RESUMEN
Galectins are multifunctional glycan-binding proteins present in various tissues that participate in multiple physiological and pathological processes and are considered as not only biomarkers of human diseases but also molecular targets for treating cancer and inflammatory illnesses in many organs. In the glycobiology field, it is crucial to determine the pattern of galectin expression and location in cells and tissues. Confocal microscopy is a powerful imaging technology that represents a unique approach to investigate the expression and location of biomolecules in various tissues and cells. The confocal microscope acquires images of the specimen through the reflected or fluorescent light from the objective's focal plane, using laser light focused on a small spot inside the tissue or cell. This technique provides high-resolution and high-contrast images without artifacts generated by conventional microscopy and enables reconstruction of virtual tridimensional images by acquiring multiple sections from several focal planes, which makes it possible to obtain the precise spatial location of any cellular structure or molecule. Furthermore, confocal microscopy is a non-invasive tissue imaging strategy used in clinical practices. We describe herein the immunofluorescence confocal method for examining galectins in frozen tissue sections and mammalian cell culture.
Asunto(s)
Galectinas , Pruebas Inmunológicas , Animales , Técnicas de Cultivo de Célula , Técnica del Anticuerpo Fluorescente , Humanos , Mamíferos , Microscopía Confocal/métodosRESUMEN
Reactive oxygen species (ROS) have been extensively studied in biology in the past years. This class of molecules can be derived from endogenous sources (e.g., phagocytic cells as neutrophils, eosinophils, monocytes, macrophages, and organelles as mitochondria and peroxisomes) and participate in physiological and pathological conditions. The beneficial and harmful effects of ROS depend on redox regulation, which establishes the balance between their production and the activity of antioxidant systems to prevent oxidative stress in vivo. Neutrophils are the immune effectors most well depicted with an intense oxidative burst in response to tissue inflammation. Several proteins and members of the galectin family are involved in this fine modulation of ROS production by neutrophils. Interestingly, studies have indicated that Galectin-1 (Gal-1) can up- or downregulate ROS production by neutrophils even when exposed to N-formyl-Met-Leu-Phe (fMLP) or Phorbol Myristate Acetate (PMA), both of which are potent neutrophil stimulants that trigger high levels of ROS production. Similarly, Galectin-3 (Gal-3) induces ROS in neutrophils from a sterile or nonsterile inflammatory environment, possibly creating a negative loop that could control ROS production. Besides, superoxide production is also induced by Galectin-8 (Gal-8) and Galectin-9 (Gal-9) in neutrophils but in a different manner. We describe herein the luminol and lucigenin-dependent chemiluminescence technique by using a luminometer as a method of assessment to measure ROS production by human neutrophils isolated and exposed to purified human recombinant Gal-1. The protocol described herein could be applied for the investigation of the role of other galectins in the modulation of ROS production by neutrophils.
Asunto(s)
Galectinas , Neutrófilos , Especies Reactivas de Oxígeno , Galectinas/genética , Galectinas/metabolismo , Galectinas/farmacología , Humanos , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/metabolismo , Estallido Respiratorio , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Skeletal muscle has the intrinsic ability to self-repair through a multifactorial process, but many aspects of its cellular and molecular mechanisms are not fully understood. There is increasing evidence that some members of the mammalian ß-galactoside-binding protein family (galectins) are involved in the muscular repair process (MRP), including galectin-3 (Gal-3). However, there are many questions about the role of this protein on muscle self-repair. Here, we demonstrate that endogenous Gal-3 is required for: (i) muscle repair in vivo by using a chloride-barium myolesion mouse model and (ii) mouse primary myoblasts myogenic programming. Injured muscle from Gal-3 knockout mice (GAL3KO) showed persistent inflammation associated with compromised muscle repair and the formation of fibrotic tissue on the lesion site. In GAL3KO mice, osteopontin expression remained high even after 7 and 14 d of the myolesion, while Myoblast differentiation transcription factor (MyoD) and myogenin had decreased their expression. In GAL3KO mouse primary myoblast cell culture, Paired Box 7 (Pax7) detection seems to sustain even when cells are stimulated to differentiation and MyoD expression is drastically reduced. The detection and temporal expression levels of these transcriptional factors appear to be altered in Gal-3-deficient myoblast. Gal-3 expression in wild-type mice for GAL3KO states, both in vivo and in vitro, in sarcoplasm/cytoplasm and myonuclei; as differentiation proceeds, Gal-3 expression is drastically reduced, and its location is confined to the sarcolemma/plasma cell membrane. We also observed a change in the temporal-spatial profile of Gal-3 expression and muscle transcription factors levels during the myolesion. Overall, these results demonstrate that endogenous Gal-3 is required for the skeletal muscle repair process.
