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Natural killer (NK) cells play a crucial role in the immune system's response against cancer. However, the challenge of obtaining the required quantity of NK cells for effective therapeutic response necessitates the development of strategies for their ex vivo expansion. This study aimed to develop a novel feeder cell line, K562.Clone1, capable of promoting the ex vivo expansion of NK cells while preserving their cytotoxic potential. he K562 leukemic cell line was transduced with mbIL-21 and 4-1BBL proteins to generate K562.Clone1 cells. NK cells were then co-cultured with these feeder cells, and their expansion rate was monitored over 14 days. The cytotoxic potential of the expanded NK cells was evaluated against acute myeloid leukemia blasts and tumor cell lines of leukemia and glial origin. Statistical analysis was performed to determine the significance of the results. The K562.Clone1 co-cultured with peripheral NK showed a significant increase in cell count, with an approximate 94-fold expansion over 14 days. Expanded NK cells demonstrated cytotoxicity against the tested tumor cell lines, indicating preservation of their cytotoxic characteristics. Additionally, the CD56, CD16, inhibitory KIRs, and activation receptors were conserved and present in a well-balanced manner. The study successfully developed a feeder cell line, K562.Clone1, that effectively promotes the expansion of NK cells ex vivo while maintaining their cytotoxic potential. This development could significantly contribute to the advancement of NK cell therapy, especially in Brazil.

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INTRODUCTION: Recently, the search for novel molecular markers in adult-type diffuse gliomas has grown substantially, yet with few novel breakthroughs. As the presence of a necrotic center is a differential diagnosis for more aggressive entities, we hypothesized that genes involved in necroptosis may play a role in tumor progression. AIM: Given that MLKL is the executioner of the necroptotic pathway, we evaluated whether this gene would help to predict prognosis of adult gliomas patients. METHODS: We analyzed a publicly available retrospective cohort (n = 530) with Kaplan Meier survival analysis (p<0.0001) and both uni- and multivariate Cox regression models. RESULTS: We determined that MLKL is an independent predictive prognostic marker for overall survival in these patients (HR: 2.56, p<0.001), even when controlled by the CNS5 gold-standard markers, namely IDH mutation and 1p/19q Codeletion (HR: 1.68, p = 0.013). These findings were confirmed in a validation cohort (n = 325), using the same cutoff value. Interestingly, higher expression of MLKL is associated with worse clinical outcome for adult-type diffuse glioma patients, which is opposite to what was found in other cell cancer types, suggesting that necroptosis undertakes an atypical detrimental role in glioma progression.

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OBJECTIVE: To better understand the immune microenvironment of pancreatic ductal adenocarcinomas (PDACs), here we explored the relevance of T and B cell compartmentalisation into tertiary lymphoid structures (TLSs) for the generation of local antitumour immunity. DESIGN: We characterised the functional states and spatial organisation of PDAC-infiltrating T and B cells using single-cell RNA sequencing (scRNA-seq), flow cytometry, multicolour immunofluorescence, gene expression profiling of microdissected TLSs, as well as in vitro assays. In addition, we performed a pan-cancer analysis of tumour-infiltrating T cells using scRNA-seq and sc T cell receptor sequencing datasets from eight cancer types. To evaluate the clinical relevance of our findings, we used PDAC bulk RNA-seq data from The Cancer Genome Atlas and the PRINCE chemoimmunotherapy trial. RESULTS: We found that a subset of PDACs harbours fully developed TLSs where B cells proliferate and differentiate into plasma cells. These mature TLSs also support T cell activity and are enriched with tumour-reactive T cells. Importantly, we showed that chronically activated, tumour-reactive T cells exposed to fibroblast-derived TGF-ß may act as TLS organisers by producing the B cell chemoattractant CXCL13. Identification of highly similar subsets of clonally expanded CXCL13 + tumour-infiltrating T cells across multiple cancer types further indicated a conserved link between tumour-antigen recognition and the allocation of B cells within sheltered hubs in the tumour microenvironment. Finally, we showed that the expression of a gene signature reflecting mature TLSs was enriched in pretreatment biopsies from PDAC patients with longer survival after receiving different chemoimmunotherapy regimens. CONCLUSION: We provided a framework for understanding the biological role of PDAC-associated TLSs and revealed their potential to guide the selection of patients for future immunotherapy trials.

