RESUMO
We investigated whether intranasal immunization with amoebic lysates plus cholera toxin modified the populations of T and B lymphocytes, macrophages and dendritic cells by flow cytometry from nose-associated lymphoid tissue (NALT), cervical lymph nodes (CN), nasal passages (NP) and spleen (SP). In all immunized groups, the percentage of CD4 was higher than CD8 cells. CD45 was increased in B cells from mice immunized. We observed IgA antibody-forming cell (IgA-AFC) response, mainly in NALT and NP. Macrophages from NP and CN expressed the highest levels of CD80 and CD86 in N. fowleri lysates with either CT or CT alone immunized mice, whereas dendritic cells expressed high levels of CD80 and CD86 in all compartment from immunized mice. These were lower than those expressed by macrophages. Only in SP from CT-immunized mice, these costimulatory molecules were increased. These results suggest that N. fowleri and CT antigens are taking by APCs, and therefore, protective immunity depends on interactions between APCs and T cells from NP and CN. Consequently, CD4 cells stimulate the differentiation from B lymphocytes to AFC IgA-positive; antibody that we previously found interacting with trophozoites in the nasal lumen avoiding the N. fowleri attachment to nasal epithelium.
Assuntos
Administração Intranasal , Antígenos de Protozoários/administração & dosagem , Naegleria fowleri/fisiologia , Mucosa Nasal/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos de Protozoários/imunologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Toxina da Cólera/administração & dosagem , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Naegleria fowleri/crescimento & desenvolvimento , Naegleria fowleri/imunologia , Mucosa Nasal/citologiaRESUMO
The aim of this study was to evaluate the in vitro lethal effect of a methanolic extract (ME) from Caesalpinia coriaria fruits against Haemonchus contortus eggs and infective larvae. The anthelmintic activity was assessed using the egg hatching inhibition assay (EHI) and the mortality test. The ME was assessed using five concentrations as follows: 6.15, 3.12, 1.56, and 0.78 mg/mL to eggs and 150, 100, 75, and 50 mg/mL to larvae, respectively. Ivermectin (5 mg/mL) was used as positive control and 4% methanol and distilled water were used as negative controls. The data of ovicidal and larvicidal effect were analyzed with a completely randomized design through ANOVA analysis using the general linear model (GLM) and lethal concentrations (LC50 and LC90) were estimated through a Probit analysis using the SAS program. A clear ME increased concentration dependence effect was observed in the EHI and mortality tests. The highest activity of the methanolic extract was observed at the highest concentration (P < 0.05) to obtain a similar effect to the positive control (ivermectin), with LC50 = 78.38 and 0.00064 mg/mL and LC90 =235.63 and 0.024 mg/mL, respectively, for larvae and eggs. The results indicate that the C. coriaria fruit ME possesses in vitro ovicidal and larvicidal properties (gallotannins: methyl gallate) against H. contortus that needs to be investigated more in vivo for the control of gastroenteric nematodes in ruminants.
Assuntos
Antinematódeos/farmacologia , Caesalpinia/química , Frutas/química , Hemoncose/tratamento farmacológico , Haemonchus/crescimento & desenvolvimento , Metanol/química , Extratos Vegetais/farmacologia , Animais , Antinematódeos/química , Larva , Extratos Vegetais/química , Zigoto/crescimento & desenvolvimentoRESUMO
Naegleria fowleri infects humans through the nasal mucosa causing a disease in the central nervous system known as primary amoebic meningoencephalitis (PAM). Polymorphonuclear cells (PMNs) play a critical role in the early phase of N. fowleri infection. Recently, a new biological defence mechanism called neutrophil extracellular traps (NETs) has been attracting attention. NETs are composed of nuclear DNA combined with histones and antibacterial proteins, and these structures are released from the cell to direct its antimicrobial attack. In this work, we evaluate the capacity of N. fowleri to induce the liberation of NETs by human PMN cells. Neutrophils were cocultured with unopsonized or IgG-opsonized N. fowleri trophozoites. DNA, histone, myeloperoxidase (MPO) and neutrophil elastase (NE) were stained, and the formation of NETs was evaluated by confocal microscopy and by quantifying the levels of extracellular DNA. Our results showed N. fowleri induce the liberation of NETs including release of MPO and NE by human PMN cells as exposure interaction time is increased, but N. fowleri trophozoites evaded killing. However, when trophozoites were opsonized, they were susceptible to the neutrophils activity. Therefore, our study suggests that antibody-mediated PMNs activation through NET formation may be crucial for antimicrobial responses against N. fowleri.
