RESUMO
Macrophages play an important role in the inflammatory response. Their various biological functions are induced by different membrane receptors, including Toll-like receptors, which trigger several intracellular signaling cascades and activate the inflammasomes, which in turn elicit the release of inflammatory mediators such as cytokines. In this study, we present a novel method for the isolation of human mature peritoneal macrophages. This method can be easily implemented by gynecologists who routinely perform laparoscopy for sterilization by tubal ligation or surgically intervene in benign gynecological pathologies. Our method confirms that macrophages are the main peritoneal leukocyte subpopulation isolated from the human peritoneum in homeostasis. We showed that primary human peritoneal macrophages present phagocytic and oxidative activities, and respond to activation of the main proinflammatory pathways such as Toll-like receptors and inflammasomes, resulting in the secretion of different proinflammatory cytokines. Therefore, this method provides a useful tool for characterizing primary human macrophages as control cells for studies of molecular inflammatory pathways in steady-state conditions and for comparing them with those obtained from pathologies involving the peritoneal cavity. Furthermore, it will facilitate advances in the screening of anti-inflammatory compounds in the human system.
Assuntos
Técnicas de Cultura de Células/métodos , Citocinas/metabolismo , Inflamassomos/metabolismo , Leucócitos/metabolismo , Macrófagos Peritoneais/metabolismo , Adulto , Feminino , Citometria de Fluxo , Humanos , Imunidade Inata , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Laparoscopia , Macrófagos Peritoneais/citologia , Fagocitose , Espécies Reativas de Oxigênio , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Crohn's disease (CD) is a complex inflammatory bowel disease whose pathogenesis appears to involve several immunologic defects causing functional impairment of the gut. Its complexity and the reported loss of effectiveness over time of standard of care together with the increase in its worldwide incidence require the application of techniques aiming to find new therapeutic strategies. Currently, systems pharmacology modeling has been gaining importance as it integrates the available knowledge of the system into a single computational model. In this work, the following workflow for robust application of systems pharmacology modeling was followed: 1) scope definition; 2) species selection and circulating plasma levels based on a search in the literature; 3) representation of model topology and parametrization of the interactions, after literature data extraction and curation, and the implementation of ordinary differential equations in SimBiology (MATLAB version R2018b); and 4) model curation and evaluation by visual comparison of simulated interleukin (IL) concentrations with the reported levels in plasma, and sensitivity analysis performed to confirm model robustness and identify the most influential parameters. Finally, 5) exposure to two dose levels of recombinant human IL10 was evaluated by simulation and comparison with reported clinical study results. In summary, we present a quantitative systems pharmacology model for the main ILs involved in CD developed using a standardized methodology and supported by a comprehensive repository summarizing the most relevant literature in the field. However, it has to be taken into account that external validation is still pending as available clinical data were primarily used for model training. SIGNIFICANCE STATEMENT: Crohn's disease (CD) is a complex heterogeneous inflammatory bowel disorder. Systems pharmacology modeling offers a great opportunity for integration of the available knowledge on the disease using a computational framework. As a result of this work, a comprehensive repository along with a quantitative systems pharmacology model for the main interleukins involved in CD is provided. This model is useful for the in silico evaluation of biomarkers and potential therapeutic targets and can be adapted to address research gaps regarding CD.
Assuntos
Doença de Crohn/imunologia , Interleucinas/sangue , Modelos Biológicos , Biomarcadores/sangue , Simulação por Computador , Doença de Crohn/sangue , HumanosRESUMO
Pteridines are aromatic compounds formed by fused pyrazine and pyrimidine rings. Many living organisms synthesize pteridines, where they act as pigments, enzymatic cofactors, or immune system activation molecules. This variety of biological functions has motivated the synthesis of a huge number of pteridine derivatives with the aim of studying their therapeutic potential. This review gathers the state-of-the-art of pteridine derivatives, describing their biological activities and molecular targets. The antitumor activity of pteridine-based compounds is one of the most studied and advanced therapeutic potentials, for which several molecular targets have been identified. Nevertheless, pteridines are also considered as very promising therapeutics for the treatment of chronic inflammation-related diseases. On the other hand, many pteridine derivatives have been tested for antimicrobial activities but, although some of them resulted to be active in preliminary assays, a deeper research is needed in this area. Moreover, pteridines may be of use in the treatment of many other diseases, such as diabetes, osteoporosis, ischemia, or neurodegeneration, among others. Thus, the diversity of the biological activities shown by these compounds highlights the promising therapeutic use of pteridine derivatives. Indeed, methotrexate, pralatrexate, and triamterene are Food and Drug Administration approved pteridines, while many others are currently under study in clinical trials.
