RESUMO
In the global context of an imminent emergence of multidrug-resistant microorganisms, the present work combined the use of nanotechnology and the therapeutic benefits of natural compounds as a strategy to potentiate antimicrobial action of the wide-spectrum antibiotic Ofloxacin (Ofx). Hybrid solid lipid nanoparticles (SLN) were synthesized by incorporation of chitosan (Chi, a cationic biopolymer with antimicrobial activity) and eugenol (Eu, a phenolic compound that interferes with bacterial quorum sensing) into a lipid matrix by hot homogenization/ultrasonication method. The developed SLN/Chi/Eu sustainably released the encapsulated Ofx for 24â¯h. Characterization by DLS, TEM, DSC, TGA and XRD revealed the presence of positively charged spherical nanoparticles with diameters around 300â¯nm and Ofx entrapped in amorphous state. The SLN exhibited an enhanced bactericidal activity against Pseudomonas aeruginosa and Staphylococcus aureus. The minimum inhibitory concentration (MIC) for free and nanoencapsulated Ofx formulations was below 1.0⯵g/ml. The MIC values decreased by 6.1- to 16.1-fold when Ofx was encapsulated in SLN/Chi/Eu. Fluorescent-labeled nanoparticles had the ability to interact with the bacterial cell membrane. Selective toxicity of SLN/Chi/Eu-Ofx was tested in the range of 0.3-30.0⯵g/ml and showed no toxicity up to 3.0⯵g/ml Ofx in human cell models (A549 and Wi-38) at 24â¯h and 48â¯h exposure. It was proved that the administration of hybrid SLN to mice by dry powder inhalation reached therapeutic Ofx levels in lungs.
Assuntos
Anti-Infecciosos , Portadores de Fármacos , Eugenol , Nanopartículas , Ofloxacino , Células A549 , Animais , Anti-Infecciosos/administração & dosagem , Anti-Infecciosos/química , Anti-Infecciosos/farmacocinética , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Liberação Controlada de Fármacos , Eugenol/administração & dosagem , Eugenol/química , Eugenol/farmacocinética , Humanos , Lipídeos/administração & dosagem , Lipídeos/química , Lipídeos/farmacocinética , Pulmão/metabolismo , Camundongos , Nanopartículas/administração & dosagem , Nanopartículas/química , Ofloxacino/administração & dosagem , Ofloxacino/química , Ofloxacino/farmacocinética , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) represent promising alternatives for drug delivery to the central nervous system. In the present work, four different nanoformulations of the antiepileptic drug Carbamazepine (CBZ) were designed and prepared by the homogenization/ultrasonication method, with encapsulation efficiencies ranging from 82.8 to 93.8%. The formulations remained stable at 4⯰C for at least 3 months. Physicochemical and microscopic characterization were performed by photon correlation spectroscopy (PCS), transmission electron microscopy (TEM), atomic force microscopy (AFM); thermal properties by differential scanning calorimetry (DSC), thermogravimetry (TGA) and X-ray diffraction analysis (XRD). The results indicated the presence of spherical shape nanoparticles with a mean particle diameter around 160â¯nm in a narrow size distribution; the entrapped CBZ displayed an amorphous state. The in vitro release profile of CBZ fitted into a Baker-Lonsdale model for spherical matrices and almost the 100% of the encapsulated drug was released in a controlled manner during the first 24â¯h. The apparent permeability of CBZ-loaded nanoparticles through a cell monolayer model was similar to that of the free drug. In vivo experiments in a mice model of seizure suggested protection by CBZ-NLC against seizures for at least 2â¯h after intraperitoneal administration. The developed CBZ-loaded lipid nanocarriers displayed optimal characteristics of size, shape and drug release and possibly represent a promising tool to improve the treatment of refractory epilepsy linked to efflux transporters upregulation.
