RESUMEN
Galectin-1 (Gal-1) and galectin-3 (Gal-3) are multifunctional glycan-binding proteins, expressed in the brain and in its limbic structures that are involved in behavioral control. Gal-1 induces the expression of the brain-derived neurotrophic factor (BDNF) and promotes adult neural stem cells proliferation, biological events impaired in stress-related psychiatric disorders, such as depression and anxiety. Despite that, there is no evidence regarding galectin involvement in emotional control during stressful situations. Thus, we analyzed the behavioral phenotype of Gal-1 or Gal-3 knock-out mice (Gal-1 KO or Gal-3 KO) in different experimental models predictive of depressive and compulsive-like behaviors. METHODS: C57BL-6 Gal-1 KO, Gal-3 KO, and wild-type mice (WT) were analyzed under the open field test (OFT) and, 6 h later, under the forced swim test (FST). Additionally, independent groups of male mice, lacking galectins or not, were exposed to the tail suspension test (TST) or to the marble burying test (MBT). The hippocampus and prefrontal cortex (PFC) of the mice submitted to MBT were dissected to access BDNF levels. RESULTS: Both Gal-1 and Gal-3 KO mice showed increased time of immobility in the FST and in the TST compared to WT animals, thus reflecting an impaired stress-coping behavior. Additionally, Gal-1 and Gal-3 KO female mice presented increased compulsive-like behavior in the MBT, without significant changes in the locomotor activity. BDNF levels were found to be decreased in the PFC of Gal-1 KO mice. DISCUSSION: Our results demonstrate that the absence of either endogenous Gal-1 and Gal-3 impairs stress-coping and increases compulsive-like behavior, suggesting that Gal-1 and Gal-3 are involved in the neurobiology of depression and obsessive-compulsive-like disorder.
Asunto(s)
Galectina 1 , Galectina 3 , Estrés Psicológico , Animales , Ansiedad , Conducta Animal , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Conducta Compulsiva , Depresión , Modelos Animales de Enfermedad , Emociones , Femenino , Galectina 1/genética , Galectina 3/genética , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Despite all the knowledge, the cellular and molecular mechanisms involved in myeloproliferative neoplasm (MPN) pathophysiology remain unclear. Authors have shown galectin-1 (Gal-1) and 3 playing roles in tumour angiogenesis and fibrosis, which were correlated with poor prognosis in patients with MPN. In the present study LGALS1 and LGALS3 were differently expressed between polycythemia vera, essential thrombocythemia (ET) and primary myelofibrosis (PMF) diseases. Increased LGALS3 expression was associated with a negative JAK2 V617F status mutation in leucocytes from PMF but not in patients with ET without this mutation. However, a positive Janus kinase 2 (JAK2) V617F cell line established from patients with ET (SET-2 cells) when treated with JAK inhibitor presented high levels of LGALS3. Additionally, high LGALS1 expression was found in CD34(+) cells but not in leucocytes from patients with PMF, in absence of JAK2 V617F mutation, and also in SET-2 cells treated with JAK inhibitor. Thus, our findings indicate that differential expression of LGALS1 and/or LGALS3 in patients with MPN is linked with JAK2 V617F status mutation in these diseases and state of cell differentiation.
