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1.
Int J Biol Macromol ; 82: 22-30, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26433176

RESUMO

Recent advances in glycobiology have revealed the essential role of lectins in deciphering the glycocodes at the cell surface to generate important biological signaling responses. ArtinM, a d-mannose-binding lectin isolated from the seeds of jackfruit (Artocarpus heterophyllus), is composed of 16 kDa subunits that are associated to form a homotetramer. Native ArtinM (n-ArtinM) exerts immunomodulatory and regenerative effects, but the potential pharmaceutical applicability of the lectin is highly limited by the fact that its production is expensive, laborious, and impossible to be scaled up. This led us to characterize a recombinant form of the lectin obtained by expression in Saccharomyces cerevisiae (y-ArtinM). In the present study, we demonstrated that y-ArtinM is similar to n-ArtinM in subunit arrangement, oligomerization and carbohydrate binding specificity. We showed that y-ArtinM can exert n-ArtinM biological activities such as erythrocyte agglutination, stimulation of neutrophil migration and degranulation, mast cell degranulation, and induction of interleukin-12 and interleukin-10 production by macrophages. In summary, the expression of ArtinM in yeast resulted in successful production of an active, recombinant form of ArtinM that is potentially useful for pharmaceutical application.


Assuntos
Carboidratos/química , Lectinas de Ligação a Manose/química , Estrutura Molecular , Proteínas Recombinantes , Animais , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Citocinas/biossíntese , Hemaglutinação , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Lectinas de Ligação a Manose/metabolismo , Lectinas de Ligação a Manose/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Polissacarídeos/química , Ligação Proteica , Receptor 2 Toll-Like , Leveduras/genética , Leveduras/metabolismo
2.
Vaccine ; 31(41): 4528-35, 2013 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-23933366

RESUMO

Virulent strains of Rhodococcus equi have a large plasmid of 80-90kb, which encodes several virulence-associated proteins (Vap), including VapA, a lipoprotein highly associated with disease. We have previously demonstrated that oral immunisation with attenuated Salmonella enterica Typhimurium strain expressing the antigen VapA (STM VapA+) induces specific and long-term humoral and cellular immunity against R. equi. It was shown that VapA activates Toll-like receptor 2 (TLR2) on macrophages by establishing an interaction that ultimately favours immunity against R. equi infection. The purpose of this study was to evaluate the immune response triggered by nasal immunisation with STM VapA+ and to determine whether TLR2 supports the vaccine effect. We developed an optimised protocol for a single nasal immunisation that conferred protection against R. equi infection in mice, which was manifested by efficient R. equi clearance in challenged animals. Nasal vaccination with STM VapA+ has also induced protection in Tlr2(-/-) mice and mice with non-functional TLR4. Moreover, spleen cells of vaccinated mice augmented T-bet expression, as well as the production of IL-12, IFN-γ, nitric oxide and hydrogen peroxide. Notably, the population of CD4(+) T cells with memory phenotype significantly increased in the spleens of vaccinated mice challenged 1 or 5 months after immunisation. In these animals, the spleen bacterial burden was also reduced. When similar experimental procedures were performed in TLR2 knockout mice, an increase in CD4(+) T cells with memory phenotype was not observed. Consequently, we conclude that nasal vaccination with attenuated Salmonella expressing the R. equi virulence factor VapA confers long-lasting protection against experimental rhodoccocosis and that TLR2 engagement was not crucial to induce this protection but may be required for a long-term immune response.


