RESUMO
Despite the considerable efficacy observed when targeting a dispensable lineage antigen, such as CD19 in B cell acute lymphoblastic leukaemia1,2, the broader applicability of adoptive immunotherapies is hampered by the absence of tumour-restricted antigens3-5. Acute myeloid leukaemia immunotherapies target genes expressed by haematopoietic stem/progenitor cells (HSPCs) or differentiated myeloid cells, resulting in intolerable on-target/off-tumour toxicity. Here we show that epitope engineering of donor HSPCs used for bone marrow transplantation endows haematopoietic lineages with selective resistance to chimeric antigen receptor (CAR) T cells or monoclonal antibodies, without affecting protein function or regulation. This strategy enables the targeting of genes that are essential for leukaemia survival regardless of shared expression on HSPCs, reducing the risk of tumour immune escape. By performing epitope mapping and library screenings, we identified amino acid changes that abrogate the binding of therapeutic monoclonal antibodies targeting FLT3, CD123 and KIT, and optimized a base-editing approach to introduce them into CD34+ HSPCs, which retain long-term engraftment and multilineage differentiation ability. After CAR T cell treatment, we confirmed resistance of epitope-edited haematopoiesis and concomitant eradication of patient-derived acute myeloid leukaemia xenografts. Furthermore, we show that multiplex epitope engineering of HSPCs is feasible and enables more effective immunotherapies against multiple targets without incurring overlapping off-tumour toxicities. We envision that this approach will provide opportunities to treat relapsed/refractory acute myeloid leukaemia and enable safer non-genotoxic conditioning.
Assuntos
Epitopos , Edição de Genes , Imunoterapia , Leucemia Mieloide Aguda , Animais , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Antígenos CD34/metabolismo , Transplante de Medula Óssea , Mapeamento de Epitopos , Epitopos/genética , Epitopos/imunologia , Hematopoese , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Xenoenxertos/imunologia , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/terapia , Receptores de Antígenos Quiméricos/imunologia , Recidiva , Linfócitos T/imunologia , Condicionamento Pré-Transplante , Evasão Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Increasing evidence in the field of bioprospection fosters the necessity of studying poorly investigated poisonous marine invertebrates to expand knowledge on animal venom biology. Among marine annelids, amphinomid fireworms are notorious for their bearded trunk equipped with a powerful stinging capacity. Here, a methodological workflow based on analytical chemistry techniques (compound isolation followed by mass spectrometry and spectroscopy analyses) was applied to gain new insights, leading to the identification and structural elucidation of an array of natural products from Mediterranean specimens of Hermodice carunculata. Eight betaine-derived unprecedented compounds, named "carunculines", were detected, bearing two terminal ammonium groups tri-and disubstituted at the Cα (A, B) and a series of different alkyl chains (I-VIII). The mixture of chemicals was found in all the body parts of H. carunculata, supporting a mechanism of action triggered by their vehiculation inside the dorsal chaetae, and subsequent injection when chaetae break off on contact. Preliminary investigations to understand adaptive features were also performed, showing a trend in carunculine abundance that fits into the evolutionary history of these worms. These findings shed light on the chemical ecology of amphinomids, giving reasons for the success of H. carunculata in benthic environments and providing promising novel metabolites for biotechnological implications.
