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The latest trends in cancer research and nanomedicine focus on using nanocarriers to target cancer stem cells (CSCs). Specifically, lipid liquid nanocapsules are usually developed as nanocarriers for lipophilic drug delivery. Here, we developed olive oil liquid NCs (O2LNCs) functionalized by covalent coupling of an anti-CD44-fluorescein isothiocyanate antibody (αCD44). First, O2LNCs are formed by a core of olive oil surrounded by a shell containing phospholipids, a nonionic surfactant, and deoxycholic acid molecules. Then, O2LNCs were coated with an αCD44 antibody (αCD44-O2LNC). The optimization of an αCD44 coating procedure, a complete physicochemical characterization, as well as clear evidence of their efficacy in vitro and in vivo were demonstrated. Our results indicate the high targeted uptake of these αCD44-O2LNCs, and the increased antitumor efficacy (up to four times) of paclitaxel-loaded-αCD44-O2LNC compared to free paclitaxel in pancreatic CSCs (PCSCs). Also, αCD44-O2LNCs were able to selectively target PCSCs in an orthotopic xenotransplant in vivo model.
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Nanocápsulas , Neoplasias Pancreáticas , Humanos , Células-Tronco Neoplásicas , Azeite de Oliva , Paclitaxel/farmacologia , Neoplasias Pancreáticas/tratamento farmacológicoRESUMO
Macrophages play an important role in the inflammatory response. Their various biological functions are induced by different membrane receptors, including Toll-like receptors, which trigger several intracellular signaling cascades and activate the inflammasomes, which in turn elicit the release of inflammatory mediators such as cytokines. In this study, we present a novel method for the isolation of human mature peritoneal macrophages. This method can be easily implemented by gynecologists who routinely perform laparoscopy for sterilization by tubal ligation or surgically intervene in benign gynecological pathologies. Our method confirms that macrophages are the main peritoneal leukocyte subpopulation isolated from the human peritoneum in homeostasis. We showed that primary human peritoneal macrophages present phagocytic and oxidative activities, and respond to activation of the main proinflammatory pathways such as Toll-like receptors and inflammasomes, resulting in the secretion of different proinflammatory cytokines. Therefore, this method provides a useful tool for characterizing primary human macrophages as control cells for studies of molecular inflammatory pathways in steady-state conditions and for comparing them with those obtained from pathologies involving the peritoneal cavity. Furthermore, it will facilitate advances in the screening of anti-inflammatory compounds in the human system.
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Técnicas de Cultura de Células/métodos , Citocinas/metabolismo , Inflamassomos/metabolismo , Leucócitos/metabolismo , Macrófagos Peritoneais/metabolismo , Adulto , Feminino , Citometria de Fluxo , Humanos , Imunidade Inata , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Laparoscopia , Macrófagos Peritoneais/citologia , Fagocitose , Espécies Reativas de Oxigênio , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Introduction: Chimeric antigen receptor-expressing T cells (CAR T cells) have revolutionized cancer treatment, particularly in B cell malignancies. However, the use of autologous T cells for CAR T therapy presents several limitations, including high costs, variable efficacy, and adverse effects linked to cell phenotype. Methods: To overcome these challenges, we developed a strategy to generate universal and safe anti-CD19 CAR T cells with a defined memory phenotype. Our approach utilizes CRISPR/Cas9 technology to target and eliminate the B2M and TRAC genes, reducing graft-versus-host and host-versus-graft responses. Additionally, we selected less differentiated T cells to improve the stability and persistence of the universal CAR T cells. The safety of this method was assessed using our CRISPRroots transcriptome analysis pipeline, which ensures successful gene knockout and the absence of unintended off-target effects on gene expression or transcriptome sequence. Results: In vitro experiments demonstrated the successful generation of functional universal CAR T cells. These cells exhibited potent lytic activity against tumor cells and a reduced cytokine secretion profile. The CRISPRroots analysis confirmed effective gene knockout and no unintended off-target effects, validating it as a pioneering tool for on/off-target and transcriptome analysis in genome editing experiments. Discussion: Our findings establish a robust pipeline for manufacturing safe, universal CAR T cells with a favorable memory phenotype. This approach has the potential to address the current limitations of autologous CAR T cell therapy, offering a more stable and persistent treatment option with reduced adverse effects. The use of CRISPRroots enhances the reliability and safety of gene editing in the development of CAR T cell therapies. Conclusion: We have developed a potent and reliable method for producing universal CAR T cells with a defined memory phenotype, demonstrating both efficacy and safety in vitro. This innovative approach could significantly improve the therapeutic landscape for patients with B cell malignancies.