Assuntos
Infecções por Actinomycetales/prevenção & controle , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Portadores de Fármacos , Rhodococcus equi/imunologia , Salmonella typhimurium/genética , Receptor 2 Toll-Like/imunologia , Infecções por Actinomycetales/imunologia , Administração Intranasal , Animais , Carga Bacteriana , Proteínas de Bactérias/genética , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Feminino , Vetores Genéticos , Memória Imunológica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Rhodococcus equi/genética , Baço/imunologia , Baço/microbiologia , Linfócitos T/imunologia , Receptor 2 Toll-Like/deficiência , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/imunologia
3.
Microbiol Immunol ; 56(8): 513-22, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22671942

RESUMO

Human infections with EHEC such as O157:H7 have been a great concern for worldwide food-industry surveillance. This pathogen is commonly associated with bloody diarrhea that can evolve to the life-threatening hemolytic uremic syndrome. Animals are the natural reservoir where this pathogen remains asymptomatically, in steps of ingestion and colonization of the bowel. The bacterium is shed in the feces, contaminating the surroundings, including water and food that are directed for human consumption. A major player in this colonization process is intimin, an outer membrane adhesion molecule encoded by the E. coli attachment and effacement (eae) gene that has been shown to be essential for intimate bacterial attachment to eukaryotic host cells. In an attempt to reduce the colonization of animal reservoirs with EHEC O157:H7, we designed a vaccine model to induce an immune response against intimin gamma. The model is based on its recombinant expression in attenuated Salmonella, used as a suitable vaccine vector because of its recognized ability to deliver recombinant antigens and to elicit all forms of immunity: mucosal, systemic, and humoral responses. To test this model, mice were orally immunized with a S. enterica serovar Typhimurium strain carrying the pYA3137eaeA vector, and challenged with E. coli O157:H7. Here we show that immunization induced the production of high levels of specific IgG and IgA antibodies and promoted reduction in the fecal shedding of EHEC after challenge. The live recombinant vaccine reported herein may contribute to the efforts of reducing animal intestinal mucosa colonization.


Assuntos
Adesinas Bacterianas/imunologia , Anticorpos Antibacterianos/imunologia , Escherichia coli O157/imunologia , Proteínas de Escherichia coli/imunologia , Vacinas contra Escherichia coli/imunologia , Imunidade nas Mucosas , Adesinas Bacterianas/genética , Administração Oral , Animais , Anticorpos Antibacterianos/análise , Anticorpos Antibacterianos/sangue , Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Vacinas contra Escherichia coli/administração & dosagem , Feminino , Vetores Genéticos , Imunoglobulina A/análise , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina G/análise , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Salmonella typhimurium/genética , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia
4.
PLoS One ; 6(11): e27892, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22132163

RESUMO

ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus (jackfruit), interacts with N-glycosylated receptors on the surface of several cells of hematopoietic origin, triggering cell migration, degranulation, and cytokine release. Because malignant transformation is often associated with altered expression of cell surface glycans, we evaluated the interaction of ArtinM with human myelocytic leukemia cells and investigated cellular responses to lectin binding. The intensity of ArtinM binding varied across 3 leukemia cell lines: NB4>K562>U937. The binding, which was directly related to cell growth suppression, was inhibited in the presence of Manα1-3(Manα1-6)Manß1, and was reverted in underglycosylated NB4 cells. ArtinM interaction with NB4 cells induced cell death (IC(50) = 10 µg/mL), as indicated by cell surface exposure of phosphatidylserine and disruption of mitochondrial membrane potential unassociated with caspase activation or DNA fragmentation. Moreover, ArtinM treatment of NB4 cells strongly induced reactive oxygen species generation and autophagy, as indicated by the detection of acidic vesicular organelles in the treated cells. NB4 cell death was attributed to ArtinM recognition of the trimannosyl core of N-glycans containing a ß1,6-GlcNAc branch linked to α1,6-mannose. This modification correlated with higher levels of N-acetylglucosaminyltransferase V transcripts in NB4 cells than in K562 or U937 cells. Our results provide new insights into the potential of N-glycans containing a ß1,6-GlcNAc branch linked to α1,6-mannose as a novel target for anti-leukemia treatment.