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INTRODUCTION: Lysosomal storage disorders (LSD) are a group of monogenic rare diseases caused by pathogenic variants in genes that encode proteins related to lysosomal function. These disorders are good candidates for gene therapy for different reasons: they are monogenic, most of lysosomal proteins are enzymes that can be secreted and cross-correct neighboring cells, and small quantities of these proteins are able to produce clinical benefits in many cases. Ex vivo gene therapy allows for autologous transplant of modified cells from different sources, including stem cells and hematopoietic precursors. AREAS COVERED: Here, we summarize the main gene therapy and genome editing strategies that are currently being used as ex vivo gene therapy approaches for lysosomal disorders, highlighting important characteristics, such as vectors used, strategies, types of cells that are modified and main results in different disorders. EXPERT OPINION: Clinical trials are already ongoing, and soon approved therapies for LSD based on ex vivo gene therapy approaches should reach the market.

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The past decades witnessed the discovery of novel modes of cell death, such as ferroptosis, pyroptosis and necroptosis, all of them presenting common necrotic traits. In this chapter, we revisit the early discoveries that unveiled necroptosis as a distinct cell death mechanism. We describe necroptosis, its main regulators and their role in maintaining cellular homeostasis and in the disease state. We conclude by discussing its phenotypic similarities with ferroptosis and the possible crosstalk between these pathways.

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Respiratory syncytial virus (RSV) is the major cause of acute bronchiolitis in infants under 2 years old. Necroptosis has been implicated in the outcomes of respiratory virus infections. We report that RSV infection triggers necroptosis in primary mouse macrophages and human monocytes in a RIPK1-, RIPK3- and MLKL-dependent manner. Moreover, necroptosis pathways are harmful to RSV clearance from alveolar macrophages. Additionally, Ripk3-/- mice were protected from RSV-induced weight loss and presented with reduced viral loads in the lungs.Alveolar macrophage depletion also protected mice from weight loss and decreased lung RSV virus load. Importantly, alveolar macrophage depletion abolished the upregulation of Ripk3 and Mlkl gene expression induced by RSV infection in the lung tissue.Autocrine tumor necrosis factor (TNF)-mediated RSV-triggered macrophage necroptosis and necroptosis pathways were also involved in TNF secretion even when macrophages were committed to cell death, which can worsen lung injury during RSV infection. In line, Tnfr1-/- mice had a marked decrease in Ripk3 and Mlkl gene expression and a sharp reduction in the numbers of necrotic alveolar macrophages in the lungs. Finally, we provide evidence that elevated nasal levels of TNF are associated with disease severity in infants with RSV bronchiolitis.We propose that targeting TNF and/or the necroptotic machinery may be valuable therapeutic approaches to reduce the respiratory morbidity caused by RSV infection in young children.

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PURPOSE: Necroptosis is a necrotic-like cell death pathway in which Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) plays a central role and may induce inflammation and immunity. Lower RIPK3 levels have been correlated with a poor prognosis in breast and colorectal cancer patients. Instead, in gliomas, the most prevalent among central nervous system cancers, necrosis concurs with a more aggressive and lethal outcome, suggesting that, in these cases, necrotic-like pathways may be linked to worse prognoses. Lower-grade gliomas (LGG) exhibit highly diverse clinical behaviors, ranging from slow-paced growth to fast progression to glioblastoma yet patient outcomes cannot be fully predicted through the available markers. To date, IDH mutational status is the most broadly used prognostic marker, albeit several candidates have been proposed to refine LGG subgrouping. Here, we aimed to assess RIPK3 role as a prognostic marker for LGG patients, independently of or in combination with IDH. METHODS: Using publicly available discovery (513 patients) and validation (134 patients) cohorts, we performed Kaplan Meier survival analysis and uni- and multivariate Cox regression models. RESULTS: RIPK3 is an independent prognostic marker in LGG patients, even when controlled by age and molecular or histological diagnostic criteria. Contrary to what was previously reported for other cancers, high RIPK3 expression levels correlates with an increased risk of death. Importantly, RIPK3 expression levels further split both the mutant and wild-type IDH patients into distinct risk groups. CONCLUSION: RIPK3 expression levels can be used in combination with IDH mutational status to better subgroup LGG patients regarding overall survival.