Asunto(s)
Galectina 3/metabolismo , Músculo Esquelético/metabolismo , Animales , Compuestos de Bario/administración & dosificación , Compuestos de Bario/farmacología , Cloruros/administración & dosificación , Cloruros/farmacología , Galectina 3/deficiencia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patologíaRESUMEN
Dystroglycanopathies are diseases characterized by progressive muscular degeneration and impairment of patient's quality of life. They are associated with altered glycosylation of the dystrophin-glycoprotein (DGC) complex components, such as α-dystroglycan (α-DG), fundamental in the structural and functional stability of the muscle fiber. The diagnosis of dystroglycanopathies is currently based on the observation of clinical manifestations, muscle biopsies and enzymatic measures, and the available monoclonal antibodies are not specific for the dystrophic hypoglycosylated muscle condition. Thus, modified α-DG mucins have been considered potential targets for the development of new diagnostic strategies toward these diseases. In this context, this work describes the synthesis of the hypoglycosylated α-DG mimetic glycopeptide NHAc-Gly-Pro-Thr-Val-Thr[αMan]-Ile-Arg-Gly-BSA (1) as a potential tool for the development of novel antibodies applicable to dystroglycanopathies diagnosis. Glycopeptide 1 was used for the development of polyclonal antibodies and recombinant monoclonal antibodies by Phage Display technology. Accordingly, polyclonal antibodies were reactive to glycopeptide 1, which enables the application of anti-glycopeptide 1 antibodies in immune reactive assays targeting hypoglycosylated α-DG. Regarding monoclonal antibodies, for the first time variable heavy (VH) and variable light (VL) immunoglobulin domains were selected by Phage Display, identified by NGS and described by in silico analysis. The best-characterized VH and VL domains were cloned, expressed in E. coli Shuffle T7 cells, and used to construct a single chain fragment variable that recognized the Glycopeptide 1 (GpαDG1 scFv). Molecular modelling of glycopeptide 1 and GpαDG1 scFv suggested that their interaction occurs through hydrogen bonds and hydrophobic contacts involving amino acids from scFv (I51, Y33, S229, Y235, and P233) and R8 and α-mannose from Glycopeptide 1.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Distroglicanos/inmunología , Glicoproteínas/inmunología , Mucinas/inmunología , Síndrome de Walker-Warburg/diagnóstico , Distroglicanos/química , Glicoproteínas/síntesis química , Humanos , Mucinas/químicaRESUMEN
Human Leucocyte Antigen-G (HLA-G) is a non classical major histocompatibility complex (MHC) molecule that through RNA splicing can encode seven isoforms which are membrane bound (-G1, -G2, -G3 and -G4) and soluble (-G5, -G6 and -G7). HLA-G is described as important immune suppressor endogenous molecule to favor maternal-fetal tolerance, transplant survival and tumor immune scape. HLA-G shows low protein variability and a unique structural complexity that is related with the expression of different isoforms followed by biochemical processes, such as, proteolytic cleavage, molecular interactions, and protein ubiquitination. Studies with HLA-G have shown difficult to assess the role of the individual isoforms. Thus, the aim of this work was to obtain a HLA-G6 recombinant form. The results indicated the production of high homogeneous preparations of soluble recombinant HLA-G6 (srHLA-G6) with molecular mass 23,603.76 Da, determined by MALD-TOF/TOF. In addition, native and denatured srHLA-G6 were detected by ELISA, using commercial monoclonal antibodies. Finally, we developed a suitable methodology to express srHLA-G6 that could contribute in structural and functional studies involving specific isoforms.
Asunto(s)
Antígenos HLA-G/química , Antígenos HLA-G/inmunología , Proteínas Recombinantes/química , Sitios de Unión , Humanos , Peso Molecular , Unión Proteica , SolubilidadRESUMEN
Histoplasma capsulatum is a dimorphic fungus that develops a yeast-like morphology in host's tissue, responsible for the pulmonary disease histoplasmosis. The recent increase in the incidence of histoplasmosis in immunocompromised patients highlights the need of understanding immunological controls of fungal infections. Here, we describe our discovery of the role of endogenous galectin-1 (Gal-1) in the immune pathophysiology of experimental histoplasmosis. All infected wild-type (WT) mice survived while only 1/3 of Lgals1-/- mice genetically deficient in Gal-1 survived 30 days after infection. Although infected Lgals1-/- mice had increased proinflammatory cytokines, nitric oxide (NO), and elevations in neutrophil pulmonary infiltration, they presented higher fungal load in lungs and spleen. Infected lung and infected macrophages from Lgals1-/- mice exhibited elevated levels of prostaglandin E2 (PGE2, a prostanoid regulator of macrophage activation) and prostaglandin E synthase 2 (Ptgs2) mRNA. Gal-1 did not bind to cell surface of yeast phase of H. capsulatum, in vitro, suggesting that Gal-1 contributed to phagocytes response to infection rather than directly killing the yeast. The data provides the first demonstration of endogenous Gal-1 in the protective immune response against H. capsulatum associated with NO and PGE2 as an important lipid mediator in the pathogenesis of histoplasmosis.
