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For advanced therapy medicinal products, the development and validation of potency assays are required, in accordance with international guidelines, to characterise the product and obtain reliable and consistent data. Our purpose was to validate the killing assay for the evaluation of autologous anti-CD19 chimeric antigen receptor (CAR) T potency. We used CD4 + and CD8 + lymphocytes or anti-CD19 CAR-T cells as effector cells and REH (CD19 +) or MOLM-13 (CD19 -) cell lines as target cells. After co-culturing target and effector cells (1:1 ratio) for 24 h, samples were labelled with 7-AAD, anti-CD3 and anti-CD19 antibodies and the frequency of CD19 + dead cells was evaluated by flow cytometry. In order to verify the CAR-T specificity for the CD19 + target, the co-culture between CAR-T and REH or MOLM-13 at different effector-to-target ratios was scheduled. Moreover, not transduced CD4 + and CD8 + lymphocytes were tested in comparison with CAR-T from the same donor to demonstrate the assay specificity. Linearity and accuracy were evaluated, and established acceptance criteria were compiled for both parameters (r2 ≥ 0.97 for linearity and average relative error ≤ 10% for accuracy). Furthermore, the method was considered robust when performed between 23 and 25 h of co-culture, and the intra-assay, inter-assay and inter-day precision was obtained. Finally, in order to verify the inter-analyst precision, the test was executed by three different operators and the intra-class correlation coefficient was > 0.4 in both cases. In conclusion, we consider this CAR-T potency assay as validated and usable in all steps of product development and quality control.
Assuntos
Antígenos CD19 , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Imunoterapia Adotiva/métodos , Antígenos CD19/imunologia , Técnicas de Cocultura , Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica , Linhagem Celular Tumoral , Linfócitos T CD4-Positivos/imunologiaRESUMO
BACKGROUND AIMS: Dendritic cells (DCs) are professional antigen-presenting cells of the mammalian immune system. Ex vivo differentiated DCs represent a unique Advanced Therapy Medicinal Product (ATMP), used in several clinical trials as personalized cancer immunotherapy. The therapy's reliability depends on its capacity to produce high-quality mature DCs (mDCs) in compliance with Good Manufacturing Practices. AIMS: From March 2010 to December 2023, 103 patients were enrolled in multiple clinical trials at the Immuno-Gene Therapy Factory at IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) "Dino Amadori". Six hundred forty-two doses were produced, and the manufacturing process was implemented to optimize production. Our study is a retrospective analysis focusing on the quality control results. METHODS: We retrospectively analyzed the results of the quality control tests carried out on each produced batch, evaluating viability, purity and phenotype of mDCs and their quality in terms of microbiological safety. The data obtained are given with median and interquartile range. RESULTS: The batches were found to be microbiologically safe in terms of sterility, mycoplasma, and endotoxins. An increase in DC maturation markers was found. The release criteria checks showed a high percentage of viability and purity was maintained during the production process. CONCLUSIONS: Our findings have confirmed that the measures implemented have ensured the safety of the products and have contributed to the establishing a robust "Pharmaceutical Quality System." This has enabled many safe mDCs to be produced for clinical trials.
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Efficient cell-to-cell communication is essential for tissue development, homeostasis, and the maintenance of cellular functions after injury. Tunneling nanotubes (TNTs) have emerged as a new important method of cell-to-cell communication. TNTs are primarily established between stressed and unstressed cells and can transport a variety of cellular components. Mitochondria are important trafficked entities through TNTs. Transcellular mitochondria transfer permits the incorporation of healthy mitochondria into the endogenous network of recipient cells, changing the bioenergetic profile and other functional properties of the recipient and may allow the recipient cells to recuperate from apoptotic processes and return to a normal operating state. Mesenchymal cells (MSCs) can form TNTs and transfer mitochondria and other constituents to target cells. This occurs under both physiological and pathological conditions, leading to changes in cellular energy metabolism and functions. This review summarizes the newly described capacity of melatonin to improve mitochondrial fusion/fission dynamics and promote TNT formation. This new evidence suggests that melatonin's protective effects could be attributed to its ability to prevent mitochondrial damage in injured cells, reduce senescence, and promote anastasis, a natural cell recovery phenomenon that rescues cells from the brink of death. The modulation of these new routes of intercellular communication by melatonin could play a key role in increasing the therapeutic potential of MSCs.