Assuntos
Pteridinas/química , Pteridinas/uso terapêutico , Aminopterina/análogos & derivados , Aminopterina/uso terapêutico , Animais , Anti-Infecciosos/uso terapêutico , Antidepressivos/uso terapêutico , Anti-Hipertensivos/uso terapêutico , Antineoplásicos/uso terapêutico , Antiparasitários/uso terapêutico , Ensaios Clínicos como Assunto , Humanos , Concentração Inibidora 50 , Metotrexato/uso terapêutico , Triantereno/uso terapêuticoRESUMO
The widespread use of drugs for unapproved purposes remains common in children, primarily attributable to practical, ethical, and financial constraints associated with pediatric drug research. Pharmacometrics, the scientific discipline that involves the application of mathematical models to understand and quantify drug effects, holds promise in advancing pediatric pharmacotherapy by expediting drug development, extending applications, and personalizing dosing. In this review, we delineate the principles of pharmacometrics, and explore its clinical applications and prospects. The fundamental aspect of any pharmacometric analysis lies in the selection of appropriate methods for quantifying pharmacokinetics and pharmacodynamics. Population pharmacokinetic modeling is a data-driven method ('top-down' approach) to approximate population-level pharmacokinetic parameters, while identifying factors contributing to inter-individual variability. Model-informed precision dosing is increasingly used to leverage population pharmacokinetic models and patient data, to formulate individualized dosing recommendations. Physiologically based pharmacokinetic models integrate physicochemical drug properties with biological parameters ('bottom-up approach'), and is particularly valuable in situations with limited clinical data, such as early drug development, assessing drug-drug interactions, or adapting dosing for patients with specific comorbidities. The effective implementation of these complex models hinges on strong collaboration between clinicians and pharmacometricians, given the pivotal role of data availability. Promising advancements aimed at improving data availability encompass innovative techniques such as opportunistic sampling, minimally invasive sampling approaches, microdialysis, and in vitro investigations. Additionally, ongoing research efforts to enhance measurement instruments for evaluating pharmacodynamics responses, including biomarkers and clinical scoring systems, are expected to significantly bolster our capacity to understand drug effects in children.
Assuntos
Medicina de Precisão , Humanos , Medicina de Precisão/métodos , Criança , Farmacocinética , Modelos Biológicos , Preparações Farmacêuticas/administração & dosagem , Relação Dose-Resposta a Droga , Desenvolvimento de Medicamentos/métodos , PediatriaRESUMO
In a recent multicenter population pharmacokinetic study of ciprofloxacin administered to children suffering from complicated urinary tract infection (cUTI), the apparent volume of distribution (V) and total plasma clearance (CL) were decreased by 83.6% and 41.5% respectively, compared to healthy children. To understand these differences, a physiologically-based pharmacokinetic model (PBPK) for ciprofloxacin was developed for cUTI children. First, a PBPK model in adults was developed, modified incorporating age-dependent functions and evaluated with paediatric data generated from a published model in healthy children. Then, the model was then adapted to a cUTI paediatric population according to the degree of renal impairment (KF) affecting renal clearance (CLRenal,) and CYP1A2 clearance (CLCYP1A2). Serum and urine samples obtained from 22 cUTI children were used for model evaluation. Lastly, a parameter sensitivity analysis identified the most influential parameters on V and CL. The PBPK model predicted the ciprofloxacin exposure in adults and children, capturing age-related pharmacokinetic changes. Plasma concentrations and fraction excreted unchanged in urine (fe) predictions improved in paediatric cUTI patients once CLrenal and CLCYP1A2 were corrected by KF. The presented PBPK model for ciprofloxacin demonstrates its adequacy to simulate different dosing scenarios to obtain PK predictions in a healthy population from 3â¯months old onwards. Model adaptation of CLRenal and CLCYP1A2 according to KF explained partially the differences seen in the plasma drug concentrations and fe vs time profiles between healthy and cUTI children. Nevertheless, it is necessary to further investigate the disease-related changes in cUTI to improve model predictions.