Asunto(s)
Galectina 1/genética , Galectina 3/genética , Janus Quinasa 2/genética , Policitemia Vera/genética , Mielofibrosis Primaria/genética , Trombocitemia Esencial/genética , Adulto , Sustitución de Aminoácidos , Antígenos CD34/genética , Proteínas Sanguíneas , Médula Ósea/patología , Neoplasias de la Médula Ósea/diagnóstico , Neoplasias de la Médula Ósea/genética , Neoplasias de la Médula Ósea/metabolismo , Línea Celular , Galectina 1/metabolismo , Galectina 3/metabolismo , Galectinas , Regulación Neoplásica de la Expresión Génica , Humanos , Janus Quinasa 2/metabolismo , Masculino , Persona de Mediana Edad , Mutación , Trastornos Mieloproliferativos/diagnóstico , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Policitemia Vera/diagnóstico , Policitemia Vera/metabolismo , Mielofibrosis Primaria/diagnóstico , Mielofibrosis Primaria/metabolismo , Trombocitemia Esencial/diagnóstico , Trombocitemia Esencial/metabolismoRESUMEN
BACKGROUND: Previous studies have established that mycobacterial infections ameliorate allergic inflammation. However, a non-infectious approach that controls allergic responses might represent a safer and more promising strategy. The 60-65 kDa heat shock protein (Hsp) family is endowed with anti-inflammatory properties, but it is still unclear whether and how single mycobacterial Hsp control allergic disorders. OBJECTIVE: Therefore, in this study we determined whether the administration of Mycobacterial leprae Hsp65 expressed by recombinant a DNA plasmid could attenuate a previously established allergic response. METHODS: We used an experimental model of airway allergic inflammation to test the effects of immunotherapy with DNA encoding Hsp65. Allergic mice, previously sensitized and challenged with ovalbumin, were treated with tree intramuscular doses of recombinant DNA encoding Hsp65. After treatment, mice received a second allergen challenge and the allergic response was measured. RESULTS: We found that immunotherapy attenuated eosinophilia, pulmonary inflammation, Th2 cytokine and mucus production. Moreover, we showed that the inhibition of allergic response is dependent on IL-10 production. Both Hsp65 and allergen-specific IL-10-producing cells contributed to this effect. Cells transferred from DNA-immunized mice to allergic mice migrated to allergic sites and down-modulated the Th2 response. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings clearly show that immunotherapy with DNA encoding Hsp65 can attenuate an established Th2 allergic inflammation through an IL-10-dependent mechanism; moreover, the migration of allergen- and Hsp65-specific cells to the allergic sites exerts a fundamental role. This work represents a novel contribution to the understanding of immune regulation by Hsp65 in allergic diseases.
Asunto(s)
Proteínas Bacterianas , Chaperonina 60 , ADN Recombinante/administración & dosificación , Inmunoterapia/métodos , Interleucina-10/metabolismo , Hipersensibilidad Respiratoria/terapia , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Chaperonina 60/genética , Chaperonina 60/inmunología , ADN Recombinante/inmunología , Modelos Animales de Enfermedad , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Interleucina-10/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Mycobacterium leprae/inmunología , Hipersensibilidad Respiratoria/inmunología , Resultado del TratamientoRESUMEN
We demonstrate here that a mannose-binding protein from Schistosoma mansoni, termed Sm60, was recovered in the mannose-eluted fraction (Man(+)) upon affinity chromatography on immobilised mannose of the soluble antigen fraction from adult worm tegument and cercariae. Sm60 was detected in the Man(+) fraction as a prominent doublet with an apparent molecular mass of 60-66 kDa by SDS-PAGE and appeared as a single band with a pI of approximately 6.9 by isoelectrofocusing. Sm60 was also detected in preparations of schistosomula extract and soluble egg antigens using a mouse polyclonal anti-Sm60 serum on immunoblotting assay. This antiserum demonstrated that Sm60 was localised on the tegument of S. mansoni adult worm. In order to determine the role of Sm60 in host-parasite interactions, we showed that Sm60 induced in vitro migration of human neutrophil in a dose-dependent manner and in vitro mast cell degranulation. Sm60 triggered these activities through its carbohydrate-binding site, since these activities were selectively inhibited by 0.2 M D-mannose, but not by 0.2 M D-galactose. Furthermore, Sm60 induced in vivo neutrophil migration. In contrast, mast cell-depleted rats presented a significant reduction of the neutrophil migration induced by Sm60 as compared with non-depleted controls. These data suggest that in vivo neutrophil migration induced by Sm60 is modulated by mast cell-dependent mechanisms. Sm60 might play a key role in the host-parasite interaction, and its characterization opens perspective to examine the role of this molecule in the biology of S. mansoni.