Assuntos
Melatonina , Células-Tronco Mesenquimais , Nanotubos , Comunicação Celular/fisiologia , Estruturas da Membrana Celular , Melatonina/metabolismo , Melatonina/farmacologia , Células-Tronco Mesenquimais/metabolismoRESUMO
Neonatal encephalopathy (NE) is a pathological condition affecting long-term neurodevelopmental outcomes. Hypothermia is the only therapeutic option, but does not always improve outcomes; hence, researchers continue to hunt for pharmaceutical compounds. Melatonin treatment has benefitted neonates with hypoxic-ischemic (HI) brain injury. However, unlike animal models that enable the study of the brain and the pathophysiologic cascade, only blood is available from human subjects. Therefore, due to the unavailability of neonatal brain tissue, assumptions about the pathophysiology in pathways and cascades are made in human subjects with NE. We analyzed animal and human specimens to improve our understanding of the pathophysiology in human neonates. A neonate with NE who underwent hypothermia and enrolled in a melatonin pharmacokinetic study was compared to HI rats treated/untreated with melatonin. MicroRNA (miRNA) analyses provided profiles of the neonate's plasma, rat plasma, and rat brain cortexes. We compared these profiles through a bioinformatics tool, identifying Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways common to HI brain injury and melatonin treatment. After evaluating the resulting pathways and the literature, to validate the method, the key proteins expressed in HI brain injury were investigated using cerebral cortexes. The upregulated miRNAs in human neonate and rat plasma helped identify two KEGG pathways, glioma and long-term potentiation, common to HI injury and melatonin treatment. A unified neonatal cerebral melatonin-sensitive HI pathway was designed and validated by assessing the expression of protein kinase Cα (PKCα), phospho (p)-Akt, and p-ERK proteins in rat brain cortexes. PKCα increased in HI-injured rats and further increased with melatonin. p-Akt and p-ERK returned phosphorylated to their basal level with melatonin treatment after HI injury. The bioinformatics analyses validated by key protein expression identified pathways common to HI brain injury and melatonin treatment. This approach helped complete pathways in neonates with NE by integrating information from animal models of HI brain injury.
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Lesões Encefálicas , Hipotermia , Hipóxia-Isquemia Encefálica , Melatonina , MicroRNAs , Animais , Animais Recém-Nascidos , Humanos , Hipotermia/tratamento farmacológico , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Hipóxia-Isquemia Encefálica/genética , Hipóxia-Isquemia Encefálica/metabolismo , Melatonina/farmacologia , Melatonina/uso terapêutico , MicroRNAs/genética , Proteína Quinase C-alfa , Proteínas Proto-Oncogênicas c-akt , RatosRESUMO
Mitochondrial dysfunction is considered one of the hallmarks of ischemia/reperfusion injury. Mitochondria are plastic organelles that undergo continuous biogenesis, fusion, and fission. They can be transferred between cells through tunneling nanotubes (TNTs), dynamic structures that allow the exchange of proteins, soluble molecules, and organelles. Maintaining mitochondrial dynamics is crucial to cell function and survival. The present study aimed to assess the effects of melatonin on mitochondrial dynamics, TNT formation, and mitochondria transfer in HT22 cells exposed to oxygen/glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin treatment during the reoxygenation phase reduced mitochondrial reactive oxygen species (ROS) production, improved cell viability, and increased the expression of PGC1α and SIRT3. Melatonin also preserved the expression of the membrane translocase proteins TOM20 and TIM23, and of the matrix protein HSP60, which are involved in mitochondrial biogenesis. Moreover, it promoted mitochondrial fusion and enhanced the expression of MFN2 and OPA1. Remarkably, melatonin also fostered mitochondrial transfer between injured HT22 cells through TNT connections. These results provide new insights into the effect of melatonin on mitochondrial network reshaping and cell survival. Fostering TNTs formation represents a novel mechanism mediating the protective effect of melatonin in ischemia/reperfusion injury.