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Antígenos CD19 , Sistemas CRISPR-Cas , Edição de Genes , Memória Imunológica , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Transcriptoma , Humanos , Imunoterapia Adotiva/métodos , Imunoterapia Adotiva/efeitos adversos , Antígenos CD19/imunologia , Antígenos CD19/genética , Edição de Genes/métodos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fenótipo , Linhagem Celular TumoralRESUMO
Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of gene therapy approaches. Generally, inducible ON systems require a chimeric transcription factor (transactivator) that becomes activated by an inductor, which is not optimal for clinical translation due to their toxicity. We generated previously the first all-in-one, transactivator-free, doxycycline (Dox)-responsive (Lent-On-Plus or LOP) lentiviral vectors (LVs) able to control transgene expression in human stem cells. Here, we have generated new versions of the LOP LVs and have analyzed their applicability for the generation of inducible advanced therapy medicinal products (ATMPs) with special focus on primary human T cells. We have shown that, contrary to all other cell types analyzed, an Is2 insulator must be inserted into the 3' long terminal repeat of the LOP LVs in order to control transgene expression in human primary T cells. Importantly, inducible primary T cells generated by the LOPIs2 LVs are responsive to ultralow doses of Dox and have no changes in phenotype or function compared with untransduced T cells. We validated the LOPIs2 system by generating inducible CAR-T cells that selectively kill CD19+ cells in the presence of Dox. In summary, we describe here the first transactivator-free, all-one-one system capable of generating Dox-inducible ATMPs.
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[This corrects the article DOI: 10.1016/j.omto.2022.05.003.].
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Autologous T cells expressing the Chimeric Antigen Receptor (CAR) have been approved as advanced therapy medicinal products (ATMPs) against several hematological malignancies. However, the generation of patient-specific CAR-T products delays treatment and precludes standardization. Allogeneic off-the-shelf CAR-T cells are an alternative to simplify this complex and time-consuming process. Here we investigated safety and efficacy of knocking out the TCR molecule in ARI-0001 CAR-T cells, a second generation αCD19 CAR approved by the Spanish Agency of Medicines and Medical Devices (AEMPS) under the Hospital Exemption for treatment of patients older than 25 years with Relapsed/Refractory acute B cell lymphoblastic leukemia (B-ALL). We first analyzed the efficacy and safety issues that arise during disruption of the TCR gene using CRISPR/Cas9. We have shown that edition of TRAC locus in T cells using CRISPR as ribonuleorproteins allows a highly efficient TCR disruption (over 80%) without significant alterations on T cells phenotype and with an increased percentage of energetic mitochondria. However, we also found that efficient TCRKO can lead to on-target large and medium size deletions, indicating a potential safety risk of this procedure that needs monitoring. Importantly, TCR edition of ARI-0001 efficiently prevented allogeneic responses and did not detectably alter their phenotype, while maintaining a similar anti-tumor activity ex vivo and in vivo compared to unedited ARI-0001 CAR-T cells. In summary, we showed here that, although there are still some risks of genotoxicity due to genome editing, disruption of the TCR is a feasible strategy for the generation of functional allogeneic ARI-0001 CAR-T cells. We propose to further validate this protocol for the treatment of patients that do not fit the requirements for standard autologous CAR-T cells administration.