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Compostos de Amônio , Anelídeos , Produtos Biológicos , Poliquetos , Animais , Betaína , Produtos Biológicos/farmacologiaRESUMO
Maladaptive eating behavior is a growing public health problem and compulsively eating excessive food in a short time, or binge eating, is a key symptom of many eating disorders. In order to investigate the binge-like eating behavior in female rats, induced by intermittent food restrictions/refeeding and frustration stress, we analyzed for the first time the metabolic profile obtained from serum of rats, through nuclear magnetic resonance (NMR) spectroscopy. In this experimental protocol, rats were exposed to chow food restricting/refeeding and frustration stress manipulation. This stress procedure consists of 15 min exposure to the odor and sight of a familiar chocolate paste, without access to it, just before offering the palatable food. In this model, a "binge-eating episode" was considered the significantly higher palatable food consumption within 2 h in restricted and stressed rats (R + S) than in the other three experimental groups: rats with no food restriction and no stress (NR + NS), only stressed rats (NR + S) or only restricted rats (R + NS). Serum samples from these four different rat groups were collected. The statistical analysis of the 1 H NMR spectral profiles of the four sets of samples pointed to O- and N-acetyl glycoproteins as the main biomarkers for the discrimination of restriction effects. Other metabolites, such as threonine, glycine, glutamine, acetate, pyruvate and lactate, showed trends that may be useful to understand metabolic pathways involved in eating disorders. This study suggested that NMR-based metabolomics is a suitable approach to detect biomarkers related to binge-eating behavior.
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Transtorno da Compulsão Alimentar/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Metabolômica , Animais , Biomarcadores/sangue , Feminino , Lipídeos/sangue , Substâncias Macromoleculares/sangue , Ratos , Ratos Sprague-DawleyRESUMO
Mendelian susceptibility to mycobacterial disease is a rare primary immunodeficiency characterized by severe infections caused by weakly virulent mycobacteria. Biallelic null mutations in genes encoding interferon gamma receptor 1 or 2 (IFNGR1 or IFNGR2) result in a life-threatening disease phenotype in early childhood. Recombinant interferon γ (IFN-γ) therapy is inefficient, and hematopoietic stem cell transplantation has a poor prognosis. Thus, we developed a hematopoietic stem cell (HSC) gene therapy approach using lentiviral vectors that express Ifnγr1 either constitutively or myeloid specifically. Transduction of mouse Ifnγr1-/- HSCs led to stable IFNγR1 expression on macrophages, which rescued their cellular responses to IFN-γ. As a consequence, genetically corrected HSC-derived macrophages were able to suppress T-cell activation and showed restored antimycobacterial activity against Mycobacterium avium and Mycobacterium bovis Bacille Calmette-Guérin (BCG) in vitro. Transplantation of genetically corrected HSCs into Ifnγr1-/- mice before BCG infection prevented manifestations of severe BCG disease and maintained lung and spleen organ integrity, which was accompanied by a reduced mycobacterial burden in lung and spleen and a prolonged overall survival in animals that received a transplant. In summary, we demonstrate an HSC-based gene therapy approach for IFNγR1 deficiency, which protects mice from severe mycobacterial infections, thereby laying the foundation for a new therapeutic intervention in corresponding human patients.
Assuntos
Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Infecções por Mycobacterium/prevenção & controle , Substâncias Protetoras , Receptores de Interferon/genética , Animais , Células Cultivadas , Transplante de Células-Tronco Hematopoéticas/métodos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium avium , Substâncias Protetoras/metabolismo , Substâncias Protetoras/uso terapêutico , Células RAW 264.7 , Receptor de Interferon gamaRESUMO
Magnetic resonance imaging (MRI) is the current gold standard for the diagnosis of brain tumors. However, despite the development of MRI techniques, the differential diagnosis of central nervous system (CNS) primary pathologies, such as lymphoma and glioblastoma or tumor-like brain lesions and glioma, is often challenging. MRI can be supported by in vivo magnetic resonance spectroscopy (MRS) to enhance its diagnostic power and multiproject-multicenter evaluations of classification of brain tumors have shown that an accuracy around 90% can be achieved for most of the pairwise discrimination problems. However, the survival rate for patients affected by gliomas is still low. The High-Resolution Magic-Angle-Spinning Nuclear Magnetic Resonance (HR-MAS NMR) metabolomics studies may be helpful for the discrimination of gliomas grades and the development of new strategies for clinical intervention. Here, we propose to use T2 -filtered, diffusion-filtered and conventional water-presaturated spectra to try to extract as much information as possible, fusing the data gathered by these different NMR experiments and applying a chemometric approach based on Multivariate Curve Resolution (MCR). Biomarkers important for glioma's discrimination were found. In particular, we focused our attention on cystathionine (Cyst) that shows promise as a biomarker for the better prognosis of glioma tumors.