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Lapachol is a plant-derived naphthoquinone that kills several types of cancer cells. Derivatives of this molecule may therefore prove to be useful chemotherapeutic agents. In this study, we explored whether glycosylation increases the cytotoxic potency of lapachol towards HL-60 human leukemia cells. Two beta-glycosides were synthesized and characterized: LA4A (lapachol-ß-glucoside) and LA4C (lapachol-N-acetylglucosamine-ß-glucoside). The sugar moieties of both novel molecules were per-acetylated to facilitate cellular uptake. The IC50 values (in µM) for LA4A (5.7) and LA4C (5.3) were lower than those for lapachol (25). LA4A and LA4C triggered typical signs of apoptosis, such as the exposure of phosphatidylserine on the outside of cells, chromatin condensation, DNA fragmentation and a decrease of the mitochondrial transmembrane potential (ΔΨm) prior to cell lysis. Moreover, DNA fragmentation triggered by the lapachol-glycosides was reduced by pre-treatment with the caspase inhibitor, z-VAD-fmk. While LA4A and LA4C activated caspases-3, -8 and -9, lapachol failed to activate these apoptotic proteases, even when used at high concentrations. Finally, the toxicity of lapachol and its derivatives was also tested on non-tumor cells. We used human peripheral neurons (PeriTox test) to evaluate the side effect potential of these compounds. LA4C was clearly less toxic than LA4A. We conclude that LA4C had the most favorable profile as drug candidate (high tumor cell toxicity, reduced neurotoxicity). In general, this study shows that the cytotoxicity of lapachol towards HL-60 can be enhanced by glycosylation, and that the therapeutic ratio may be modified by the type of sugar added.

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In vitro drug screening is widely used in the development of new drugs, because they constitute a cost-effective approach to select compounds with more potential for therapy. They are also an attractive alternative to in vivo testing. However, most of these assays are done in two-dimensional culture models, where cells are grown on a polystyrene or glass flat surface. In order to develop in vitro models that would more closely resemble physiological conditions, three-dimensional models have been developed. Here, we introduce two novel fully synthetic scaffolds produced using the polymer polyhydroxybutyrate (PHB): a Solvent-Casting Particle-Leaching (SCPL) membrane; and an electrospun membrane, to be used for 3D cultures of B16 F10 murine melanoma cells and 4T1 murine breast cancer cells. A 2D cell culture system in regular tissue culture plates and a classical 3D model where cells are grown on a commercially available gel derived from Engelbreth-Holm Swarm (EHS) tumor were used for comparison with the synthetic scaffolds. Cells were also collected from in vivo tumors grown as grafts in syngeneic mice. Morphology, cell viability, response to chemotherapy and gene expression analysis were used to compare all systems. In the electrospun membrane model, cells were grown on nanometer-scale fibers and in the SCPL membrane, which provides a foam-like structure for cell growth, pore sizes varied. Cells grown on all 3D models were able to form aggregates and spheroids, allowing for increased cell-cell contact when compared with the 2D system. Cell morphology was also more similar between 3D systems and cells collected from the in vivo tumors. Cells grown in 3D models showed an increase in resistance to dacarbazine, and cisplatin. Gene expression analysis also revealed similarities among all 3D platforms. The similarities between the two synthetic systems to the classic EHS gel model highlight their potential application as cost effective substitutes in drug screening, in which fully synthetic models could represent a step towards higher reproducibility. We conclude PHB synthetic membranes offer a valuable alternative for 3D cultures.

