormal mitochondrial dynamics via Drp1 SUMO/deSUMOylation in cerebral ischemia-reperfusion injury.

OBJECTIVES: Astragaloside IV (AST IV) and ligustrazine (Lig), the main ingredients of Astragali Radix and Chuanxiong Rhizoma respectively, have demonstrated significant benefits in treatment of cerebral ischemia -reperfusion injury (CIRI); however, the mechanisms underlying its benificial effects remain unclear. SUMO-1ylation and deSUMO-2/3ylation of dynamin-related protein 1 (Drp1) results in mitochondrial homeostasis imbalance following CIRI, which subsequently aggravates cell damage. This study investigates the mechanisms by which AST IV combined with Lig protects against CIRI, focusing on the involvement of SUMOylation in mitochondrial dynamics. METHODS: Rats were administrated AST IV and Lig for 7 days, and middle cerebral artery occlusion was established to mimic CIRI. Neural function, cerebral infarction volume, cerebral blood flow, cognitive function, cortical pathological lesions, and mitochondrial morphology were measured. SH-SY5Y cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) injury. Mitochondrial membrane potential and lactic dehydrogenase (LDH), reactive oxygen species (ROS), and adenosine triphosphate (ATP) levels were assessed with commercial kits. Moreover, co-immunoprecipitation (Co-IP) was used to detect the binding of SUMO1 and SUMO2/3 to Drp1. The protein expressions of Drp1, Fis1, MFF, OPA1, Mfn1, Mfn2, SUMO1, SUMO2/3, SENP1, SENP2, SENP3, SENP5, and SENP6 were measured using western blot. RESULTS: In rats with CIRI, AST IV and Lig improved neurological and cognitive functions, restored CBF, reduced brain infarct volume, and alleviated cortical neuron and mitochondrial damage. Moreover, in SH-SY5Y cells, the combination of AST IV and Lig enhanced cellular viability, decreased release of LDH and ROS, increased ATP content, and improved mitochondrial membrane potential. Furthermore, AST IV combined with Lig reduced the binding of Drp1 with SUMO1, increased the binding of Drp1 with SUMO2/3, suppressed the expressions of Drp1, Fis1, MFF, and SENP3, and increased the expressions of OPA1, Mfn1, Mfn2, SENP1, SENP2, and SENP5. SUMO1 overexpression promoted mitochondrial fission and inhibited mitochondrial fusion, whereas SUMO2/3 overexpression suppressed mitochondrial fission. AST IV combined with Lig could reverse the effects of SUMO1 overexpression while enhancing those of SUMO2/3 overexpression. CONCLUSIONS: This study posits that the combination of AST IV and Lig has the potential to reduce the SUMO-1ylation of Drp1, augment the SUMO-2/3ylation of Drp1, and thereby exert a protective effect against CIRI.

Choosing T-cell sources determines CAR-T cell activity in neuroblastoma.

Introduction: The clinical success of chimeric antigen receptor-modified T cells (CAR-T cells) for hematological malignancies has not been reproduced for solid tumors, partly due to the lack of cancer-type specific antigens. In this work, we used a novel combinatorial approach consisting of a versatile anti-FITC CAR-T effector cells plus an FITC-conjugated neuroblastoma (NB)-targeting linker, an FITC-conjugated monoclonal antibody (Dinutuximab) that recognizes GD2. Methods: We compared cord blood (CB), and CD45RA-enriched peripheral blood leukapheresis product (45RA) as allogeneic sources of T cells, using peripheral blood (PB) as a control to choose the best condition for anti-FITC CAR-T production. Cells were manufactured under two cytokine conditions (IL-2 versus IL-7+IL-15+IL-21) with or without CD3/CD28 stimulation. Immune phenotype, vector copy number, and genomic integrity of the final products were determined for cell characterization and quality control assessment. Functionality and antitumor capacity of CB/45RA-derived anti-FITC CAR-T cells were analyzed in co-culture with different anti-GD2-FITC labeled NB cell lines. Results: The IL-7+IL-15+IL-21 cocktail, in addition to co-stimulation signals, resulted in a favorable cell proliferation rate and maintained less differentiated immune phenotypes in both CB and 45RA T cells. Therefore, it was used for CAR-T cell manufacturing and further characterization. CB and CD45RA-derived anti-FITC CAR-T cells cultured with IL-7+IL-15+IL-21 retained a predominantly naïve phenotype compared with controls. In the presence of the NB-FITC targeting, CD4+ CB-derived anti-FITC CAR-T cells showed the highest values of co-stimulatory receptors OX40 and 4-1BB, and CD8+ CAR-T cells exhibited high levels of PD-1 and 4-1BB and low levels of TIM3 and OX40, compared with CAR-T cells form the other sources studied. CB-derived anti-FITC CAR-T cells released the highest amounts of cytokines (IFN-γ and TNF-α) into co-culture supernatants. The viability of NB target cells decreased to 30% when co-cultured with CB-derived CAR-T cells during 48h. Conclusion: CB and 45RA-derived T cells may be used as allogeneic sources of T cells to produce CAR-T cells. Moreover, ex vivo culture with IL-7+IL-15+IL-21 could favor CAR-T products with a longer persistence in the host. Our strategy may complement the current use of Dinutuximab in treating NB through its combination with a targeted CAR-T cell approach.

Mitotic gene regulation by the N-MYC-WDR5-PDPK1 nexus.