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Isquemia Encefálica/patologia , Estruturas da Membrana Celular/efeitos dos fármacos , Melatonina/farmacologia , Mitocôndrias/efeitos dos fármacos , Neurônios/ultraestrutura , Animais , Linhagem Celular , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Hipocampo/ultraestrutura , Camundongos , Mitocôndrias/metabolismo , Nanotubos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Traumatismo por Reperfusão/patologiaRESUMO
For many years, oncological clinical trials have taken advantage of dendritic cells (DC) for the design of DC-based cellular therapies. This has required the design of suitable quality control assays to evaluate the potency of these products. The purpose of our work was to develop and validate a novel bioassay that uses flow cytometry as a read-out measurement. In this method, CD3+ cells are labeled with a fluorescent dye and the DC costimulatory activity is measured by the degree of T cell proliferation caused by the DC-T cell interaction. The validation of the method was achieved by the evaluation of essential analytical parameters defined by international guidelines. Our results demonstrated that the method could be considered specific, selective, and robust. The comparison between measured values and estimated true values confirmed a high level of accuracy and a lack of systematic error. Repeated experiments have shown the reproducibility of the assay and the proportionality between the potency and the DC amount has proven its linearity. Our results suggest that the method is compliant with the guidelines and could be adopted as a quality control assay or batch-release testing within GMP facilities.
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Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Biomarcadores , Vacinas Anticâncer/uso terapêutico , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. Chemotherapy, the treatment of choice in non-operable cases, achieves a dismal success rate, raising the need for new therapeutic options. In about 25% of NSCLC, the activating mutations of the KRAS oncogene define a subclass that cannot benefit from tyrosine kinase inhibitors (TKIs). The tumor suppressor miR-16 is downregulated in many human cancers, including NSCLC. The main objectives of this study were to evaluate miR-16 treatment to restore the TKI sensitivity and compare its efficacy to MEK inhibitors in KRAS-mutated NSCLC. METHODS: We performed in vitro and in vivo studies to investigate whether miR-16 could be exploited to overcome TKI resistance in KRAS-mutated NSCLC. We had three goals: first, to identify the KRAS downstream effectors targeted by mir-16, second, to study the effects of miR-16 restoration on TKI resistance in KRAS-mutated NSCLC both in vitro and in vivo, and finally, to compare miR-16 and the MEK inhibitor selumetinib in reducing KRAS-mutated NSCLC growth in vitro and in vivo. RESULTS: We demonstrated that miR-16 directly targets the three KRAS downstream effectors MAPK3, MAP2K1, and CRAF in NSCLC, restoring the sensitivity to erlotinib in KRAS-mutated NSCLC both in vitro and in vivo. We also provided evidence that the miR-16-erlotinib regimen is more effective than the selumetinib-erlotinib combination in KRAS-mutated NSCLC. CONCLUSIONS: Our findings support the biological preclinical rationale for using miR-16 in combination with erlotinib in the treatment of NSCLC with KRAS-activating mutations.
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Carcinoma Pulmonar de Células não Pequenas , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares , MAP Quinase Quinase Quinases , MicroRNAs , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras) , RNA Neoplásico , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/biossíntese , MicroRNAs/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This study was conducted to evaluate the expression of fibrinogen receptors on platelets of Philadelphia-negative chronic myeloproliferative neoplasm (MPN) patients. We collected blood samples from 40 consecutive MPN patients and healthy volunteers. We performed flow cytometry analysis of P-selectin expression and integrin beta-3, activation of glycoprotein (GP) IIb/IIIa and fibrinogen receptor exposure (PAC-1 binding). Surprisingly, we found a very low PAC-1 binding capacity in MPN patients; however, the expression of PAC-1 was almost completely recovered with aspirin intake. We hypothesize that the hypercoagulable states observed in MPN patients could depend on a primarily plasma-driven impairment of fibrin turnover and thrombin generation.