Assuntos
Leucemia Mieloide/patologia , Lectinas de Ligação a Manose/metabolismo , Polissacarídeos/metabolismo , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Leucemia Mieloide/enzimologia , Lectinas de Ligação a Manose/farmacologia , Modelos Biológicos , Espécies Reativas de Oxigênio/metabolismo
5.
Vet Immunol Immunopathol ; 140(3-4): 317-22, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21281969

RESUMO

Macrophages are fundamental cells of the innate immune system, which, through phagocytosis and nitric oxide production, eliminate pathogens. The aim of the present study was to determine if macrophages from chicken families divergently selected to high and low antibodies response differ in nitric oxide production and phagocytic capacity. Blood monocytes derived macrophages were activated with lipopolysaccharide and supernatant from chicken spleen lymphocytes cultured with Concanavalin A (containing chicken interferon). Nitric oxide production was evaluated in culture supernatants. Phagocytic capacity of activated and non-activated macrophages was assayed using yeasts and IgY opsonized sheep red blood cells. Activated and non-activated macrophages from the high antibodies response family produced higher nitric oxide levels, internalized more yeast and significantly more opsonized sheep red blood cells than macrophages from the low antibodies response family. Moreover, activated macrophages became more elongated and widely spread. These findings indicate that macrophages from the high antibodies response family were more active suggesting that the differences in antibody response also depend on macrophage function.


Assuntos
Galinhas/imunologia , Galinhas/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Animais , Formação de Anticorpos , Forma Celular , Células Cultivadas , Imunidade Inata , Ativação de Macrófagos , Macrófagos/citologia , Óxido Nítrico/metabolismo , Fagocitose
6.
Protein Expr Purif ; 59(2): 197-202, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18358740

RESUMO

Dps, found in many eubacterial and archaebacterial species, appears to protect cells from oxidative stress and/or nutrient-limited environment. Dps has been shown to accumulate during the stationary phase, to bind to DNA non-specifically, and to form a crystalline structure that compacts and protects the chromosome. Our previous results have indicated that Dps is glycosylated at least for a certain period of the bacterial cell physiology and this glycosylation is thought to be orchestrated by some factors not yet understood, explaining our difficulties in standardizing the Dps purification process. In the present work, the open reading frame of the dps gene, together with all the upstream regulatory elements, were cloned into a PCR cloning vector. As a result, the expression of dps was also controlled by the plasmid system introduced in the bacterial cell. The gene was then over-expressed regardless of the growth phase of the culture and a glycosylated fraction was purified to homogeneity by lectin-immobilized chromatography assay. Unlike the high level expression of Dps in Salmonella cells, less than 1% of the recombinant protein was purified by affinity chromatography using jacalin column. Sequencing and mass spectrometry data confirmed the identity of the dps gene and the protein, respectively. In spite of the low level of purification of the jacalin-binding Dps, this work shall aid further investigations into the mechanism of Dps glycosylation.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Salmonella typhimurium/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Vetores Genéticos/genética , Glicosilação , Salmonella typhimurium/genética
7.
Fertil Steril ; 86(3): 758-61, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16831436

RESUMO

The present study describes the temporal expression of complement regulatory molecules membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, and CD59 in the human endometrium throughout the normal menstrual cycle and in patients submitted to ovarian hyperstimulation. During its proliferative phase, the endometrium expresses MCP, with increased expression during the secretory phase. Phase-dependent expression also was observed for DAF and CD59, mainly in the secretory phase. Expression of CR1 was not detected. These results suggest the presence of complement system activity during the menstrual cycle, with greater expression of regulatory molecules during the secretory phase to protect the epithelial integrity of human endometrium.


Assuntos
Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Proteínas do Sistema Complemento/metabolismo , Proteína Cofatora de Membrana/metabolismo , Ciclo Menstrual/metabolismo , Indução da Ovulação , Receptores de Complemento 3b/metabolismo , Adulto , Feminino , Expressão Gênica , Humanos , Ovulação/metabolismo
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