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Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioma/metabolismo , Glioma/patologia , Metabolômica , Adulto , Idoso , Análise Discriminante , Humanos , Análise dos Mínimos Quadrados , Metaboloma , Pessoa de Meia-Idade , Gradação de Tumores , Análise de Componente Principal , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
Hereditary pulmonary alveolar proteinosis due to GM-CSF receptor deficiency (herPAP) constitutes a life-threatening lung disease characterized by alveolar deposition of surfactant protein secondary to defective alveolar macrophage function. As current therapeutic options are primarily symptomatic, we have explored the potential of hematopoietic stem cell-based gene therapy. Using Csf2rb-/- mice, a model closely reflecting the human herPAP disease phenotype, we here demonstrate robust pulmonary engraftment of an alveolar macrophage population following intravenous transplantation of lentivirally corrected hematopoietic stem and progenitor cells. Engraftment was associated with marked improvement of critical herPAP disease parameters, including bronchoalveolar fluid protein, cholesterol and cytokine levels, pulmonary density on computed tomography scans, pulmonary deposition of Periodic Acid-Schiff+ material as well as respiratory mechanics. These effects were stable for at least nine months. With respect to engraftment and alveolar macrophage differentiation kinetics, we demonstrate the rapid development of CD11c+/SiglecF+ cells in the lungs from a CD11c-/SiglecF+ progenitor population within four weeks after transplantation. Based on these data, we suggest hematopoietic stem cell-based gene therapy as an effective and cause-directed treatment approach for herPAP.
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Proteinose Alveolar Pulmonar , Animais , Modelos Animais de Doenças , Terapia Genética , Células-Tronco Hematopoéticas , Macrófagos Alveolares , Camundongos , Proteinose Alveolar Pulmonar/genética , Proteinose Alveolar Pulmonar/terapiaRESUMO
Asbestos is a commercial term indicating six natural silicates with asbestiform crystal habit. Of these, five are double-chain silicates (amphibole) and one is a layer silicate (serpentine asbestos or chrysotile). Although all species are classified as human carcinogens, their degree of toxicity is still a matter of debate. Amphibole asbestos species are biopersistent in the human lungs and exert their chronic toxic action for decades, whereas chrysotile is not biopersistent and transforms into an amorphous silica structure prone to chemical/physical clearance when exposed to the acidic environment created by the alveolar macrophages. There is evidence in the literature of the toxicity of chrysotile, but its limited biopersistence is thought to explain the difference in toxicity with respect to amphibole asbestos. To date, no comprehensive model describing the toxic action of chrysotile in the lungs is available, as the structure and toxic action of the product formed by the biodissolution of chrysotile are unknown. This work is aimed at fulfilling this gap and explaining the toxic action in terms of structural, chemical, and physical properties. We show that chrysotile's fibrous structure induces cellular damage, mainly through physical interactions. Based on our previous work and novel findings, we propose the following toxicity model: inhaled chrysotile fibers exert their toxicity in the alveolar space by physical and biochemical action. The fibers are soon leached by the intracellular acid environment into a product with residual toxicity, and the dissolution process liberates toxic metals in the intracellular and extracellular environment.