Asunto(s)
Proteínas del Helminto/aislamiento & purificación , Lectina de Unión a Manosa/aislamiento & purificación , Schistosoma mansoni/química , Animales , Degranulación de la Célula/efectos de los fármacos , Quimiotaxis de Leucocito/efectos de los fármacos , Cromatografía de Afinidad , Relación Dosis-Respuesta a Droga , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas del Helminto/metabolismo , Proteínas del Helminto/farmacología , Interacciones Huésped-Parásitos , Masculino , Lectina de Unión a Manosa/metabolismo , Lectina de Unión a Manosa/farmacología , Mastocitos/fisiología , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas WistarRESUMEN
OBJECTIVE AND DESIGN: To study the neutrophil migration and aggregation induced by euphorbin, a D-galactose binding lectin from Euphorbia milii var. milli latex. MATERIALS AND METHODS: Euphorbin-induced neutrophil migration was evaluated in vivo and in vitro, in the absence or presence of soluble D-galactose. Neutrophil aggregation induced in vitro by euphorbin was determined by light microscopy. RESULTS: The neutrophil migration inducing activity of euphorbin was dose-dependent and inhibited by soluble D-galactose. Neutrophil aggregation was rapidly reversed when provoked by 0.1 mg/ml euphorbin. In higher concentrations, euphorbin caused persistent and more extensive neutrophil aggregation. CONCLUSIONS: Euphorbin induced neutrophil migration through its sugar recognition property. The transitory neutrophil aggregation, induced by a euphorbin quantity similar to that able to cause maximal chemotactic response, is characteristic of homotypic neutrophil adhesion, whereas persistent aggregation, provoked by higher euphorbin quantities, corresponds to cell agglutination by a multivalent lectin.
Asunto(s)
Euphorbiaceae/química , Lectinas/farmacología , Neutrófilos/efectos de los fármacos , Animales , Agregación Celular/efectos de los fármacos , Quimiotaxis de Leucocito/efectos de los fármacos , Galactosa/farmacología , Látex/química , Masculino , Cavidad Peritoneal/citología , Lectinas de Plantas , Ratas , Ratas WistarRESUMEN
Chemokine IL-8 attracts neutrophils by a haptotactic gradient, made possible by its interaction with proteoglycans of the extracellular matrix. Heparan sulfate, but not heparin, potentiates the attraction exerted in vitro by IL-8. In the present study we first confirmed this in vitro phenomenon, observing that IL-8 activity was potentiated 100% by heparan sulfate, but not by heparin. Then, we evaluated the interference of heparan sulfate or heparin on in vivo neutrophil migration induced by IL-8. The activity of rat IL-8 (3.5 microg/animal) preincubated with heparan sulfate (50 microg/animal) or heparin (77 microg/animal) was assayed on the rat dorsal air pouch. Contrary to in vitro experiments, heparin, but not heparan sulfate, potentiated the in vivo IL-8 activity two-fold. We investigated the relationship between this observation and that reported by others, that IL-8-induced migration depends on the presence of mast cells, which contain heparin-rich granules. We studied the neutrophil migration induced by IL-8 (3.5 microg/animal) into the rat peritoneal cavity depleted of mast cells. Neutrophil migration was reduced by 32% when compared to that observed in normal animals. The response of depleted rats was reconstituted by preincubation of IL-8 with heparin (77 microg/animal). These data suggest that heparin released from cytoplasmic granules may be the contribution of mast cells to IL-8-induced neutrophil migration.
Asunto(s)
Quimiotaxis de Leucocito/efectos de los fármacos , Heparina/farmacología , Interleucina-8/farmacología , Neutrófilos/efectos de los fármacos , Animales , Células Cultivadas , Heparitina Sulfato/farmacología , Mastocitos/citología , Neutrófilos/citología , RatasRESUMEN
KM+, a lectin purified from Artocarpusintegrifolia seeds, is an attractant for neutrophils, and has properties similar to fMLP, IL-8 and MNCF. The endogenous lectin MNCF, inhibits carrageenan-induced neutrophil migration when intravenously administered in rats. In an attempt to mimic the activity of MNCF with KM+, we determined the effect of intravenous (iv) injection of KM+ (5 microg) on neutrophil migration to the peritoneal cavity of Wistar rats induced by KM+ (50 microg, intraperitoneal, ip), fMLP (5 ng, ip) and carrageenan (300 microg, ip). Initially we evaluated the effect of the time interval between intravenous and intraperitoneal administration of KM+. The intervals ranged from 20 to 120 min and progressively stronger inhibition was observed with increasing time intervals up to a maximum of 60 min, with effect decreasing thereafter. With injections at the optimum interval of 60 min, we observed that KM+ inhibited KM+- and carrageenan-induced neutrophil migration by 72%, and fMLP-induced migration by 56%. White cell counts for Wistar rats that only received KM+iv, performed at 0 to 120 min intervals after injection, revealed early neutropenia lasting 60 min, followed by a marked increase in circulating neutrophils that reached a maximum of twice the initial levels within 90 min and after 120 min returned to levels near to that observed before intravenous administration of KM+. These results indicate that when KM+ is present in the intravascular space, it produces an inhibitory effect on neutrophil migration similar to that caused by the intravenous administration of other chemoattractants, regardless of whether they act through a mechanism independent of carbohydrate recognition, as does IL-8, or are dependent on carbohydrate recognition, like MNCF.