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Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/genética , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Linfócitos T , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Linfoma de Células B/etiologiaRESUMO
Anti-CD19 chimeric antigen receptor (CAR)-T cells have achieved impressive outcomes for the treatment of relapsed and refractory B-lineage neoplasms. However, important limitations still remain due to severe adverse events (i.e., cytokine release syndrome and neuroinflammation) and relapse of 40%-50% of the treated patients. Most CAR-T cells are generated using retroviral vectors with strong promoters that lead to high CAR expression levels, tonic signaling, premature exhaustion, and overstimulation, reducing efficacy and increasing side effects. Here, we show that lentiviral vectors (LVs) expressing the transgene through a WAS gene promoter (AW-LVs) closely mimic the T cell receptor (TCR)/CD3 expression kinetic upon stimulation. These AW-LVs can generate improved CAR-T cells as a consequence of their moderate and TCR-like expression profile. Compared with CAR-T cells generated with human elongation factor α (EF1α)-driven-LVs, AW-CAR-T cells exhibited lower tonic signaling, higher proportion of naive and stem cell memory T cells, less exhausted phenotype, and milder secretion of tumor necrosis factor alpha (TNF-α) and interferon (IFN)-É£ after efficient destruction of CD19+ lymphoma cells, both in vitro and in vivo. Moreover, we also showed their improved efficiency using an in vitro CD19+ pancreatic tumor model. We finally demonstrated the feasibility of large-scale manufacturing of AW-CAR-T cells in guanosine monophosphate (GMP)-like conditions. Based on these data, we propose the use of AW-LVs for the generation of improved CAR-T products.
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Integration-deficient lentiviral vectors (IDLVs) have recently generated increasing interest, not only as a tool for transient gene delivery, but also as a technique for detecting off-target cleavage in gene-editing methodologies which rely on customized endonucleases (ENs). Despite their broad potential applications, the efficacy of IDLVs has historically been limited by low transgene expression and by the reduced sensitivity to detect low-frequency off-target events. We have previously reported that the incorporation of the chimeric sequence element IS2 into the long terminal repeat (LTR) of IDLVs increases gene expression levels, while also reducing the episome yield inside transduced cells. Our study demonstrates that the effectiveness of IDLVs relies on the balance between two parameters which can be modulated by the inclusion of IS2 sequences. In the present study, we explore new IDLV configurations harboring several elements based on IS2 modifications engineered to mediate more efficient transgene expression without affecting the targeted cell load. Of all the insulators and configurations analysed, the insertion of the IS2 into the 3'LTR produced the best results. After demonstrating a DAPI-low nuclear gene repositioning of IS2-containing episomes, we determined whether, in addition to a positive effect on transcription, the IS2 could improve the capture of IDLVs on double strand breaks (DSBs). Thus, DSBs were randomly generated, using the etoposide or locus-specific CRISPR-Cas9. Our results show that the IS2 element improved the efficacy of IDLV DSB detection. Altogether, our data indicate that the insertion of IS2 into the LTR of IDLVs improved, not only their transgene expression levels, but also their ability to be inserted into existing DSBs. This could have significant implications for the development of an unbiased detection tool for off-target cleavage sites from different specific nucleases.
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Transplant of gene-modified autologous hematopoietic progenitors cells has emerged as a new therapeutic approach for Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency with microthrombocytopenia and abnormal lymphoid and myeloid functions. Despite the clinical benefits obtained in ongoing clinical trials, platelet restoration is suboptimal. The incomplete restoration of platelets in these patients can be explained either by a low number of corrected cells or by insufficient or inadequate WASP expression during megakaryocyte differentiation and/or in platelets. We therefore used in vitro models to study the endogenous WASP expression pattern during megakaryocytic differentiation and compared it with the expression profiles achieved by different therapeutic lentiviral vectors (LVs) driving WAS cDNA through different regions of the WAS promoter. Our data showed that all WAS promoter-driven LVs mimic very closely the endogenous WAS expression kinetic during megakaryocytic differentiation. However, LVs harboring the full-length (1.6-kb) WAS-proximal promoter (WW1.6) or a combination of the WAS alternative and proximal promoters (named AW) had the best behavior. Finally, all WAS-driven LVs restored the WAS knockout (WASKO) mice phenotype and functional defects of hematopoietic stem and progenitor cells (HSPCs) from a WAS patient with similar efficiency. In summary, our data back up the use of WW1.6 and AW LVs as physiological gene transfer tools for WAS therapy.