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Asbestos Serpentinas/metabolismo , Asbestos Serpentinas/toxicidade , Pulmão/química , Pulmão/efeitos dos fármacos , Asbestos Serpentinas/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Teoria da Densidade Funcional , Humanos , Pulmão/metabolismo , Modelos Moleculares , Estrutura Molecular , Difração de Pó , Células THP-1RESUMO
RATIONALE: Although the transplantation of induced pluripotent stem cell (iPSC)-derived cells harbors enormous potential for the treatment of pulmonary diseases, in vivo data demonstrating clear therapeutic benefits of human iPSC-derived cells in lung disease models are missing. OBJECTIVES: We have tested the therapeutic potential of iPSC-derived macrophages in a humanized disease model of hereditary pulmonary alveolar proteinosis (PAP). Hereditary PAP is caused by a genetic defect of the GM-CSF (granulocyte-macrophage colony-stimulating factor) receptor, which leads to disturbed macrophage differentiation and protein/surfactant degradation in the lungs, subsequently resulting in severe respiratory insufficiency. METHODS: Macrophages derived from human iPSCs underwent intrapulmonary transplantation into humanized PAP mice, and engraftment, in vivo differentiation, and therapeutic efficacy of the transplanted cells were analyzed. MEASUREMENTS AND MAIN RESULTS: On intratracheal application, iPSC-derived macrophages engrafted in the lungs of humanized PAP mice. After 2 months, transplanted cells displayed the typical morphology, surface markers, functionality, and transcription profile of primary human alveolar macrophages. Alveolar proteinosis was significantly reduced as demonstrated by diminished protein content and surfactant protein D levels, decreased turbidity of the BAL fluid, and reduced surfactant deposition in the lungs of transplanted mice. CONCLUSIONS: We here demonstrate for the first time that pulmonary transplantation of human iPSC-derived macrophages leads to pulmonary engraftment, their in situ differentiation to an alveolar macrophage phenotype, and a reduction of alveolar proteinosis in a humanized PAP model. To our knowledge, this finding presents the first proof-of-concept for the therapeutic potential of human iPSC-derived cells in a pulmonary disease and may have profound implications beyond the rare disease of PAP.
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Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Macrófagos Alveolares/metabolismo , Proteinose Alveolar Pulmonar/metabolismo , Proteinose Alveolar Pulmonar/terapia , Animais , Humanos , Camundongos , Reação em Cadeia da PolimeraseRESUMO
We used 1H, 13C HRMAS and genomic analysis to investigate regionally the transition from oxidative to glycolytic phenotype and its relationship with altered gene expression in adjacent biopsies through the brain of rats bearing C6 gliomas. Tumor-bearing animals were anesthetized and infused with a solution of [1-13C]-glucose, and small adjacent biopsies were obtained spanning transversally from the contralateral hemisphere (regions I and II), the right and left peritumoral areas (regions III and V, respectively), and the tumor core (region IV). These biopsies were analyzed by 1H, 13C HRMAS and by quantitative gene expression techniques. Glycolytic metabolism, as reflected by the [3-13C]-lactate content, increased clearly from regions I to IV, recovering partially to physiological levels in region V. In contrast, oxidative metabolism, as reflected by the [4-13C]-glutamate labeling, decreased in regions I-IV, recovering partially in region V. This metabolic shift from normal to malignant metabolic phenotype paralleled changes in the expression of HIF1α, HIF2α, HIF3α genes, downstream transporters, and regulatory glycolytic, oxidative, and anaplerotic genes in the same regions. Together, our results indicate that genetic and metabolic alterations occurring in the brain of rats bearing C6 gliomas colocalize in situ and the profile of genetic alterations in every region can be inferred from the metabolomic profiles observed in situ by multinuclear HRMAS.