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Aspirina/uso terapêutico , Fibrinogênio/uso terapêutico , Transtornos Mieloproliferativos/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Aspirina/farmacologia , Plaquetas , Doença Crônica , Fibrinogênio/farmacologia , Humanos , Pessoa de Meia-IdadeRESUMO
INTRODUCTION: Neonates with hypoxic-ischemic encephalopathy (HIE) undergoing hypothermia may benefit from adjunctive therapy with melatonin. However, melatonin safety, pharmacokinetics (PK), and dosage in this sensitive population are still unknown. METHODS AND RESULTS: This study assessed the PK and safety of melatonin enteral administration to neonates with HIE undergoing hypothermia. Melatonin was infused at 0.5 mg/kg in five neonates with HIE undergoing hypothermia. Infusion started 1 hour after the neonates reached the target temperature of 33.5°C. Blood samples were collected before and at selective times after melatonin infusion. Abdominal complications or clinically significant changes in patients' vital signs were not found during or after melatonin. The peak plasma concentration reached 0.25 µg/mL. The area under the curve in 24 hours was 4.35 µg/mL*h. DISCUSSION: Melatonin half-life and clearance were prolonged, and the distribution volume decreased compared to adults. In silico simulation estimated that the steady state can be reached after four infusions. Hypothermia does not affect melatonin PK. In humans high blood concentrations with lower doses can be achieved compared to animal experimentation, although intravenous administration is advised in the neonate population. Our study is a preparatory step for future clinical studies aimed at assessing melatonin efficacy in HIE.
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Hipotermia Induzida , Melatonina/farmacocinética , Feminino , Humanos , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/terapia , Recém-Nascido , Masculino , Melatonina/uso terapêuticoRESUMO
AIMS: Glycogen synthase kinase-3 beta (GSK-3ß) is a serine/threonine kinase involved in glycogen metabolism, cell cycle progression, differentiation, embryogenesis, migration, metabolism, survival and cellular senescence. Its main biological function is to inhibit ß-catenin by sequestration and promotion of its proteasomal degradation in the Wnt canonical pathway; however, GSK-3ß interacts with multiple signalling pathways, and aberrant expression of the enzyme was reported in many solid neoplasms. This study aimed to investigate the biological relevance of GSK-3ß in classical Hodgkin lymphomas (cHL). METHODS AND RESULTS: We analysed the functional status of GSK-3ß enzyme in cHL by using antibodies raised against fixation-resistant epitopes of phospho Y216 GSK-3ß (active form), phospho S9 GSK-3ß (inactive form) and ß-catenin protein. We first detected the pY216 GSK-3ß active form of the enzyme in 100 of 100 (100%) of the cases, and in line with the latter expression profile, the ß-catenin protein was found in only 12 of 100 (12%) of the samples. As reported previously in bladder cancer, pancreatic adenocarcinoma and chronic lymphocytic leukaemia, we showed an aberrant nuclear localization in the neoplastic clone of active pY216 GSK-3ß in 78 of 100 (78%) of cHL cases. CONCLUSIONS: We demonstrated the activation of GSK-3ß in cHL resulting in inhibition of the Wnt/ß-catenin signal cascade and the aberrant accumulation of its activated form in nuclei of Hodgkin Reed-Sternberg and Hodgkin cells. These findings may be relevant for future clinical studies, identifying GSK-3ß as a potential therapeutic target for cHL.
Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Doença de Hodgkin/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , TranscriptomaRESUMO
Increasing evidence indicates that melatonin possesses protective effects toward different kinds of damage in various organs, including the brain. In a neonatal model of hypoxia-ischemia (HI), melatonin was neuroprotective and preserved the expression of the silent information regulator 1 (SIRT1) 24 hours after the insult. This study aimed to gain more insight into the role of SIRT1 in the protective effect of melatonin after HI by studying the early (1 hour) modulation of SIRT1 and its downstream targets, and the consequences on necrosis, apoptosis, autophagy, and glial cell activation. We found that melatonin administered 5 minutes after the ischemic insult significantly reduced necrotic cell death assessed 1 hour after its administration. In parallel, we found a reduced activation of the early phases of intrinsic apoptosis, detected by reduced BAX translocation to the mitochondria and preservation of the mitochondrial expression of cytochrome C, indicating a reduced outer mitochondrial membrane permeabilization in the melatonin-treated ischemic animals. These effects were concomitant to increased expression and activity of SIRT1, reduced expression and acetylation of p53, and increased autophagy activation. Melatonin also reduced HI-induced glial cells activation. SIRT1 was expressed in neurons after HI and melatonin but not in reactive glial cells expressing GFAP. Colocalization between SIRT1 and GFAP was found in some cells in control conditions. In summary, our results provide more insight into the connection between SIRT1 and melatonin in neuroprotection. The possibility that melatonin-induced SIRT1 activity might contribute to differentiate neuronal progenitor cells during the neurodegenerative process needs to be further investigated.
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Isquemia Encefálica/metabolismo , Melatonina/metabolismo , Sirtuína 1/metabolismo , Animais , Animais Recém-Nascidos , Isquemia Encefálica/patologia , Morte Celular , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Neurônios/metabolismo , Gravidez , Ratos Sprague-Dawley , Proteína Supressora de Tumor p53/metabolismoRESUMO
Melatonin possesses potential efficacy in perinatal brain injuries, and has been proposed as adjunctive pharmacological therapy in combination with hypothermia in the clinical setting. However, the pharmacokinetics of melatonin in preterm and term newborns is still unknown. The aim of this study was to analyze the pharmacokinetics of melatonin after intragastric administration in preterm infants. Preterm newborns were enrolled 24-72 h after birth, and randomly assigned to three groups receiving a single bolus of 0.5 mg·kg-1 melatonin, or 3 boluses of 1 or 5 mg·kg-1 of melatonin at 24-h intervals. Blood samples were collected before and at selective times after melatonin administration. The half-life of melatonin in plasma ranged from 7.98 to 10.94 h, and the area under the curve (AUC) from 10.48 to 118.17 µg·mL-1·h-1. Our results indicate a different pharmacokinetic profile in premature newborns, compared to adults and experimental animals. The high peak plasma concentrations and the long half-life indicate that in the neonatal clinical setting, it is possible to obtain and maintain high serum concentrations using a single administration of melatonin repeated every 12/24 h.
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Melatonina/farmacocinética , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacocinética , Administração Oral , Adulto , Área Sob a Curva , Terapia Combinada , Relação Dose-Resposta a Droga , Feminino , Meia-Vida , Humanos , Lactente , Recém-Nascido , Recém-Nascido Prematuro , Melatonina/administração & dosagem , Melatonina/sangue , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/sangue , GravidezRESUMO
We herein report the results from a comparative study of arsenite toxicity in respiration-proficient (RP) and -deficient (RD) U937 cells. An initial characterization of these cells led to the demonstration that the respiration-deficient phenotype is not associated with apparent changes in mitochondrial mass and membrane potential. In addition, similar levels of superoxide (O2(.-)) were generated by RP and RD cells in response to stimuli specifically triggering respiratory chain-independent mitochondrial mechanisms or extramitochondrial, NADPH-oxidase dependent, mechanisms. At the concentration of 2.5µM, arsenite elicited selective formation of O2(.-) in the respiratory chain of RP cells, with hardly any contribution of the above mechanisms. Under these conditions, O2(.-) triggered downstream events leading to endoplasmic reticulum (ER) stress, autophagy and apoptosis. RD cells challenged with similar levels of arsenite failed to generate O2(.-) because of the lack of a functional respiratory chain and were therefore resistant to the toxic effects mediated by the metalloid. Their resistance, however, was lost after exposure to four fold greater concentrations of arsenite, coincidentally with the release of O2(.-) mediated by NADPH oxidase. Interestingly, extramitochondrial O2(.-) triggered the same downstream events and an identical mode of death previously observed in RP cells. Taken together, the results obtained in this study indicate that arsenite toxicity is strictly dependent on O2(.-) availability that, regardless of whether generated in the mitochondrial or extramitochondrial compartments, triggers similar downstream events leading to ER stress, autophagy and apoptosis.