Assuntos
Anticorpos Antiprotozoários/imunologia , Armadilhas Extracelulares/imunologia , Imunoglobulina G/imunologia , Naegleria fowleri/imunologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Trofozoítos/imunologia , Animais , Técnicas de Cocultura , DNA/metabolismo , Histonas/metabolismo , Humanos , Elastase de Leucócito/metabolismo , Meningoencefalite/imunologia , Meningoencefalite/parasitologia , Microscopia Confocal , Mucosa Nasal/parasitologia , Peroxidase/metabolismo , Fagocitose/imunologiaRESUMO
The neuraminidase (NA) epitope from the Mexican AH1N1 influenza virus was identified by using sequences registered at the GenBank during the peak of a pandemic (from April 2009 to October 2010). First, NA protein sequences were submitted for multiple alignment analysis, and their three-dimensional models (3-D) were then built by using homology modeling. The most common sequence (denominated wild-type) and its mutants were submitted to linear and nonlinear epitope predictors, which included the major histocompatibility complex type II (MHC II) and B-cell peptides. The epitope prediction was in accordance with evolutionary behavior and some protein structural properties. The latter included a low NA mutation rate, NA 3-D surface exposure, and the presence of high hindrance side chain residues. After selecting the epitope, docking studies and molecular dynamics (MD) simulations were used to explore interactions between the epitope and MHC II. Afterward, several experimental assays were performed to validate the theoretical study by using antibodies from humans (infected by pandemic H1N1) and rabbits (epitope vaccination). The results show 119 complete sequences that were grouped into 28 protein sequences according to their identity (one wild-type and 27 representative mutants (1-5 mutations)). The predictors yielded several epitopes, with the best fit being the one located in the C-terminal region. Theoretical methods demonstrated that the selected epitope reached the P4, P6, P7, and P9 pockets of MHC II, whereas the experimental evidence indicates that the epitope is recognized by human antibodies and also by rabbit antibodies immunized with the peptide.
Assuntos
Mapeamento de Epitopos/métodos , Epitopos de Linfócito B/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/imunologia , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/metabolismo , Biologia Computacional , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Influenza Humana/diagnóstico , México , Modelos Animais , Dados de Sequência Molecular , Mutação/genética , Neuraminidase/genética , Neuraminidase/imunologia , Ligação Proteica , Conformação Proteica , Coelhos , VacinaçãoRESUMO
Genome analysis of Entamoeba histolytica predicts the presence of acetyl-CoA carboxylase. Using Western blot, histochemistry, and confocal microscopy, we demonstrated the presence of a biotin-containing protein in the cytoplasm of E. histolytica, with a molecular weight of 136 kDa and biotin-carboxylase activity. This protein probably corresponds to a transcarboxylase that catalyzes the rate-limiting reaction leading to fatty acid elongation.
Assuntos
Carboxil e Carbamoil Transferases/metabolismo , Entamoeba histolytica/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas de Protozoários/metabolismo , Animais , Carboxil e Carbamoil Transferases/genética , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Genoma de Protozoário , Proteínas de Protozoários/genéticaRESUMO
According to previous reports, intranasal administration of the Cry1Ac protein alone or with amoebic lysates increases protection against Naegleria fowleri meningoencephalitis in mice, apparently by eliciting IgA responses in the nasal mucosa. In the current study, we performed an immunohistochemical analysis of IgA in the nasal mucosa of mice immunized intranasally with Cry1Ac, and amoebic lysates or a combination of both. The animals were sacrificed 24 h after the last immunization or after an intranasal lethal challenge with N. fowleri. Our results indicate that all of the intranasal immunizations provoked an increase in areas with metaplasia in the olfactory epithelium, allowing for secretion of IgA. As a result, IgA antibodies were found interacting with trophozoites in the nasal lumen, and there was a marked increase of IgA in the metaplasic epithelium. On the other hand in nonimmunized mice trophozoites were observed invading the nasal mucosa, which was not the case for immunized mice. Our results suggest that intranasal immunization provokes cellular changes in the olfactory epithelium, leading to greater protection against N. fowleri that is probably caused by an increased secretion of IgA. The increased IgA response induced in the nasal mucosa by immunization probably impedes both amoebic adhesion and subsequent invasion of the parasite to the nasal epithelium.