Assuntos
Neoplasias Encefálicas/genética , Reprogramação Celular , Glioma/genética , Glicólise/genética , Fosforilação Oxidativa , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biópsia , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Isótopos de Carbono , Núcleo Caudado/diagnóstico por imagem , Núcleo Caudado/metabolismo , Núcleo Caudado/patologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glioma/diagnóstico por imagem , Glioma/metabolismo , Glioma/patologia , Glucose/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ácido Láctico/metabolismo , Imageamento por Ressonância Magnética/métodos , Transplante de Neoplasias , Ratos , Ratos Wistar , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transplante HeterólogoRESUMO
Humanized mice engrafted with human hematopoietic stem cells and developing functional human T-cell adaptive responses are in critical demand to test human-specific therapeutics. We previously showed that humanized mice immunized with long-lived induced-dendritic cells loaded with the pp65 viral antigen (iDCpp65) exhibited a faster development and maturation of T cells. Herein, we evaluated these effects in a long-term (36 weeks) nonclinical model using two stem cell donors to assess efficacy and safety. Relative to baseline, iDCpp65 immunization boosted the output of effector memory CD4+ T cells in peripheral blood and lymph nodes. No weight loss, human malignancies, or systemic graft-versus-host (GVH) disease were observed. However, for one reconstitution cohort, some mice immunized with iDCpp65 showed GVH-like signs on the skin. Histopathology analyses of the inflamed skin revealed intrafollicular and perifollicular human CD4+ cells near F4/80+ mouse macrophages around hair follicles. In spleen, CD4+ cells formed large clusters surrounded by mouse macrophages. In plasma, high levels of human T helper 2-type inflammatory cytokines were detectable, which activated in vitro the STAT5 pathway of murine macrophages. Despite this inflammatory pattern, human CD8+ T cells from mice with GVH reacted against the pp65 antigen in vitro. These results uncover a dynamic cross-species interaction between human memory T cells and mouse macrophages in the skin and lymphatic tissues of humanized mice.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Macrófagos/imunologia , Pele/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Antígenos CD34/análise , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Comunicação Celular/imunologia , Linhagem Celular , Citocinas/sangue , Proteínas do Citoesqueleto , Células Dendríticas/transplante , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/patologia , Transplante de Células-Tronco Hematopoéticas , Xenoenxertos , Camundongos Endogâmicos NOD , Proteínas dos Microfilamentos , Fosfoproteínas/imunologia , Pele/patologiaRESUMO
Lung surfactant is a complex mixture of lipids and proteins lining the alveolar epithelium. At the air-liquid interface, surfactant lowers surface tension, avoiding alveolar collapse and reducing the work of breathing. The essential role of lung surfactant in breathing and therefore in life, is highlighted by surfactant deficiency in premature neonates, which causes neonatal respiratory distress syndrome and results in early death after birth. In addition, defects in surfactant metabolism alter lung homeostasis and lead to disease. Special attention should be paid to two important key cells responsible for surfactant metabolism: alveolar epithelial type II cells (AE2C) and alveolar macrophages (AM). On the one hand, surfactant deficiency coming from abnormal AE2C function results in high surface tension, promoting alveolar collapse and mechanical stress in the epithelium. This epithelial injury contributes to tissue remodeling and lung fibrosis. On the other hand, impaired surfactant catabolism by AM leads to accumulation of surfactant in air spaces and the associated altered lung function in pulmonary alveolar proteinosis (PAP). We review here two recent cell therapies that aim to recover the activity of AE2C or AM, respectively, therefore targeting the restoring of surfactant metabolism and lung homeostasis. Applied therapies successfully show either transplantation of healthy AE2C in fibrotic lungs, to replace injured AE2C cells and surfactant, or transplantation of bone marrow-derived macrophages to counteract accumulation of surfactant lipid and proteinaceous material in the alveolar spaces leading to PAP. These therapies introduce an alternative treatment with great potential for patients suffering from lung diseases.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Doença , Pulmão/metabolismo , Surfactantes Pulmonares/metabolismo , Animais , Endocitose , Humanos , Macrófagos Alveolares/metabolismoRESUMO
Efficient photoelectrochemical production of hydrogen from water is the aim of many studies in recent decades. Typically, one observes that the electric potential required to initiate the process significantly exceeds the thermodynamic limit. It was suggested that by controlling the spins of the electrons that are transferred from the solution to the anode, and ensuring that they are co-aligned, the threshold voltage for the process can be decreased to that of the thermodynamic voltage. In the present study, by using anodes coated with chiral conductive polymer, the hydrogen production from water is enhanced and the threshold voltage is reduced, as compared with anodes coated with achiral polymer. When CdSe quantum dots were embedded within the polymer, the current density was doubled. These new results point to a possible new direction for producing inexpensive, environmental friendly, efficient water splitting photoelectrochemical cells.