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Autophagy and inflammasome activation are cell-autonomous and cross-regulated processes involved in host resistance against infections. Our group previously described that NLRP3 inflammasome is required for the control of Trypanosoma cruzi, the causative agent of Chagas disease. However, the involvement of autophagy in this process was unclear. Here, we demonstrated that T. cruzi was able to induce an increase in LC3-II expression as well as autophagosome and autolysosome formation in peritoneal macrophages (PMs) from C57BL/6 wild-type mice. Moreover, the pharmacologic inhibition of autophagic machinery impaired the ability of PMs to control T. cruzi replication. Importantly, NLRP3 was required for the induction of a regular autophagic flux in response to T. cruzi, an effect mediated by its participation in the autolysosomes formation. Together, these results indicate autophagy as an effector mechanism mediated by NLRP3 to control T. cruzi infection.

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Trypanossoma cruzi (T. cruzi), the causative protozoan of Chagas disease (CD) invades many cell types, including central nervous system (CNS) cells triggering local lesions and neurological impact. Previous work from our group described NLRP3 inflammasomes as central effectors for the parasite control by macrophages. Recent evidences demonstrate that NLRP3 can be activated in CNS cells with controversial consequences to the control of infections and inflammatory pathologies. However, the relative contribution of NLRP3 in different cell types remains to be elucidated. In this article, we described an effector response mediated by NLRP3 that works on microglia but not on astrocytes to control T. cruzi infection. Despite T. cruzi ability to invade astrocytes and microglia, astrocytes were clearly more permissive to parasite replication. Moreover, the absence of NLRP3 renders microglia but not astrocytes more permissive to T. cruzi replication. In fact, microglia but not astrocytes were able to secrete NLRP3-dependent IL-1ß and NO in response to T. cruzi. Importantly, the pharmacological inhibition of iNOS with aminoguanidine resulted in a significant increase in the numbers of amastigotes found in microglia from wild-type but not from NLRP3-/- mice, indicating the importance of NLRP3-mediated NO secretion to the infection control by these cells. Taken together, our findings revealed that T. cruzi differentially activates NLRP3 inflammasomes in astrocytes and microglia and established a role for these platforms in the control of a protozoan infection by glial cells from CNS.

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Pattern Recognition Receptors (PRRs) are proteins capable of recognizing molecules frequently found in pathogens (the so-called Pathogen-Associated Molecular Patterns-PAMPs), or molecules released by damaged cells (the Damage-Associated Molecular Patterns-DAMPs). They emerged phylogenetically prior to the appearance of the adaptive immunity and, therefore, are considered part of the innate immune system. Signals derived from the engagement of PRRs on the immune cells activate microbicidal and pro-inflammatory responses required to eliminate or, at least, to contain infectious agents. Molecularly controlled forms of cell death are also part of a very ancestral mechanism involved in key aspects of the physiology of multicellular organism, including the elimination of unwanted, damaged or infected cells. Interestingly, each form of cell death has its particular effect on inflammation and on the development of innate and adaptive immune responses. In this review article, we discuss some aspects of the molecular interplay between the cell death machinery and signals initiated by the activation of PRRs by PAMPs and DAMPs.

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In the early 2000s, receptor-interacting serine/threonine protein kinase 1 (RIPK1), a molecule already recognized as an important regulator of cell survival, inflammation and disease, was attributed an additional function: the regulation of a novel cell death pathway that came to be known as necroptosis. Subsequently, the related kinase RIPK3 and its substrate mixed-lineage kinase domain-like protein (MLKL) were also implicated in the necroptotic pathway, and links between this pathway and apoptosis were established. In this Timeline article, we outline the discoveries that have helped to identify the roles of RIPK1, RIPK3, MLKL and other regulators of necroptosis, and how they interact to determine cell fate.