Asunto(s)
Citocinas/metabolismo , Dinoprostona/metabolismo , Galectina 1/metabolismo , Histoplasma/patogenicidad , Óxido Nítrico/metabolismo , Animales , Citometría de Flujo , Galectina 1/genética , Histoplasmosis/metabolismo , Histoplasmosis/microbiología , Humanos , Pulmón/metabolismo , Pulmón/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Galectin-3 is associated with the development and malignancy of several types of tumor, mediating important tumor-related functions, such as tumorigenesis, neoplastic transformation, tumor cell survival, angiogenesis, tumor metastasis and regulation of apoptosis. Therefore, synthetic galectin-3 inhibitors are of utmost importance for development of new antitumor therapeutic strategies. In this review we present an updated selection of synthetic glycoconjugates inhibitors of tumor-related galectin-3, properly addressed as monosaccharide- and disaccharide-based inhibitors, and multivalent-based inhibitors, disclosuring relevant methods for their synthesis along with their inhibitory activities towards galectin-3. In general, Cu(I)-assisted 1,3-dipolar azide-alkyne cycloaddition (CuAAC) reactions were predominantly applied for the synthesis of the described inhibitors, which had their inhibitory activities against galectin-3 evaluated by fluorescence polarization, surface plasmon resonance (SPR), hemagglutination, ELISA and cell imaging assays. Overall, the presented synthetic glycoconjugates represent frontline galectin-3 inhibitors, finding important biomedical applications in cancer.
Asunto(s)
Galectina 3/antagonistas & inhibidores , Glicoconjugados , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Animales , Proteínas Sanguíneas , Galectina 3/metabolismo , Galectinas , Glicoconjugados/síntesis química , Glicoconjugados/química , Glicoconjugados/uso terapéutico , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismoRESUMEN
Confocal microscopy provides a unique modality to examine the expression and localization of biomolecules in a variety of settings. Using this technique, an image is acquired from the focal plane of the objective using focused laser light, making it possible to work within the resolution limit of the optical system. In addition, by acquiring multiple images from a variety of focal planes, stacked series of images can provide clear spatial localization of a probed structure or protein. We describe herein the immunofluorescence methods for galectin staining in frozen sections of tissue for galectin localization using confocal microscopy.
Asunto(s)
Galectinas/metabolismo , Microscopía Confocal/métodos , Técnica del Anticuerpo Fluorescente , Galectinas/análisis , Vidrio/química , Transporte de ProteínasRESUMEN
5-Lipoxygenase-derived products have been implicated in both the inhibition and promotion of chronic infection. Here, we sought to investigate the roles of endogenous 5-lipoxygenase products and exogenous leukotrienes during Histoplasma capsulatum infection in vivo and in vitro. 5-LO deficiency led to increased lung CFU, decreased nitric oxide production and a deficient primary immune response during active fungal infection. Moreover, H. capsulatum-infected 5-LO(-/-) mice showed an intense influx of neutrophils and an impaired ability to generate and recruit effector T cells to the lung. The fungal susceptibility of 5-LO(-/-) mice correlated with a lower rate of macrophage ingestion of IgG-H. capsulatum relative to WT macrophages. Conversely, exogenous LTB4 and LTC4 restored macrophage phagocytosis in 5-LO deficient mice. Our results demonstrate that leukotrienes are required to control chronic fungal infection by amplifying both the innate and adaptive immune response during histoplasmosis.
Asunto(s)
Araquidonato 5-Lipooxigenasa/fisiología , Histoplasma/inmunología , Histoplasmosis/inmunología , Pulmón/inmunología , Macrófagos Peritoneales/inmunología , Linfocitos T/inmunología , Animales , Proliferación Celular , Células Cultivadas , Citotoxicidad Inmunológica , Citometría de Flujo , Histoplasmosis/microbiología , Histoplasmosis/mortalidad , Inmunidad Humoral , Inmunidad Innata , Leucotrieno B4/metabolismo , Leucotrieno C4/metabolismo , Pulmón/microbiología , Activación de Linfocitos , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/microbiología , Ratones , Ratones Noqueados , Óxido Nítrico/metabolismo , Fagocitosis , Tasa de Supervivencia , Linfocitos T/microbiologíaRESUMEN
Myofiber degeneration, inflammation, and fibrosis are remarkable features of Duchenne muscular dystrophy. We hypothesized that the administration of imatinib mesylate, an inhibitor of tyrosine kinase and TGF-beta pro-fibrogenic activity, could improve the muscular conditions in mdx mice. Four-week old mdx mice were treated and exercised for 6 weeks. Gastrocnemius and diaphragm histopathology, strength, creatine kinase, and cytokine levels were evaluated. The treated group presented increased muscular strength and decreased CK levels, injured myofibers, and inflammatory infiltrates. Pro-inflammatory cytokines and TGF-beta were also reduced, while IL-10 was increased, suggesting an immunomodulatory effect of imatinib, which can ameliorate the dystrophic phenotype in mdx mice.