RESUMO
An interlaboratory comparison (ILC) was organized with the aim to set up quality control indicators suitable for multicomponent quantitative analysis by nuclear magnetic resonance (NMR) spectroscopy. A total of 36 NMR data sets (corresponding to 1260 NMR spectra) were produced by 30 participants using 34 NMR spectrometers. The calibration line method was chosen for the quantification of a five-component model mixture. Results show that quantitative NMR is a robust quantification tool and that 26 out of 36 data sets resulted in statistically equivalent calibration lines for all considered NMR signals. The performance of each laboratory was assessed by means of a new performance index (named Qp-score) which is related to the difference between the experimental and the consensus values of the slope of the calibration lines. Laboratories endowed with a Qp-score falling within the suitable acceptability range are qualified to produce NMR spectra that can be considered statistically equivalent in terms of relative intensities of the signals. In addition, the specific response of nuclei to the experimental excitation/relaxation conditions was addressed by means of the parameter named NR. NR is related to the difference between the theoretical and the consensus slopes of the calibration lines and is specific for each signal produced by a well-defined set of acquisition parameters.
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RATIONALE: Hereditary pulmonary alveolar proteinosis (hPAP) caused by granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor α-chain (CSF2RA) deficiency is a rare, life-threatening lung disease characterized by accumulation of proteins and phospholipids in the alveolar spaces. The disease is caused by a functional insufficiency of alveolar macrophages, which require GM-CSF signaling for terminal differentiation and effective degradation of alveolar proteins and phospholipids. Therapeutic options are extremely limited, and the pathophysiology underlying the defective protein degradation in hPAP alveolar macrophages remains poorly understood. OBJECTIVES: To further elucidate the cellular mechanisms underlying hPAP and evaluate novel therapeutic strategies, we here investigated the potential of hPAP patient-derived induced pluripotent stem cell (PAP-iPSCs) derived monocytes and macrophages. METHODS: Patient-specific PAP-iPSCs were generated from CD34(+) bone marrow cells of a CSF2RA-deficient patient with PAP. We assessed pluripotency, chromosomal integrity, and genetic correction of established iPSC lines. On hematopoietic differentiation, genetically corrected or noncorrected monocytes and macrophages were investigated in GM-CSF-dependent assays. MEASUREMENTS AND MAIN RESULTS: Although monocytes and macrophages differentiated from noncorrected PAP-iPSCs exhibited distinct defects in GM-CSF-dependent functions, such as perturbed CD11b activation, phagocytic activity, and STAT5 phosphorylation after GM-CSF exposure and lack of GM-CSF uptake, these defects were fully repaired on lentiviral gene transfer of a codon-optimized CSF2RA-cDNA. CONCLUSIONS: These data establish PAP-iPSC-derived monocytes and macrophages as a valid in vitro disease model of CSF2RA-deficient PAP, and introduce gene-corrected iPSC-derived monocytes and macrophages as a potential autologous cell source for innovative therapeutic strategies. Transplantation of such cells to patients with hPAP could serve as a paradigmatic proof for the potential of iPSC-derived cells in clinical gene therapy.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/terapia , Terapia Genética , Células-Tronco Pluripotentes Induzidas , Proteinose Alveolar Pulmonar/terapia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/deficiência , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Pré-Escolar , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Humanos , Macrófagos Alveolares/metabolismo , Modelos Biológicos , Monócitos/metabolismo , Proteinose Alveolar Pulmonar/genética , Proteinose Alveolar Pulmonar/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Skeletal muscles are heterogenous tissues composed of different myofiber types that can be classified as slow oxidative, fast oxidative, and fast glycolytic which are distinguished on the basis of their contractile and metabolic properties. Improving oxidative metabolism in skeletal muscles can prevent metabolic diseases and plays a protective role against muscle wasting in a number of neuromuscular diseases. Therefore, achieving a detailed understanding of the factors that regulate myofiber metabolic properties might provide new therapeutic opportunities for these diseases. Here, we investigated whether peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) is involved in the control of myofiber metabolic behaviors. Indeed, PIN1 controls glucose and lipid metabolism in a number of tissues, and it is also abundant in adult skeletal muscles; however, its role in the control of energy homeostasis in this tissue is still to be defined. To start clarifying this topic, we compared the metabolome of the tibialis anterior muscle (mainly glycolytic) and soleus muscle (oxidative) in wild-type and Pin1 knockout mice with High-Resolution Magic Angle Spinning (HR-MAS) NMR on intact tissues. Our analysis reveals a clear demarcation between the metabolomes in the two types of muscles and allows us to decode a signature able to discriminate the glycolytic versus oxidative muscle phenotype. We also detected some changes in Pin1-depleted muscles that suggest a role for PIN1 in regulating the metabolic phenotype of skeletal muscles.