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Arsenitos/toxicidade , Respiração Celular , NADPH Oxidases/metabolismo , Superóxidos/metabolismo , Apoptose/efeitos dos fármacos , Autofagia , Sobrevivência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Consumo de Oxigênio , Células U937RESUMO
Maternal infection/inflammation represents one of the most important factors involved in the etiology of brain injury in newborns. We investigated the modulating effect of prenatal melatonin on the neonatal brain inflammation process resulting from maternal intraperitoneal (i.p.) lipopolysaccharide (LPS) injections. LPS (300 µg/kg) was administered to pregnant rats at gestational days 19 and 20. Melatonin (5 mg/kg) was administered i.p. at the same time as LPS. Melatonin counteracted the LPS sensitization to a second ibotenate-induced excitotoxic insult performed on postnatal day (PND) 4. As melatonin succeeded in reducing microglial activation in neonatal brain at PND1, pathways previously implicated in brain inflammation regulation, such as endoplasmic reticulum (ER) stress, autophagy and silent information regulator 1 (SIRT1), a melatonin target, were assessed at the same time-point in our experimental groups. Results showed that maternal LPS administrations resulted in an increase in CHOP and Hsp70 protein expression and eIF2α phosphorylation, indicative of activation of the unfolded protein response consequent to ER stress, and a slighter decrease in the autophagy process, determined by reduced lipidated LC3 and increased p62 expression. LPS-induced inflammation also reduced brain SIRT1 expression and affected the expression of miR-34a, miR146a, and miR-126. All these effects were blocked by melatonin. Cleaved-caspase-3 apoptosis pathway did not seem to be implicated in the noxious effect of LPS on the PND1 brain. We conclude that melatonin is effective in reducing maternal LPS-induced neonatal inflammation and related brain injury. Its role as a prophylactic/therapeutic drug deserves to be investigated by clinical studies.
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Autofagia/efeitos dos fármacos , Encéfalo/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Melatonina/farmacologia , MicroRNAs/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Encéfalo/patologia , Feminino , Inflamação/metabolismo , Inflamação/patologia , Lipopolissacarídeos/toxicidade , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/patologia , Ratos , Ratos WistarRESUMO
BACKGROUND: Acute myeloid leukemia (AML) is an incurable disease with fatal infections or relapse being the main causes of death in most cases. In particular, the severe infections occurring in these patients before or during any treatment suggest an intrinsic alteration of the immune system. In this respect, IL-17-producing T helper (Th17) besides playing a key role in regulating inflammatory response, tumor growth and autoimmune diseases, have been shown to protect against bacterial and fungal pathogens. However, the role of Th17 cells in AML has not yet been clarified. METHODS: T cell frequencies were assessed by flow cytometry in the peripheral blood of 30 newly diagnosed AML patients and 30 age-matched healthy volunteers. Cytokine production was determined before and after culture of T cells with either Candida Albicans or AML blasts. Statistical analyses were carried out using the paired and unpaired two-tailed Student's t tests and confirmed with the non parametric Wilcoxon signed-rank test. RESULTS: A strong increase of Th17 cells producing immunosuppressive IL-10 was observed in AML patients compared with healthy donors. In addition, stimulation of AML-derived T cells with a Candida albicans antigen induced significantly lower IFN-γ production than that observed in healthy donors; intriguingly, depletion of patient Th17 cells restored IFN-γ production after stimulation. To address the role of AML blasts in inducing Th17 alterations, CD4+ cells from healthy donors were co-cultured with CD33+ blasts: data obtained showed that AML blasts induce in healthy donors levels of IL-10-producing Th17 cells similar to those observed in patients. CONCLUSIONS: In AML patients altered Th17 cells actively cause an immunosuppressive state that may promote infections and probably tumor escape. Th17 cells could thus represent a new target to improve AML immunotherapy.