Assuntos
Antibacterianos/farmacocinética , Antibacterianos/uso terapêutico , Ciprofloxacina/farmacocinética , Ciprofloxacina/uso terapêutico , Modelos Biológicos , Infecções Urinárias/tratamento farmacológico , Administração Oral , Adolescente , Adulto , Criança , Pré-Escolar , Ciprofloxacina/sangue , Humanos , Injeções IntravenosasRESUMO
Pteridines are bicyclic heterocyclic compounds with a pyrazino[2,3-d]pyrimidine nucleus that have shown a wide range of therapeutic utilities. Concretely, 4-aminopteridine derivatives have demonstrated both anti-inflammatory and anti-cancer properties, and some of them, such as methotrexate, are profusely used in medical practice. We have recently synthesized and tested the biological activity of a novel series of 4-amino-2-aryl-6,9-dichlorobenzo[g]pteridines, finding that they present anti-inflammatory properties, as they were able to inhibit in vitro the production of pro-inflammatory cytokines TNF-α and IL-6. Now, we have evaluated the anti-tumor potential of these compounds on HL-60 and K562 leukemia cell lines. Cells growing at exponential rate were exposed to decreasing doses of each compound, from 50 to 0.39 µM, for 24, 48, and 72 h. Cell viability was tested by MTT assay and cell death fashion determined by annexin V/propidium iodide assay. The cytotoxicity of the compounds was determined in differentiated macrophage-like HL-60 cells and in human peripheral blood mononuclear cells to evaluate the potential side effects on quiescent tumor cells and normal cells, respectively. Among the series, compounds 1a, 1b, 1g, 1j, and 1k showed anti-proliferative activity. Compounds 1j and 1k were active against both HL-60 and K562 cells, with a lower IC50 against HL-60 cells. Compounds 1a, 1b, and 1g had a great cytotoxic activity against HL-60, but they were far less potent against K562 cells. None had side effects in differentiated tumor cells or in human peripheral blood mononuclear cells. In conclusion, our results demonstrate that some compounds of this series of 4-amino-2-aryl-6,9-dichlorobenzo[g]pteridines have anti-cancer properties in vitro.
Assuntos
Antineoplásicos/farmacologia , Leucemia/tratamento farmacológico , Pteridinas/farmacologia , Células HL-60 , Humanos , Células K562 , Leucócitos Mononucleares/efeitos dos fármacosRESUMO
BACKGROUND AND AIM: The presumed role of the inhibitory receptor LAIR-1 (CD305) in the inflammatory response suggests that it might contribute to the pathophysiology of chronic inflammatory diseases such as liver cirrhosis. We studied the LAIR-1 expression on liver macrophages and blood monocytes related to the progression of liver cirrhosis. METHODS: The expression of LAIR-1 was analyzed by immunohistochemistry, flow cytometry, and Western blot. RESULTS: We found a decreased number of macrophages expressing LAIR-1 in cirrhotic liver that could be due to a high presence of collagen, ligand of LAIR-1, in the fibrotic tissue which could downregulate its expression or interfere with the immunostaining. The expression of LAIR-1 decreased after cell differentiation, and the total content, but not the cell surface expression, increased after activation in the HL-60 human macrophage in vitro model. Blood monocytes exhibited higher LAIR-1 expression levels in cirrhotic patients, which were evident even in early clinical stages in all monocyte subsets, and greater in the "intermediate" inflammatory monocyte subpopulation. The in vitro activation of human blood monocytes did not increase its expression on the cell surface suggesting that the in vivo increase of LAIR-1 must be the result of a specific combination of stimuli present in cirrhotic patients. This represents an exclusive feature of liver cirrhosis, since blood monocytes from other chronic inflammatory pathologies showed similar or lower LAIR-1 levels compared with those of healthy controls. CONCLUSIONS: These results may indicate that monocyte LAIR-1 expression is a new biomarker to early detect liver damage caused by chronic inflammation in liver cirrhosis.