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Immunotherapy is a very promising therapeutic approach against cancer that is particularly effective when combined with gene therapy. Immuno-gene therapy approaches have led to the approval of four advanced therapy medicinal products (ATMPs) for the treatment of p53-deficient tumors (Gendicine and Imlygic), refractory acute lymphoblastic leukemia (Kymriah) and large B-cell lymphomas (Yescarta). In spite of these remarkable successes, immunotherapy is still associated with severe side effects for CD19+ malignancies and is inefficient for solid tumors. Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of immunotherapy. The aim is to develop smart immunogene therapy-based-ATMPs, which can be controlled by the addition of innocuous drugs or agents, allowing the clinicians to manage the intensity and durability of the therapy. In the present manuscript, we will review the different inducible, versatile and externally controlled gene delivery systems that have been developed and their applications to the field of immunotherapy. We will highlight the advantages and disadvantages of each system and their potential applications in clinics.
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Terapia Genética , Imunoterapia , Animais , Biomarcadores , Regulação da Expressão Gênica , Terapia Genética/métodos , Terapia Genética/normas , Humanos , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Imunoterapia/normas , Terapia de Alvo Molecular , Transgenes , Pesquisa Translacional BiomédicaRESUMO
Genome editing technologies not only provide unprecedented opportunities to study basic cellular system functionality but also improve the outcomes of several clinical applications. In this review, we analyze various gene editing techniques used to fine-tune immune systems from a basic research and clinical perspective. We discuss recent advances in the development of programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases. We also discuss the use of programmable nucleases and their derivative reagents such as base editing tools to engineer immune cells via gene disruption, insertion, and rewriting of T cells and other immune components, such natural killers (NKs) and hematopoietic stem and progenitor cells (HSPCs). In addition, with regard to chimeric antigen receptors (CARs), we describe how different gene editing tools enable healthy donor cells to be used in CAR T therapy instead of autologous cells without risking graft-versus-host disease or rejection, leading to reduced adoptive cell therapy costs and instant treatment availability for patients. We pay particular attention to the delivery of therapeutic transgenes, such as CARs, to endogenous loci which prevents collateral damage and increases therapeutic effectiveness. Finally, we review creative innovations, including immune system repurposing, that facilitate safe and efficient genome surgery within the framework of clinical cancer immunotherapies.
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Vacinas Anticâncer/imunologia , Edição de Genes/métodos , Rejeição de Enxerto/imunologia , Doença Enxerto-Hospedeiro/terapia , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Antígenos Quiméricos/genética , Animais , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Terapia Genética , Humanos , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Nucleases de Dedos de Zinco/metabolismoRESUMO
Over recent decades, gene therapy, which has enabled the treatment of several incurable diseases, has undergone a veritable revolution. Cell therapy has also seen major advances in the treatment of various diseases, particularly through the use of adult stem cells (ASCs). The combination of gene and cell therapy (GCT) has opened up new opportunities to improve advanced therapy medicinal products for the treatment of several diseases. Despite the considerable potential of GCT, the use of retroviral vectors has major limitations with regard to oncogene transactivation and the lack of physiological expression. Recently, gene therapists have focused on genome editing (GE) technologies as an alternative strategy. In this review, we discuss the potential benefits of using GE technologies to improve GCT approaches based on ASCs. We will begin with a brief summary of different GE platforms and techniques and will then focus on key therapeutic approaches that have been successfully used to treat diseases in animal models. Finally, we discuss whether ASC GE could become a real alternative to retroviral vectors in a GCT setting.