Assuntos
Anticonvulsivantes/química , Carbamazepina/química , Lipídeos/química , Nanopartículas/química , Nanoestruturas/química , Animais , Anticonvulsivantes/administração & dosagem , Anticonvulsivantes/farmacocinética , Varredura Diferencial de Calorimetria , Carbamazepina/administração & dosagem , Carbamazepina/farmacocinética , Cães , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos , Células Madin Darby de Rim Canino , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Nanopartículas/ultraestrutura , Nanoestruturas/ultraestrutura , Tamanho da Partícula , Termogravimetria , Difração de Raios XRESUMO
Hematopoietic cell transplant (HCT) recipients have a substantial risk of developing secondary solid cancers (SSCs). The aim of this retrospective study was to compare the incidence of SSC in a monocentric cohort of thalassemia major (TM) patients (n=122) who received HCT versus an hematopoietic cell donor monocentric cohort (n=122) and versus a large multicenter cohort of age- and sex-matched TM patients (n=244) who received conventional therapy. With a median follow-up of 24 years, 8 transplanted patients were diagnosed with SSC at a median of 18 years after HCT and at a median age of 33 years. Three patients died of cancer progression and 5 are living after a follow-up ranging from 10 months to 16 years after SSC diagnosis. The 30-year cumulative incidence of developing SSC was 13.24%. The occurrence of solid cancers in the hematopoietic cell donor cohort was limited to only one case for a significantly lower cumulative incidence (3.23%, P=0.02) and to 3 cases in the cohort of nontransplant patients for a significantly lower cumulative incidence (1.32%, P=0.005). This study shows that the magnitude of increased risk of SST is fourfold to sixfold for patients treated with HCT as compared with hematopoietic cell donors and nontransplant patients.
Assuntos
Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Segunda Neoplasia Primária/etiologia , Condicionamento Pré-Transplante/efeitos adversos , Talassemia beta/complicações , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Segunda Neoplasia Primária/patologiaRESUMO
The discovery of T regulatory cells has been one of the most important advances in basic immunology and has opened the door to the development of innovative therapeutic strategies for improving the outcome of solid organ and hematopoietic stem cell transplantation. Basic immunology is rapidly elucidating the complex biology of these cells even though the difficulties in purifying or even expanding them in vitro represent a major limitation to the development of clinical studies. The clinical benefit potentially associated with this therapeutic approach remains to be demonstrated. Meanwhile, several drugs used for the treatment of hematologic malignancies or for other purposes have been shown to upregulate the number and function of Tregs in vivo. In the near future, both ex vivo or in vivo expanded T cells are likely to enter the therapeutic armamentarium of clinical transplantation.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Linfócitos T Reguladores/metabolismo , Condicionamento Pré-Transplante/métodos , Transplante Homólogo/métodos , Animais , HumanosAssuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Receptor Notch1/genética , Alelos , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Frequência do Gene , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Notch1/metabolismoRESUMO
UNLABELLED: Adult haematopoietic stem/progenitor cells (HSPCs) constitute the lifespan reserve for the generation of all the cellular lineages in the blood. Although massive progress in identifying the cluster of master genes controlling self-renewal and multipotency has been achieved in the past decade, some aspects of the physiology of HSPCs still need to be clarified. In particular, there is growing interest in the metabolic profile of HSPCs in view of their emerging role as determinants of cell fate. Indeed, stem cells and progenitors have distinct metabolic profiles, and the transition from stem to progenitor cell corresponds to a critical metabolic change, from glycolysis to oxidative phosphorylation. In this review, we summarize evidence, reported in the literature and provided by our group, highlighting the peculiar ability of HSPCs to adapt their mitochondrial oxidative/bioenergetic metabolism to survive in the hypoxic microenvironment of the endoblastic niche and to exploit redox signalling in controlling the balance between quiescence versus active cycling and differentiation. Especial prominence is given to the interplay between hypoxia inducible factor-1, globins and NADPH oxidases in managing the mitochondrial dioxygen-related metabolism and biogenesis in HSPCs under different ambient conditions. A mechanistic model is proposed whereby 'mitochondrial differentiation' is a prerequisite in uncommitted stem cells, paving the way for growth/differentiation factor-dependent processes. Advancing the understanding of stem cell metabolism will, hopefully, help to (i) improve efforts to maintain, expand and manipulate HSPCsâ ex vivo and realize their potential therapeutic benefits in regenerative medicine; (ii) reprogramme somatic cells to generate stem cells; and (iii) eliminate, selectively, malignant stem cells. LINKED ARTICLES: This article is part of a themed section on Emerging Therapeutic Aspects in Oncology. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-8.
Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Mitocôndrias/metabolismo , Adulto , Diferenciação Celular , Linhagem da Célula , Metabolismo Energético , Glicólise , Humanos , Modelos Biológicos , Oxirredução , Fosforilação OxidativaRESUMO
Systemic sclerosis (SSc) is a chronic disease, with early activation of the immune system. The aim of our work was to address how SSc-mesenchymal stem cells (MSCs), although senescent, might preserve specific immunomodulatory abilities during SSc. MSCs were obtained from 10 SSc patients and 10 healthy controls (HC). Senescence was evaluated by assessing cell cycle, ß-galactosidase (ß-Gal) activity, p21 and p53 expression; doxorubicin was used as acute senescence stimulus to evaluate their ability to react in stressed conditions. Immunomodulatory abilities were studied co-culturing MSCs with peripheral blood mononuclear cells (PBMCs) and CD4(+) cells, in order to establish both their ability to block proliferation in mixed lymphocyte reaction and in regulatory T cells (Tregs) induction. SSc-MSC showed an increase of senescence biomarkers. Eighty per cent of MSCs were in G0-G1 phase, without significant differences between SSc and HC. SSc-MSCs showed an increased positive ß-Gal staining and higher p21 transcript level compared to HC cells. After doxorubicin, ß-Gal staining increased significantly in SSc-MSCs. On the contrary, doxorubicin abolished p21 activation and elicited p53 induction both in SSc- and HC-MSCs. Interleukin (IL)-6 and transforming growth factor (TGF)-ß-related transcripts and their protein levels were significantly higher in SSc-MSCs. The latter maintained their immunosuppressive effect on lymphocyte proliferation and induced a functionally regulatory phenotype on T cells, increasing surface expression of CD69 and restoring the regulatory function which is impaired in SSc. Increased activation of the IL-6 pathway observed in our cells might represent an adaptive mechanism to senescence, but preserving some specific cellular functions, including immunosuppression.
Assuntos
Células-Tronco Mesenquimais/imunologia , Escleroderma Sistêmico/imunologia , Linfócitos T Reguladores/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Proliferação de Células/efeitos dos fármacos , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Senescência Celular/imunologia , Técnicas de Cocultura , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Doxorrubicina/farmacologia , Humanos , Imunomodulação , Interleucina-6/genética , Interleucina-6/metabolismo , Lectinas Tipo C/metabolismo , Escleroderma Sistêmico/terapia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Proteína Supressora de Tumor p53/metabolismo , beta-Galactosidase/metabolismoRESUMO
CNS drug development is characterized by an especially high attrition rate, despite clear unmet medical needs in the field of neuro-pharmacology and significant investment in R of novel CNS drug treatments. Here, we overview the issues underlying the intrinsic difficulty of CNS drugs development, including obstacles of pharmacokinetic nature and lack of predictivity of preclinical tests. We highlight current efforts to overcome these limitations, with an emphasis on modeling opportunities towards early recognition of CNS candidates (stressing the possibilities of multi-target directed ligands or "magic shotguns") and different approaches to improve CNS bioavailability.