BACKGROUND: During mitosis the cell depends on proper attachment and segregation of replicated chromosomes to generate two identical progeny. In cancers defined by overexpression or dysregulation of the MYC oncogene this process becomes impaired, leading to genomic instability and tumor evolution. Recently it was discovered that the chromatin regulator WDR5-a critical MYC cofactor-regulates expression of genes needed in mitosis through a direct interaction with the master kinase PDPK1. However, whether PDPK1 and WDR5 contribute to similar mitotic gene regulation in MYC-overexpressing cancers remains unclear. Therefore, to characterize the influence of WDR5 and PDPK1 on mitotic gene expression in cells with high MYC levels, we performed a comparative transcriptomic analysis in neuroblastoma cell lines defined by MYCN-amplification, which results in high cellular levels of the N-MYC protein. RESULTS: Using RNA-seq analysis, we identify the genes regulated by N-MYC and PDPK1 in multiple engineered CHP-134 neuroblastoma cell lines and compare them to previously published gene expression data collected in CHP-134 cells following inhibition of WDR5. We find that as expected N-MYC regulates a multitude of genes, including those related to mitosis, but that PDPK1 regulates specific sets of genes involved in development, signaling, and mitosis. Analysis of N-MYC- and PDPK1-regulated genes reveals a small group of commonly controlled genes associated with spindle pole formation and chromosome segregation, which overlap with genes that are also regulated by WDR5. We also find that N-MYC physically interacts with PDPK1 through the WDR5-PDPK1 interaction suggesting regulation of mitotic gene expression may be achieved through a N-MYC-WDR5-PDPK1 nexus. CONCLUSIONS: Overall, we identify a small group of genes highly enriched within functional gene categories related to mitotic processes that are commonly regulated by N-MYC, WDR5, and PDPK1 and suggest that a tripartite interaction between the three regulators may be responsible for setting the level of mitotic gene regulation in N-MYC amplified cell lines. This study provides a foundation for future studies to determine the exact mechanism by which N-MYC, WDR5, and PDPK1 converge on cell cycle related processes.

Extracellular mixed histones are neurotoxic and modulate select neuroimmune responses of glial cells.

Although histone proteins are widely known for their intranuclear functions where they organize DNA, all five histone types can also be released into the extracellular space from damaged cells. Extracellular histones can interact with pattern recognition receptors of peripheral immune cells, including toll-like receptor 4 (TLR4), causing pro-inflammatory activation, which indicates they may act as damage-associated molecular patterns (DAMPs) in peripheral tissues. Very limited information is available about functions of extracellular histones in the central nervous system (CNS). To address this knowledge gap, we applied mixed histones (MH) to cultured cells modeling neurons, microglia, and astrocytes. Microglia are the professional CNS immunocytes, while astrocytes are the main support cells for neurons. Both these cell types are critical for neuroimmune responses and their dysregulated activity contributes to neurodegenerative diseases. We measured effects of extracellular MH on cell viability and select neuroimmune functions of microglia and astrocytes. MH were toxic to cultured primary murine neurons and also reduced viability of NSC-34 murine and SH-SY5Y human neuron-like cells in TLR4-dependent manner. MH did not affect the viability of resting or immune-stimulated BV-2 murine microglia or U118 MG human astrocytic cells. When applied to BV-2 cells, MH enhanced secretion of the potential neurotoxin glutamate, but did not modulate the release of nitric oxide (NO), tumor necrosis factor-α (TNF), C-X-C motif chemokine ligand 10 (CXCL10), or the overall cytotoxicity of lipopolysaccharide (LPS)- and/or interferon (IFN)-γ-stimulated BV-2 microglial cells towards NSC-34 neuron-like cells. We demonstrated, for the first time, that MH downregulated phagocytic activity of LPS-stimulated BV-2 microglia. However, MH also exhibited protective effect by ameliorating the cytotoxicity of LPS-stimulated U118 MG astrocytic cells towards SH-SY5Y neuron-like cells. Our data demonstrate extracellular MH could both damage neurons and alter neuroimmune functions of glial cells. These actions of MH could be targeted for treatment of neurodegenerative diseases.

An anthocyanin-rich extract from Zea mays L. var. ceratina alleviates neuronal cell death caused by hydrogen peroxide-induced cytotoxicity in SH-SY5Y cells.

The incidence of dementia is rising, with neuronal cell death from oxidative stress and apoptosis recognized as a significant contributor to its development. However, effective strategies to combat this condition are lacking, necessitating further investigation. This study aimed to assess the potential of an anthocyanin-rich extract from Zea mays L. var. ceratina (AZC) in alleviating neuronal cell death.Neurotoxicity was induced in SH-SY5Y cells using hydrogen peroxide (H2O2) at a concentration of 200 µM. Cells were pretreated with varying doses (31.25 and 62.5 µg/mL) of AZC. Cell viability was assessed using the MTT assay, and molecular mechanisms including reactive oxygen species (ROS) levels, antioxidant enzyme activities (catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px)), malondialdehyde (MDA) levels for oxidative stress, and the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), cAMP response element-binding protein (CREB), and apoptotic factors (B-cell lymphoma 2 (Bcl-2), caspase 3) were explored.Results showed that AZC significantly improved cell viability, reduced ROS production and MDA levels, and downregulated caspase 3 expression. It enhanced CAT, SOD, and GSH-Px activities, activated ERK1/2 and CREB, and upregulated Bcl-2 expression. These findings support the neuroprotective effects of AZC, suggesting it activates ERK1/2, leading to CREB activation and subsequent upregulation of Bcl-2 expression while suppressing caspase 3. AZC may mitigate neuronal cell death by reducing ROS levels through enhanced scavenging enzyme activities.In conclusion, this study underscores the potential of AZC as a neuroprotective agent against neuronal cell death. However, further investigations including toxicity assessments, in vivo studies, and clinical trials are necessary to validate its benefits in neuroprotection.