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Candidíase/imunologia , Terapia de Imunossupressão , Interleucina-10/biossíntese , Interleucina-17/biossíntese , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/microbiologia , Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Crise Blástica/imunologia , Candida albicans/imunologia , Candidíase/complicações , Candidíase/microbiologia , Técnicas de Cocultura , Citocinas/metabolismo , Feminino , Humanos , Interferon gama/biossíntese , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/complicações , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Células Th17/imunologiaRESUMO
The present study deals with the preparation of albumin nanocapsules containing fenretinide and their evaluation in experimental models of human non-small cell lung cancer. These nanocapsules showed enhanced antitumor activity with respect to free fenretinide due to the solubilization effect of albumin on the hydrophobic drug, known to improve bioavailability. The high expression of caveolin-1 on the A549 cell surface further enhanced the antitumor activity of the nanoencapsulated fenretinide. Caveolin-1 favored albumin uptake and improved the efficacy of the fenretinide-loaded albumin nanocapsules, especially in 3-D cultures where the densely packed 3-D structures impaired drug diffusibility and severely reduced the activity of the free drug. The efficacy of the fenretinide albumin nanocapsules was further confirmed in tumor xenograft models of A549 by the significant delay in tumor progression observed with respect to control after intravenous administration of the novel formulation. FROM THE CLINICAL EDITOR: This study describes the preparation of fenretinide containing albumin nanocapsules and their evaluation in experimental models of non-small cell lung cancer, showing enhanced antitumor activity compared to free fenretinide.
Assuntos
Antineoplásicos/química , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Fenretinida/administração & dosagem , Nanocápsulas/administração & dosagem , Albuminas/administração & dosagem , Albuminas/química , Animais , Antineoplásicos/administração & dosagem , Disponibilidade Biológica , Linhagem Celular Tumoral , Humanos , Camundongos , Nanocápsulas/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although combination chemotherapy and radiotherapy have become the standard of care in numerous tumors, the mechanisms of interaction are often still unclear. The purpose of this study was to analyze the efficacy of radiation treatment and cisplatin sequences and to investigate their mechanisms of interaction. Three melanoma cell lines were used to evaluate in vitro radiation-induced cytotoxicity before and after cisplatin treatment. Expression levels of a panel of genes were determined by real-time RT-PCR. Cytotoxic effect was evaluated by flow cytometry analysis and Comet assay. We also used normal human dermal fibroblasts (HUDE) to evaluate the cytotoxicity of the two treatments by clonogenic assay. Radiation and cisplatin used singly were not particularly effective in reducing proliferation in melanoma cells. Conversely, radiation treatment followed by cisplatin showed a strong synergistic interaction in all cell lines, with a ratio index ranging from 16 to >100. The synergistic effect was accompanied by apoptosis induction (up to 40%) and an increase in the percentage of comet-shaped nucleoids from 85% to 99%. In parallel, our results also showed that radiation treatment of HUDE fibroblasts followed by cisplatin only induced weak cytotoxicity. Our findings highlight the efficacy of the sequence radiation â cisplatin in reducing cell proliferation and in inducing apoptosis in melanoma cell lines. This sequence also modulated a network of proteins involved in DNA damage repair.