Assuntos
Fármacos do Sistema Nervoso Central/química , Fármacos do Sistema Nervoso Central/farmacologia , Doenças do Sistema Nervoso Central/tratamento farmacológico , Descoberta de Drogas/métodos , Animais , Sistema Nervoso Central/efeitos dos fármacos , Desenho Assistido por Computador , HumanosRESUMO
Non-Hodgkin lymphoma (NHL) has been associated with immunological defects, chronic inflammatory and autoimmune conditions. Given the link between immune dysfunction and NHL, genetic variants in toll-like receptors (TLRs) have been regarded as potential predictive factors of susceptibility to NHL. Adequate anti-tumoral responses are known to depend on TLR9 function, such that the use of its synthetic ligand is being targeted as a therapeutic strategy. We investigated the association between the functional rs5743836 polymorphism in the TLR9 promoter and risk for B-cell NHL and its major subtypes in three independent case-control association studies from Portugal (1160 controls, 797 patients), Italy (468 controls, 494 patients) and the US (972 controls, 868 patients). We found that the rs5743836 polymorphism was significantly overtransmitted in both Portuguese (odds ratio (OR), 1.85; P=7.3E-9) and Italian (OR, 1.84; P=6.0E-5) and not in the US cohort of NHL patients. Moreover, the increased transcriptional activity of TLR9 in mononuclear cells from patients harboring rs5743836 further supports a functional effect of this polymorphism on NHL susceptibility in a population-dependent manner.
Assuntos
Linfoma não Hodgkin/genética , Polimorfismo Genético , Receptor Toll-Like 9/genética , Feminino , Genética Populacional , Humanos , Linfoma não Hodgkin/epidemiologia , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
T helper (Th) 17 cells have emerged as important mediators in infectious and inflammatory diseases and, recently, in transplant rejection. We analyzed the associations between five common genetic variants in the IL-23/Th17 signaling pathway, namely in IL17A, IL17F and IL23R genes, and clinical outcome in T cell-depleted allogeneic SCT (allo-SCT). In the multivariate analysis, variants in IL23R and IL17A genes were the most important prognostic factors. Thus, patient GA genotype at rs11209026 in IL23R was associated with improved overall survival (hazard ratio (HR)=0.48; P=0.028) and, in donor, with decreased risk of fungal infections (P=0.05). In contrast, patient TC and CC genotypes at rs8193036 in IL17A gene were associated with increased risk of CMV infection (HR=3.68; P=0.011) and patient acute GVHD (HR=7.08; P=0.008), respectively. These results suggest that genetic variants in the IL-23/Th17 inflammatory pathway are important prognostic factors for the clinical outcome of allo-SCT. Although validation studies are ultimately required, our results would suggest the potential usefulness of IL-23/Th17 genotyping in donor selection and patient evaluation.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Interleucina-17/genética , Interleucina-23/genética , Depleção Linfocítica , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Criança , Intervalo Livre de Doença , Feminino , Genótipo , Humanos , Interleucina-17/metabolismo , Interleucina-23/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Linfócitos T/citologia , Adulto JovemRESUMO
Although CD4(+)/CD25(+) T regulatory cells (T(regs)) are a potentially powerful tool in bone marrow transplantation, a prerequisite for clinical use is a cell-separation strategy complying with good manufacturing practice guidelines. We isolated T(regs) from standard leukapheresis products using double-negative selection (anti-CD8 and anti-CD19 monoclonal antibodies) followed by positive selection (anti-CD25 monoclonal antibody). The final cell fraction (CD4(+)/CD25(+)) showed a mean purity of 93.6% +/- 1.1. Recovery efficiency was 81.52% +/- 7.4. The CD4(+)/CD25(+bright) cells were 28.4% +/- 6.8. The CD4(+)/CD25(+) fraction contained a mean of 51.9% +/- 15.1 FoxP3 cells and a mean of 18.9% +/- 11.5 CD127 cells. Increased FoxP3 and depleted CD127 mRNAs in CD4(+)CD25(+)FoxP3(+) cells were in line with flow cytometric results. In Vbeta spectratyping the complexity scores of CD4(+)/CD25(+) cells and CD4(+)/CD25(-) cells were not significantly different, indicating that T(regs) had a broad T cell receptor repertoire. The inhibition assay showed that CD4(+)/CD25(+) cells inhibited CD4(+)/CD25(-) cells in a dose-dependent manner (mean inhibition percentages: 72.4 +/- 8.9 [ratio of T responder (T(resp)) to T(regs), 1:2]; 60.8% +/- 20.5 (ratio of T(resp) to T(regs), 1:1); 25.6 +/- 19.6 (ratio of T(resp) to T(regs), 1:0.1)). Our study shows that negative/positive T(reg) selection, performed using the CliniMACS device and reagents, enriches significantly CD4(+)CD25(+)FoxP3(+) cells endowed with immunosuppressive capacities. The CD4(+)CD25(+)FoxP3(+) population is a source of natural T(reg) cells that are depleted of CD8(+) and CD4(+)/CD25(-) reacting clones which are potentially responsible for triggering graft-versus-host disease (GvHD). Cells isolated by means of this approach might be used in allogeneic haematopoietic cell transplantation to facilitate engraftment and reduce the incidence and severity of GvHD without abrogating the potential graft-versus-tumour effect.