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Primary Sjögren's Syndrome (pSS) is a multi-system autoimmune disease that involves the exocrine glands. Lymphocytes infiltrate the gland tissue, leading to anatomical modification and hypofunction. Even if the prognosis of pSS is favorable, quality of life is typically reduced due to the diverse manifestations of the disease. The aim of this study is to compare the salivary metabolomes of pSS with healthy controls (HCs). Seven cases were selected from a cohort of pSS patients, and six age- and sex-matched HCs were recruited from a cohort of volunteers. Whole unstimulated saliva was collected for NMR analysis. Our metabolomic analysis focused on 360 ms total echo 1D 1H NMR CPMG spectra. Metabolites detected with CPMG NMR spectra were assigned through 2D NMR spectra (COSY, TOCSY, and HSQC). About 50 metabolites were detected and assigned. Unsupervised exploratory PCA returned partial clustering, and PLS-DA improved the separation between pSS and HCs, highlighting a pool of metabolites distinctly describing each group. Despite the limited number of samples, the presented preliminary data are promising. PLS-DA indicated well-defined group separation, suggesting that the application of 1H-NMR metabolomics is suitable for the study of pSS.
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BACKGROUND AND AIMS: Mutations in IL10 or the IL10 receptor lead to very early onset [VEO] inflammatory bowel disease [IBD], a life-threatening disease which is often unresponsive to conventional medication. Recent studies have demonstrated that defective IL-10 receptor signalling in innate immune cells is a key driver of severe intestinal inflammation in VEO-IBD. Specifically, IL10 unresponsiveness of macrophages, which govern the tight balance between pro- and anti-inflammatory responses in the intestinal system, plays a central role in the events leading to excessive inflammatory responses and the development of IBD. METHODS AND RESULTS: We here evaluated haematopoietic stem cell gene therapy in a VEO-IBD mouse model and demonstrated that the therapeutic response closely correlates with gene correction of the IL10 signalling pathway in intestinal macrophages. This finding prompted us to evaluate the therapeutic efficacy of macrophage transplantation in the Il10rb-/- VEO-IBD mouse model. A 6-week regimen employing a combination of depletion of endogenous hyperinflammatory macrophages followed by intraperitoneal administration of wild-type [WT] macrophages significantly reduced colitis symptoms. CONCLUSIONS: In summary, we show that the correction of the IL10 receptor defect in macrophages, either by genetic therapy or transfer of WT macrophages to the peritoneum, can ameliorate disease-related symptoms and potentially represent novel treatment approaches for VEO-IBD patients.