Assuntos
Progressão da Doença , Cirrose Hepática/diagnóstico , Monócitos/imunologia , Receptores Imunológicos/genética , Adulto , Idoso , Biomarcadores/análise , Diferenciação Celular , Feminino , Citometria de Fluxo , Células HL-60 , Humanos , Inflamação/diagnóstico , Inflamação/etiologia , Lipopolissacarídeos , Fígado/imunologia , Cirrose Hepática/imunologia , Macrófagos/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
There is growing concern about the limitations of rodent models with regard to recapitulation of human disease pathogenesis. Computational modeling of data from humans and animals sharing similar diseases provides an opportunity for parallel drug development in human and veterinary medicine. This "reverse translational" approach needs to be supported by continuing efforts to refine the in silico tools that allow extrapolation of results between species.
Assuntos
Modelos Animais de Doenças , Desenvolvimento de Medicamentos/métodos , Pesquisa Translacional Biomédica/métodos , Medicina Veterinária/métodos , Animais , Simulação por Computador , Humanos , Saúde Única , Roedores , Especificidade da EspécieRESUMO
Peritoneal macrophages play a critical role in the control of infectious and inflammatory diseases. Although recent progress on murine peritoneal macrophages has revealed multiple aspects on their origin and mechanisms involved in their maintenance in this compartment, little is known on the characteristics of human peritoneal macrophages in homeostasis. Here, we have studied by flow cytometry several features of human peritoneal macrophages obtained from the peritoneal cavity of healthy women. Three peritoneal monocyte/macrophage subsets were established on the basis of CD14/CD16 expression (CD14++CD16-, CD14++CD16+ and CD14highCD16high), and analysis of CD11b, CD11c, CD40, CD62L, CD64, CD80, CD86, CD116, CD119, CD206, HLA-DR and Slan was carried out in each subpopulation. Intracellular expression of GATA6 and cytokines (pro-inflammatory IL-6 and TNF-α, anti-inflammatory IL-10) as well as their phagocytic/oxidative activities were also analyzed, in an attempt to identify genuine resident peritoneal macrophages. Results showed that human peritoneal macrophages are heterogeneous regarding their phenotype, cell complexity and functional abilities. A direct relationship of CD14/CD16 expression, intracellular content of GATA6, and activation/maturation markers like CD206 and HLA-DR, support that the CD14highCD16high subset represents the mature phenotype of steady-state human resident peritoneal macrophages. Furthermore, increased expression of CD14/CD16 is also related to the phagocytic activity.
Assuntos
Citocinas/metabolismo , Fator de Transcrição GATA6/metabolismo , Homeostase , Macrófagos Peritoneais/metabolismo , Monócitos/metabolismo , Fagocitose , Adulto , Antígenos CD/metabolismo , Feminino , Humanos , Oxirredução , FenótipoRESUMO
MOTIVATION: The literature on complex diseases is abundant but not always quantitative. This is particularly so for Inflammatory Bowel Disease (IBD), where many molecular pathways are qualitatively well described but this information cannot be used in traditional quantitative mathematical models employed in drug development. We propose the elaboration and validation of a logic network for IBD able to capture the information available in the literature that will facilitate the identification/validation of therapeutic targets. RESULTS: In this article, we propose a logic model for Inflammatory Bowel Disease (IBD) which consists of 43 nodes and 298 qualitative interactions. The model presented is able to describe the pathogenic mechanisms of the disorder and qualitatively describes the characteristic chronic inflammation. A perturbation analysis performed on the IBD network indicates that the model is robust. Also, as described in clinical trials, a simulation of anti-TNFα, anti-IL2 and Granulocyte and Monocyte Apheresis showed a decrease in the Metalloproteinases node (MMPs), which means a decrease in tissue damage. In contrast, as clinical trials have demonstrated, a simulation of anti-IL17 and anti-IFNγ or IL10 overexpression therapy did not show any major change in MMPs expression, as corresponds to a failed therapy. The model proved to be a promising in silico tool for the evaluation of potential therapeutic targets, the identification of new IBD biomarkers, the integration of IBD polymorphisms to anticipate responders and non-responders and can be reduced and transformed in quantitative model/s.