Assuntos
Linfócitos T CD4-Positivos/citologia , Fatores de Transcrição Forkhead/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Adulto , Linfócitos T CD4-Positivos/imunologia , Fatores de Transcrição Forkhead/genética , Humanos , Separação Imunomagnética , Imunofenotipagem/métodos , Subunidade alfa de Receptor de Interleucina-2/análise , Subunidade alfa de Receptor de Interleucina-5/análise , Subunidade alfa de Receptor de Interleucina-7/genética , Leucaférese/métodos , Pessoa de Meia-Idade , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Cariotipagem Espectral/métodosAssuntos
Análise Mutacional de DNA/métodos , Transtornos Mieloproliferativos/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Análise Mutacional de DNA/instrumentação , Humanos , Janus Quinase 2 , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Mutação PuntualRESUMO
In this case, originally reported as primary eyelid plasmacytoma, the tumor recurred on the same eyelid within 2 years of surgery. No plasma cell infiltration was observed at bone marrow biopsy. No serum or urinary monoclonal component was detected at immunofixation. Histology and immunohistochemistry confirmed plasma cell infiltration. Tumor cell clonality was determined by immunohistological staining; cells were positive for kappa light chain like the first eyelid tumor. Surgery was followed by radiotherapy. Twenty months later, biopsy of one enlarged right cervical lymph node showed massive diffuse infiltration of atypical plasma cells (CD20(-), CD79a(+), CD138(+), MUM1/IRF4(+)). Given the rapid diffusion to lymph nodes and the appearance of the monoclonal component, the lymph node was removed surgically. No adjuvant chemotherapy was given. Unexpectedly, the serum monoclonal component normalized. No plasma cell infiltration was observed at bone marrow biopsy. As this case might be a particularly slow-progressing extra-medullary plasmacytoma, this study recommends closely monitored follow-ups so that the aggressive form can be treated in time.
Assuntos
Neoplasias Palpebrais/patologia , Recidiva Local de Neoplasia/patologia , Plasmocitoma/patologia , Adulto , Biópsia , Neoplasias Palpebrais/radioterapia , Neoplasias Palpebrais/cirurgia , Seguimentos , Humanos , Metástase Linfática , Masculino , Recidiva Local de Neoplasia/radioterapia , Recidiva Local de Neoplasia/cirurgia , Plasmocitoma/radioterapia , Plasmocitoma/cirurgia , Indução de Remissão , Resultado do TratamentoRESUMO
T cell-depleted allogeneic stem cell transplantation is associated with delayed immunological reconstitution. Bone marrow stroma and interleukin 7 (IL-7) regulate homeostasis of T lymphocytes. We engineered human stromal cells with a retroviral vector containing the IL-7 gene and studied in vitro effects on T cells. Human stromal cells were successfully transduced and generated a layer that was morphologically and phenotypically normal. IL-7-engineered stromal cells conserve the biological properties of unmanipulated stromal cells. Through their production of IL-7, they enhance survival and homeostatic proliferation of naive T cells. Because of this cytokine production, they might be an ideal vehicle for gene therapy aimed at supporting lymphopoiesis in the T cell-deficient host.