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Células-Tronco Adultas/metabolismo , Edição de Genes , Terapia Genética , Adulto , Animais , Ensaios Clínicos como Assunto , Humanos , Memória ImunológicaRESUMO
Exosomes are extracellular vesicles released by the vast majority of cell types both in vivo and ex vivo, upon the fusion of multivesicular bodies (MVBs) with the cellular plasma membrane. Two main functions have been attributed to exosomes: their capacity to transport proteins, lipids and nucleic acids between cells and organs, as well as their potential to act as natural intercellular communicators in normal biological processes and in pathologies. From a clinical perspective, the majority of applications use exosomes as biomarkers of disease. A new approach uses exosomes as biologically active carriers to provide a platform for the enhanced delivery of cargo in vivo. One of the major limitations in developing exosome-based therapies is the difficulty of producing sufficient amounts of safe and efficient exosomes. The identification of potential proteins involved in exosome biogenesis is expected to directly cause a deliberate increase in exosome production. In this review, we summarize the current state of knowledge regarding exosomes, with particular emphasis on their structural features, biosynthesis pathways, production techniques and potential clinical applications.
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Mesenchymal stromal cell (MSC) therapy has produced very promising results for multiple diseases in animal models, with over 780 clinical trials on going or completed. However, most of the human clinical trials have not been as successful as trials using preclinical models. To improve the therapeutic potential of MSCs, different research groups have used gene transfer vectors to express factors involved in migration, survival, differentiation, and immunomodulation. The ideal gene transfer vector for most applications should achieve long-term, stable (constitutive or inducible) transgene expression in MSCs and their progeny. Given their efficiency and low impact on transduced cells, lentiviral vectors (LVs) are the vectors of choice. In this chapter we will describe a detailed protocol for the generation of genetically modified MSCs using lentiviral vectors (LVs). Although this protocol has been optimized for MSC lentiviral transduction, it can be easily adapted to other stem cells by changing culture conditions while maintaining volumes and incubation times.
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Técnicas de Cultura de Células/métodos , Lentivirus/genética , Células-Tronco Mesenquimais/citologia , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Técnicas de Transferência de Genes , Vetores Genéticos , Células HEK293 , Humanos , TransgenesRESUMO
Peritoneal macrophages play a critical role in the control of infectious and inflammatory diseases. Although recent progress on murine peritoneal macrophages has revealed multiple aspects on their origin and mechanisms involved in their maintenance in this compartment, little is known on the characteristics of human peritoneal macrophages in homeostasis. Here, we have studied by flow cytometry several features of human peritoneal macrophages obtained from the peritoneal cavity of healthy women. Three peritoneal monocyte/macrophage subsets were established on the basis of CD14/CD16 expression (CD14++CD16-, CD14++CD16+ and CD14highCD16high), and analysis of CD11b, CD11c, CD40, CD62L, CD64, CD80, CD86, CD116, CD119, CD206, HLA-DR and Slan was carried out in each subpopulation. Intracellular expression of GATA6 and cytokines (pro-inflammatory IL-6 and TNF-α, anti-inflammatory IL-10) as well as their phagocytic/oxidative activities were also analyzed, in an attempt to identify genuine resident peritoneal macrophages. Results showed that human peritoneal macrophages are heterogeneous regarding their phenotype, cell complexity and functional abilities. A direct relationship of CD14/CD16 expression, intracellular content of GATA6, and activation/maturation markers like CD206 and HLA-DR, support that the CD14highCD16high subset represents the mature phenotype of steady-state human resident peritoneal macrophages. Furthermore, increased expression of CD14/CD16 is also related to the phagocytic activity.