Assuntos
Algoritmos , Doenças Inflamatórias Intestinais/metabolismo , Metaloproteinases da Matriz/metabolismo , Modelos Biológicos , Simulação por Computador , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Interferon gama/antagonistas & inibidores , Interferon gama/metabolismo , Interleucina-17/antagonistas & inibidores , Interleucina-17/metabolismo , Terapia de Alvo Molecular/métodos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare aggressive myeloid neoplasm which shows a high rate of central nervous system (CNS) recurrence and overall survival (OS) of <1 year. Despite this, screening for CNS involvement is not routinely performed at diagnosis and intrathecal (IT) prophylaxis is not regularly administered in BPDCN. Here, we prospectively evaluated 13 consecutive BPDCN patients for the presence of CNS involvement by flow cytometry. Despite none of the patients presented with neurological symptoms, occult CNS involvement was detected in 6/10 cases evaluated at diagnosis and 3/3 studied at relapse/progression. BPDCN patients evaluated at diagnosis received IT treatment -either CNS prophylaxis (n = 4) or active therapy (n = 6)- and all but one remain alive (median follow-up of 20 months). In contrast, all three patients assessed at relapse/progression died. The potential benefit of IT treatment administered early at diagnosis on OS and CNS recurrence-free survival of BPDCN was further confirmed in a retrospective cohort of another 23 BPDCN patients. Our results show that BPDCN patients studied at diagnosis frequently display occult CNS involvement; moreover, they also indicate that treatment of occult CNS disease might lead to a dramatically improved outcome of BPDCN.
Assuntos
Neoplasias do Sistema Nervoso Central/secundário , Neoplasias do Sistema Nervoso Central/terapia , Sistema Nervoso Central/patologia , Células Dendríticas/patologia , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/terapia , Adolescente , Adulto , Idoso , Criança , Feminino , Neoplasias Hematológicas/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Estudos Prospectivos , Estudos Retrospectivos , Resultado do Tratamento , Adulto JovemRESUMO
Objetivo: Determinar la frecuencia de β-lactamasas de espectro extendido (BLEE) en Escherichia coli y Klebsiella pneumoniae y la frecuencia de CTX-M en las productoras de BLEE en el Instituto Nacional de Salud del Niño - Breña (INSN-B). Material y métodos: Se analizaron enterobacterias productoras de BLEE del INSN-B entre los meses de agosto de 2012 y enero del 2013. Se incluyeron 724 aislamientos de Escherichia coli y 181 aislamientos de Klebsiella pneumoniae, consecutivos no repetidos, de pacientes hospitalizados y de la comunidad. La identificación se realizó por bioquímica convencional. la detección fenotípica de BLEE se hizo por el método de Jarlier y la detección genotÃpica de CTX-M mediante reacción en cadena de la polimerasa (PCR). Resultados: 281 (31%) de los aislamientos de ambas enterobacterias fueron productoras de BLEE; 207/724 (28,6%) E. coli y 74/181 (40,9%) K. pneumoniae. Se detectó el gen bla en 256 de los aislamientos productores de BLEE (91,1%). Conclusiones: Las BLEE de tipo CTX-M están presentes en nuestra institución, a pesar que nuestros datos representan una sola institución, brinda parte del panorama nacional sobre la resistencia a los antimicrobianos; por lo tanto, el enfoque de epidemiológico molecular es importante para desarrollar más y mejores estrategias de control y manejo de estos patógenos en nuestro país.
Objective: To determine the frequency of extended spectrum β-lactamases (ESBL) in clinical infections caused by Escherichia coli y Klebsiella pneumoniae and the frequency of CTX-M among them at the Instituto Nacional de Salud del Niño-Breña, Lima, Peru. Methods: ESBL producing strains of E. coli and K. pneumoniae collected from August 2012 and January 2013 were analyzed; a total of 724 E. coli and 181 K. pneumoniae consecutive, non- repeated isolates from community and hospital acquired infections were included. Identification was performed by conventional biochemistry, ESBL phenotype was detected following the Jarlier´s method and PCR was used to detect CTX-M. Results: overall prevalence of ESBL was 31% (281 strains); 207/724 (28.6%) E. coli and 74/181 (40.9%) K. pneumoniae. The bla gene was detected in 256 of ESBL producing strains (91.1%). Conclusions: The CTX-M phenotype of ESBL producing strains is present in our institution. Despite of showing information of a single institution, these data bring a glance of what the antimicrobial resistance pattern may be at a national level and underscores the utility of molecular biology in designing preventing measures.