Asunto(s)
Quimiotaxis de Leucocito/efectos de los fármacos , Lectinas/farmacología , Neutrófilos/efectos de los fármacos , Animales , Inyecciones Intravenosas , Lectinas/administración & dosificación , Recuento de Leucocitos , Masculino , Neutrófilos/citología , Cavidad Peritoneal/citología , Ratas , Ratas WistarRESUMEN
A neutrophil migration-inducing protein has been isolated from the saline extract of Artocarpus integrifolia seeds by successive sugar affinity chromatography steps during which the protein was not absorbed by D-galactose resin, and then was absorbed to and eluted from D-mannose resin by 0.1 M D-mannose. Gel filtration on Superdex 75 HR indicated a molecular mass of 52 kDa when 0.1 M D-mannose was present in the elution buffer. A single band of apparent molecular mass of 13 kDa was demonstrable by SDS-PAGE only after heating, both in the presence and absence of reducing agent, suggesting that the molecule is a tetramer formed by the noncovalent association of 13 kDa chains. Isoelectric forms corresponding to isoelectric points of 4.0, 4.2, 5.0, and 5.2 were demonstrable by isoelectric focusing-PAGE, and four active forms having the same isoelectric points were separated by chromatofocusing. The minimal m.w. calculated from amino acid analysis data was 13,193. The protein, denoted KM+, stimulated neutrophil migration in the rat peritoneal cavity assay in a dose-related manner in the range of 1 to 300 micrograms per rat. The dose-response curve of the in vitro chemotactic activity of KM+ was bell shaped and its ascending limb was dose dependent in the range of 1 ng to 10 micrograms/well. D-Mannose (0.1 M) inhibited the in vitro (80%) and in vivo (60%) neutrophil migration-inducing activities of KM+ and also its hemmaglutinating activity. The chemotactic activity was shown to be caused by haptotaxis rather than chemokinesis. The physical and biologic properties of KM+ suggest that this lectin may attract neutrophils by a mechanism involving a haptotactic gradient as has been proposed for IL-8. KM+ might be used as tool to study protein-carbohydrate interactions during neutrophil migration through the extracellular matrix.
Asunto(s)
Proteínas Portadoras/química , Quimiotaxis de Leucocito/efectos de los fármacos , Lectinas/química , Neutrófilos/efectos de los fármacos , Fitohemaglutininas/química , Proteínas de Plantas/aislamiento & purificación , Aminoácidos/química , Animales , Proteínas Portadoras/farmacología , Lectinas/farmacología , Masculino , Lectinas de Unión a Manosa , Peso Molecular , Fitohemaglutininas/farmacología , Lectinas de Plantas , Proteínas de Plantas/farmacología , Ratas , Ratas Wistar , SemillasRESUMEN
We have previously reported that rat peritoneal macrophages stimulated with LPS release a factor (MNCF) which induces neutrophil migration that is not blocked by glucocorticoids. The supernatant of macrophage monolayers stimulated with LPS was submitted to affinity chromatography on immobilized sugar columns. We observed that the D-gal binding fraction retained MNCF activity. This fraction, consisting of four protein components, was submitted to chromatography on Superdex 75, yielding a homogeneous preparation of the active component. MNCF has a MW of 54 KDa (gel filtration and SDS-PAGE) and pI < 4.0 (isoelectrofocusing and chromatofocusing). D-gal did not interfere with the behaviour of known interleukins (IL-1 beta, IL-6, IL-8 TNF-alpha), but blocked MNCF activity in an in vitro migration assay. The present results reinforce our previous suggestion that MNCF may correspond to a novel monokine which induces neutrophil migration through a direct mechanism involving the D-gal binding site of the molecule.