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OBJECTIVE: Nitro-fatty acids (NO(2)-FAs) are emerging as a new class of cell signaling mediators. Because NO(2)-FAs are found in the vascular compartment and their impact on vascularization remains unknown, we aimed to investigate the role of NO(2)-FAs in angiogenesis. METHODS AND RESULTS: The effects of nitrolinoleic acid and nitrooleic acid were evaluated on migration of endothelial cell (EC) in vitro, EC sprouting ex vivo, and angiogenesis in the chorioallantoic membrane assay in vivo. At 10 µmol/L, both NO(2)-FAs induced EC migration and the formation of sprouts and promoted angiogenesis in vivo in an NO-dependent manner. In addition, NO(2)-FAs increased intracellular NO concentration, upregulated protein expression of the hypoxia inducible factor-1α (HIF-1α) transcription factor by an NO-mediated mechanism, and induced expression of HIF-1α target genes, such as vascular endothelial growth factor, glucose transporter-1, and adrenomedullin. Compared with typical NO donors such as spermine-NONOate and deta-NONOate, NO(2)-FAs were slightly less potent inducers of EC migration and HIF-1α expression. Short hairpin RNA-mediated knockdown of HIF-1α attenuated the induction of vascular endothelial growth factor mRNA expression and EC migration stimulated by NO(2)-FAs. CONCLUSION: Our data disclose a novel physiological role for NO(2)-FAs, indicating that these compounds induce angiogenesis in an NO-dependent mechanism via activation of HIF-1α.

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Multidrug resistance (MDR) is multifactorial and may be mediated by overexpression of anti-apoptotic proteins. This paper investigated whether pomolic acid (PA) was able to overcome resistance mediated by overexpression of Bcl-2 or BcL-xL. The results obtained showed that overexpression of these proteins partially inhibited the PA-induced apoptosis, loss of mitochondrial membrane potential and caspase -3 and -9 activation observed in HL-60/neo. Since Bcl-2 transfected cell lines were shown to be quite resistant to a series of chemotherapeutic agents, the data presented call attention to the possible clinical significance of PA as an anti-MDR drug.

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One of the major goals in chemotherapy is to circumvent anti-apoptotic strategies developed by tumor cells. In a previous paper, we showed that pomolic acid (PA) is able to kill the leukemia cell line K562 and its MDR derivative, Lucena 1. Here, we demonstrated that PA-induced apoptosis of HL-60 cells is dependent on the activation of caspases-3 and -9 and dissipation of the mitochondrial transmembrane potential (Deltapsim). Disruption of Deltapsim precedes caspase activation and is not inhibited by zVAD-fmk indicating mitochondria as the main target of PA. Our data pointed to the potential use of PA to overcome apoptosis resistance.

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One of the major critical factors for cancer proneness is the cell response to DNA damage. In this work, we used human DNA repair deficient cell lines to investigate the responses to ultraviolet irradiation that lead to apoptosis, and the influence of maintaining the cells resting in confluent state. UV-induced apoptosis is prevented in photolyase-proficient HeLa cells when cyclobutane pyrimidine dimers (CPDs) are removed by photorepair. At the same time, we show recovery of RNA synthesis, thus indicating that blockage of RNA transcription may trigger apoptosis in human cells. On the other hand, confluent primary XPC and trichothiodystrophy (TTD)/XPD cell lines, related to xeroderma pigmentosum and trichothiodystrophy repair syndromes, had a reduced and delayed apoptosis when compared to non-confluent cells. In contrast, XPA cells were similarly sensitive in both the confluent and non-confluent growing state. The effect of cellular confluence on UV-mediated apoptosis in CSB cells, related to Cockayne's syndrome, was unclear. Thus, these results indicate that the induction of apoptosis by UV light may also be affected by DNA replication. In addition, they argue for the use of confluent primary cells in studies of induction of apoptosis by UV, a condition close to skin cells in vivo.

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Ectopic expression of Bcr-Abl, Bcl-2 or Bcl-x(L) in HL-60 cells conferred resistance to apoptosis against a variety of death-inducing agents. Bcr-Abl-mediated interference with mitochondrial events was confirmed by the analysis of the loss of mitochondrial transmembrane potential and cytochrome c release. HL-60.Bcr-Abl cells were extremely resistant to all apoptogenic stimuli tested, even in circumstances where HL-60.Bcl-2 or HL-60.Bcl-x(L) cells were only partially protected from apoptosis. The levels of Mcl-1, Bax, Bid, Akt, c-IAP-1, c-IAP-2, XIAP and c-FLIP were compared in all HL-60 lines. Our findings show that Bcr-Abl is a more powerful anti-apoptotic molecule than Bcl-2 or Bcl-x(L).

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