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Transferência Adotiva , Doenças Inflamatórias Intestinais/fisiopatologia , Doenças Inflamatórias Intestinais/terapia , Subunidade beta de Receptor de Interleucina-10/fisiologia , Macrófagos/transplante , Animais , Modelos Animais de Doenças , Doenças Inflamatórias Intestinais/etiologia , CamundongosRESUMO
The association between the metabolic profile and inflammatory cytokines in psoriasis is poorly understood. We analyzed the metabolic and cytokine/chemokine profiles in serum and skin from patients with new-onset psoriasis and healthy subjects (n = 7/group) by HR-MAS NMR and Bio-Plex immunoassay. Immuno-metabolic correlation matrix was analyzed in skin and serum to identify a potential immune-metabolic signature. Metabolomics analysis showed a significant increase in ascorbate and a decrease in scyllo-inositol, and a trend towards an increase in eight other metabolites in psoriatic skin. In serum, there was a significant increase of dimethylglycine and isoleucine. In parallel, psoriatic skin exhibited an increase of early inflammatory cytokines (IL-6, IL-8, TNF-α, IL-1ß) and correlation analysis highlighted some major clusters of immune-metabolic correlations. A cluster comprising scyllo-inositol and lysine showed correlations with T-cell cytokines; a cluster comprising serine and taurine showed a negative correlation with early inflammatory cytokines (IL-6, G-CSF, CCL3). A strong positive correlation was enlightened between glutathione and inflammatory cytokines/angiogenesis promoters of psoriasis. The integration of metabolic and immune data indicated a molecular signature constituted by IL-6, IL1-ra, DMG, CCL4, Ile, Gly and IL-8, which could discriminate patients and healthy subjects and could represent a candidate tool in the diagnosis of new-onset psoriasis.
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Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Metabolômica , Estudos de Casos e Controles , Citocinas/sangue , Humanos , Mediadores da Inflamação/sangue , Isoleucina/sangue , Espectroscopia de Ressonância Magnética/métodos , Sarcosina/análogos & derivados , Sarcosina/sangue , Pele/metabolismoRESUMO
The immunosuppressive microenvironment surrounding tumor cells represents a key cause of treatment failure. Therefore, immunotherapies aimed at reprogramming the immune system have largely spread in the past years. We employed gene transfer into hematopoietic stem and progenitor cells to selectively express anti-tumoral cytokines in tumor-infiltrating monocytes/macrophages. We show that interferon-γ (IFN-γ) reduced tumor progression in mouse models of B-cell acute lymphoblastic leukemia (B-ALL) and colorectal carcinoma (MC38). Its activity depended on the immune system's capacity to respond to IFN-γ and drove the counter-selection of leukemia cells expressing surrogate antigens. Gene-based IFN-γ delivery induced antigen presentation in the myeloid compartment and on leukemia cells, leading to a wave of T cell recruitment and activation, with enhanced clonal expansion of cytotoxic CD8+ T lymphocytes. The activity of IFN-γ was further enhanced by either co-delivery of tumor necrosis factor-α (TNF-α) or by drugs blocking immunosuppressive escape pathways, with the potential to obtain durable responses.
Assuntos
Leucemia , Neoplasias , Animais , Apresentação de Antígeno , Interferon gama , Camundongos , Células Mieloides , Microambiente Tumoral , Fator de Necrose Tumoral alfaRESUMO
Background-Actinic keratoses (AKs) are the most common sun-induced precancerous lesions that can progress to squamocellular carcinoma (SCC). Recently, the grade-independent association between AKs and SCC has been suggested; however, the molecular bases of this potential association have not been investigated. This study has assessed the metabolomic fingerprint of AK I, AK II, AK III and SCC using high resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy in order to evaluate the hypothesis of grade-independent association between AK and SCC. Association between AKs and SCCs has also been evaluated by histopathology. Methods-Metabolomic data were obtained through HR-MAS NMR spectroscopy. The whole spectral profiles were analyzed through multivariate statistical analysis using MetaboAnalyst 5.0. Histologic examination was performed on sections stained with hematoxylin and eosin; statistical analysis was performed using STATA software version 14. Results-A group of 35 patients affected by AKs and/or SCCs and 10 healthy controls were enrolled for metabolomics analysis. Histopathological analysis was conducted on 170 specimens of SCCs and AKs (including the ones that underwent metabolomic analysis). SCCs and AK I were found to be significantly associated in terms of the content of some metabolites. Moreover, in the logistic regression model, the presence of parakeratosis in AKs appeared to be less frequently associated with SCCs, while AKs with hypertrophy had a two-fold higher risk of being associated with SCC. Conclusions-Our findings, derived from metabolomics and histopathological data, support the notion that AK I are different from healthy skin and share some different features with SCCs. This may further support the expanding notion that all AKs should be treated independently from their clinical appearance or histological grade because they may be associated with SCC.