Assuntos
Antineoplásicos/farmacologia , Quimiorradioterapia/métodos , Cisplatino/farmacologia , Melanoma/patologia , Neoplasias Cutâneas/patologia , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quimiorradioterapia/efeitos adversos , Cisplatino/toxicidade , Ensaio Cometa , Dano ao DNA , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Esquema de Medicação , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Concentração Inibidora 50 , Melanoma/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/genética , Fatores de TempoRESUMO
Conditions that interfere with the endoplasmic reticulum (ER) functions cause accumulation of unfolded proteins in the ER lumen, referred to as ER stress, and activate a homeostatic signaling network known as unfolded protein response (UPR). We have previously shown that in neonatal rats subjected to hypoxia-ischemia (HI), melatonin administration significantly reduces brain damage. This study assessed whether attenuation of ER stress is involved in the neuroprotective effect of melatonin after neonatal HI. We found that the UPR was strongly activated after HI. Melatonin significantly reduced the neuron splicing of XBP-1 mRNA, the increased phosphorylation of eIF2α, and elevated expression of chaperone proteins GRP78 and Hsp70 observed after HI in the brain. CHOP, which plays a convergent role in the UPR, was reduced as well. Melatonin also completely prevented the depletion of SIRT-1 induced by HI, and this effect was observed in the same neurons that over-express CHOP. These results demonstrate that melatonin reduces ER stress induced by neonatal HI and preserves SIRT-1 expression, suggesting that SIRT-1, due to its action in the modulation of a wide variety of signaling pathways involved in neuroprotection, may play a key role in the reduction of ER stress and neuroprotection observed after melatonin.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Hipóxia-Isquemia Encefálica/metabolismo , Melatonina/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Sirtuína 1/metabolismo , Animais , Animais Recém-Nascidos , RatosRESUMO
Promoting neural cell proliferation may represent an important strategy for enhancing brain repair after developmental brain injury. The present study aimed to assess the effects of melatonin on cell proliferation after an ischemic injury in the developing hippocampus, focusing on cell cycle dynamics. After in vivo neonatal hypoxia-ischemia (HI), hippocampal cell cycle dynamics were assessed by flow cytometry, together with histological evaluation of dentate gyrus cellularity and proliferation. Melatonin significantly increased the number of proliferating cells in the G2/M phase as well as the proliferating cell nuclear antigen (PCNA) and doublecortin (DCX) labeling reduced by HI. In vivo BrdU labeling revealed a higher BrdU-positivity in the dentate gyrus of ischemic rats treated with melatonin, an effect followed by increased cellularity and preserved hippocampal tissue integrity. These results indicate that the protective effect of melatonin after ischemic injury in neonatal rats may rely on the modulation of cell cycle dynamics of newborn hippocampal cells and increased cell proliferation.
Assuntos
Animais Recém-Nascidos , Ciclo Celular , Proliferação de Células , Proteína Duplacortina , Hipocampo , Melatonina , Animais , Melatonina/farmacologia , Melatonina/uso terapêutico , Hipocampo/patologia , Hipocampo/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Ratos Wistar , Hipóxia-Isquemia Encefálica/patologia , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Ratos , MasculinoRESUMO
Oxidative stress (OS) and inflammation play a key role in the development of hypoxic-ischemic (H-I) induced brain damage. Following H-I, rapid neuronal death occurs during the acute phase of inflammation, and activation of the oxidant-antioxidant system contributes to the brain damage by activated microglia. So far, in an animal model of perinatal H-I, it was showed that neuroprostanes are present in all brain damaged areas, including the cerebral cortex, hippocampus and striatum. Based on the interplay between inflammation and OS, it was demonstrated in the same model that inflammation reduced brain sirtuin-1 expression and affected the expression of specific miRNAs. Moreover, through proteomic approach, an increased expression of genes and proteins in cerebral cortex synaptosomes has been revealed after induction of neonatal H-I. Administration of melatonin in the experimental treatment of brain damage and neurodegenerative diseases has produced promising therapeutic results. Melatonin protects against OS, contributes to reduce the generation of pro-inflammatory factors and promotes tissue regeneration and repair. Starting from the above cited aspects, this educational review aims to discuss the inflammatory and OS main pathways in H-I brain injury, focusing on the role of melatonin as neuroprotectant and providing current and emerging evidence.