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Citocinas/metabolismo , Fator de Transcrição GATA6/metabolismo , Homeostase , Macrófagos Peritoneais/metabolismo , Monócitos/metabolismo , Fagocitose , Adulto , Antígenos CD/metabolismo , Feminino , Humanos , Oxirredução , FenótipoRESUMO
Integration-defective lentiviral vectors (IDLVs) have become an important alternative tool for gene therapy applications and basic research. Unfortunately, IDLVs show lower transgene expression as compared to their integrating counterparts. In this study, we aimed to improve the expression levels of IDLVs by inserting the IS2 element, which harbors SARs and HS4 sequences, into their LTRs (SE-IS2-IDLVs). Contrary to our expectations, the presence of the IS2 element did not abrogate epigenetic silencing by histone deacetylases. In addition, the IS2 element reduced episome levels in IDLV-transduced cells. Interestingly, despite these negative effects, SE-IS2-IDLVs outperformed SE-IDLVs in terms of percentage and expression levels of the transgene in several cell lines, including neurons, neuronal progenitor cells, and induced pluripotent stem cells. We estimated that the IS2 element enhances the transcriptional activity of IDLV LTR circles 6- to 7-fold. The final effect the IS2 element in IDLVs will greatly depend on the target cell and the balance between the negative versus the positive effects of the IS2 element in each cell type. The better performance of SE-IS2-IDLVs was not due to improved stability or differences in the proportions of 1-LTR versus 2-LTR circles but probably to a re-positioning of IS2-episomes into transcriptionally active regions.
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Inflammation is part of a complex biological response directed by the immune system to fight pathogens and maintain homeostasis. Dysregulation of the inflammatory process leads to development of chronic inflammatory or autoimmune diseases. Several cell types, such as macrophages, and cytokines such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) are involved in the regulation of inflammation. The important role played by these cytokines as mediators of the inflammatory process and the side effects of current therapies have promoted the search of new therapeutic alternatives. Quinoxalines are important compounds allowing a wide range of chemical modifications in order to provide an extensive repertoire of biological activities. We have previously shown that a series of 4-alkoxy-6,9-dichloro[1,2,4]triazolo[4,3-a]quinoxalines exhibit potent anti-inflammatory activity, inhibiting the production of TNF-α and IL-6. Our aim here was to study the mechanism thereby this series of compounds act upon different intracellular signaling pathways to uncover their potential molecular targets. By using immunoblotting assays, we found that these compounds inhibit ERK 1/2 and JNK/c-Jun cascades, and reduce c-Fos expression, while activate the anti-inflammatory PI3K/Akt route. These results provide further information on their effect upon the intracellular signal transduction mechanisms leading to inhibition of TNF-α and IL-6 secretion. Our results may be of great interest for the pharmaceutical industry, and could be used as a starting point for the development of new and more potent anti-inflammatory drugs derived from the quinoxaline core.
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Anti-Inflamatórios/farmacologia , Inflamação/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Quinoxalinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Células HL-60 , Humanos , Inflamação/metabolismo , Interleucina-6/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The aim of this study was to characterize monocyte-derived macrophages (M-DM) from blood and ascites of cirrhotic patients comparatively with those obtained from blood of healthy controls. The phenotypic profile based on CD14/CD16 expression was analyzed by flow cytometry. Cells were isolated and stimulated in vitro with LPS and heat killed Candida albicans. Phosphorylation of ERK, c-Jun, p38 MAPK, and PKB/Akt was analyzed by Western blotting. A novel CD14(high)CD16(high) M-DM subpopulation is present in ascites (â¼33%). The CD14(++)CD16(+) intermediate subset is increased in the blood of cirrhotic patients (â¼from 4% to 11%) and is predominant in ascites (49%), while the classical CD14(++)CD16(-) subpopulation is notably reduced in ascites (18%). Basal hyperactivation of ERK and JNK/c-Jun pathways observed in ascites M-DM correlates with CD14/CD16 high expressing subsets, while PI3K/PKB does it with the CD16 low expressing cells. In vitro LPS treatment highly increases ERK1/2, PKB/Akt and c-Jun phosphorylation, while that of p38 MAPK is decreased in M-DM from ascites compared to control blood M-DM. Stimulation of healthy blood M-DM with LPS and C. albicans induced higher phosphorylation levels of p38 than those from ascites. Regarding cytokines secretion, in vitro activated M-DM from ascites of cirrhotic patients produced significantly higher amounts of IL-6, IL-10 and TNF-α, and lower levels of IL-1ß and IL-12 than control blood M-DM. In conclusion, a new subpopulation of CD14(high)CD16(high) peritoneal M-DM has been identified in ascites of cirrhotic patients, which is very sensitive to LPS stimulation.
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Cirrose Hepática/imunologia , Macrófagos Peritoneais/imunologia , Adulto , Idoso , Ascite/imunologia , Candida albicans/imunologia , Citocinas/metabolismo , Feminino , Humanos , Receptores de Lipopolissacarídeos/imunologia , Lipopolissacarídeos/imunologia , Masculino , Pessoa de Meia-Idade , Receptores de IgG/imunologia , Transdução de Sinais , Adulto JovemRESUMO
This review focuses on new findings about the inflammatory status involved in the development of human liver cirrhosis induced by the two main causes, hepatitis C virus (HCV) infection and chronic alcohol abuse, avoiding results obtained from animal models. When liver is faced to a persistent and/or intense local damage the maintained inflammatory response gives rise to a progressive replacement of normal hepatic tissue by non-functional fibrotic scar. The imbalance between tissue regeneration and fibrosis will determine the outcome toward health recovery or hepatic cirrhosis. In all cases progression toward liver cirrhosis is caused by a dysregulation of mechanisms that govern the balance between activation/homeostasis of the immune system. Detecting differences between the inflammatory status in HCV-induced vs alcohol-induced cirrhosis could be useful to identify specific targets for preventive and therapeutic intervention in each case. Thus, although survival of patients with alcoholic cirrhosis seems to be similar to that of patients with HCV-related cirrhosis (HCV-C), there are important differences in the altered cellular and molecular mechanisms implicated in the progression toward human liver cirrhosis. The predominant features of HCV-C are more related with those that allow viral evasion of the immune defenses, especially although not exclusively, inhibition of interferons secretion, natural killer cells activation and T cell-mediated cytotoxicity. On the contrary, the inflammatory status of alcohol-induced cirrhosis is determined by the combined effect of direct hepatotoxicity of ethanol metabolites and increases of the intestinal permeability, allowing bacteria and bacterial products translocation, into the portal circulation, mesenteric lymph nodes and peritoneal cavity. This phenomenon generates a stronger pro-inflammatory response compared with HCV-related cirrhosis. Hence, therapeutic intervention in HCV-related cirrhosis must be mainly focused to counteract HCV-immune system evasion, while in the case of alcohol-induced cirrhosis it must try to break the inflammatory loop established at the gut-mesenteric lymph nodes-peritoneal-systemic axis.
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Hepacivirus/imunologia , Hepatite C/complicações , Mediadores da Inflamação/imunologia , Cirrose Hepática Alcoólica/imunologia , Cirrose Hepática/imunologia , Fígado/imunologia , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Translocação Bacteriana , Diagnóstico Diferencial , Hepacivirus/patogenicidade , Hepatite C/diagnóstico , Hepatite C/imunologia , Hepatite C/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação/metabolismo , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Cirrose Hepática/diagnóstico , Cirrose Hepática/metabolismo , Cirrose Hepática/virologia , Cirrose Hepática Alcoólica/diagnóstico , Cirrose Hepática Alcoólica/metabolismo , Valor Preditivo dos Testes , Prognóstico , Fatores de Risco , Transdução de SinaisRESUMO
The study of quinoxalines has increased immeasurably during the last two decades, due firstly to their relatively simple chemical synthesis, which has generated a vast variety of compounds with diverse structural modifications, and secondly, to the wide therapeutic potential and biological activities exhibited by this family of compounds. Quinoxalines constitute a rising biomedical class of low-molecular weight heterocyclic compounds with potential functions as antitumour, anti-inflammatory, antibacterial, antiviral, antifungal, antiparasitic and antidiabetic agents, as well as being of interest for the potential treatment of glaucoma, insomnia, cardiovascular and neurological diseases, among others. However, a deeper knowledge of the molecular targets of quinoxalines that fulfil a key role in certain pathologies is required for the development of new and more specific drugs through a rational design strategy to avoid undesirable side effects. In the present review, we summarize the most important molecular targets of the quinoxaline derivatives discovered to date, thus providing a first reference index for researchers to identify the potential targets of their quinoxalines derived collections, which could facilitate the development of new quinoxaline- based therapies.