RESUMEN
Naegleria fowleri is a protozoan that causes primary amebic meningoencephalitis (PAM). The infection occurs when the trophozoites enter the nasal cavity, adhere to the nasal mucosa, invade the epithelium, and migrate until they reach the olfactory bulb. Like other pathogens, there is evidence that the adhesion of N. fowleri to host cells is an important factor in the process of cytopathogenicity and disease progression. However, the factors involved in the adhesion of the pathogen to the cells of the nasal epithelium have not been characterized. The objective of this study was to identify a protein on the surface of N. fowleri, which could act as adhesin to the mouse nasal epithelium in the PAM model. The interaction between proteins of extracts of N. fowleri and cells of the nasal epithelium of BALB/c mice was analyzed using overlay and Western blot assays. A 72-kDa band of N. fowleri interacted directly with epithelial cell proteins, this polypeptide band was purified and analyzed by mass spectrometry. Analysis revealed that polypeptide bands of 72 kDa contained peptides that matched the membrane protein, actin 1 and 2, and Hsp70. Moreover, the N. fowleri extracts resolved in 2D-SDS-PAGE showed that 72-kDa spot interacted with proteins of mouse epithelial cells, which include characteristics of the theoretical data of molecular weight and pH obtained in the analysis by mass spectrometry. Immunofluorescence tests showed that this protein is located on the surface of trophozoites and plays an important role in the adhesion of amoeba either in vitro or in vivo assays, suggesting that this protein contributes during the N. fowleri invasion and migration to the brain, causing primary amoebic meningoencephalitis.
Asunto(s)
Infecciones Protozoarias del Sistema Nervioso Central , Ratones Endogámicos BALB C , Naegleria fowleri , Mucosa Nasal , Proteínas Protozoarias , Trofozoítos , Animales , Ratones , Mucosa Nasal/parasitología , Proteínas Protozoarias/metabolismo , Infecciones Protozoarias del Sistema Nervioso Central/parasitología , Western Blotting , Adhesión Celular , Femenino , Amebiasis/parasitologíaRESUMEN
The prevalence of diabetes is rapidly increasing. The current number of diagnosed cases is ~422 million, expected to reach ~640 million by 2040. Type 2 diabetes, which constitutes ~95% of the cases, is characterized by insulin resistance and a progressive loss of ß-cell function. Despite intense research efforts, no treatments are yet able to cure the disease or halt its progression. Since all existing animal models of type 2 diabetes have serious drawbacks, one is needed that represents the complete pathogenesis, is low cost and non-obese, and can be developed relatively quickly. The aim of this study was to evaluate a low-cost, non-obese model of type 2 diabetes engendered by administering a daily high dose of tacrolimus (an immunosuppressant) to Wistar rats for 4 weeks. The biochemical and antioxidant markers were measured at basal and after the 4-week tacrolimus treatment. At week 4, the values of these parameters closely resembled those observed in human type 2 diabetes, including fasting blood glucose at 141.5 mg/dL, blood glucose greater than 200 mg/dL at 120 min of the glucose tolerance test, blood glucose at varied levels in the insulin tolerance test, and elevated levels of cholesterol and triglyceride. The tacrolimus treatment produced hypoinsulinemia and sustained hyperglycemia, probably explained by the alteration found in pancreatic ß-cell function and morphology. This model should certainly be instrumental for evaluating possible type 2 diabetes treatments, and for designing new immunosuppressants that do not cause pancreatic damage, type 2 diabetes, or new-onset diabetes after transplantation (NODAT).
Asunto(s)
Diabetes Mellitus Tipo 2 , Tacrolimus , Animales , Glucemia , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Prueba de Tolerancia a la Glucosa , Ratas , Ratas Wistar , Tacrolimus/uso terapéuticoRESUMEN
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.
Asunto(s)
Administración Intranasal , Antígenos de Protozoos/administración & dosificación , Naegleria fowleri/fisiología , Mucosa Nasal/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos de Protozoos/inmunología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Toxina del Cólera/administración & dosificación , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos BALB C , Naegleria fowleri/crecimiento & desarrollo , Naegleria fowleri/inmunología , Mucosa Nasal/citologíaRESUMEN
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.
Asunto(s)
Antinematodos/farmacología , Caesalpinia/química , Frutas/química , Hemoncosis/tratamiento farmacológico , Haemonchus/crecimiento & desarrollo , Metanol/química , Extractos Vegetales/farmacología , Animales , Antinematodos/química , Larva , Extractos Vegetales/química , Cigoto/crecimiento & desarrolloRESUMEN
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.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Trampas Extracelulares/inmunología , Inmunoglobulina G/inmunología , Naegleria fowleri/inmunología , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Trofozoítos/inmunología , Animales , Técnicas de Cocultivo , ADN/metabolismo , Histonas/metabolismo , Humanos , Elastasa de Leucocito/metabolismo , Meningoencefalitis/inmunología , Meningoencefalitis/parasitología , Microscopía Confocal , Mucosa Nasal/parasitología , Peroxidasa/metabolismo , Fagocitosis/inmunologíaRESUMEN
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.
Asunto(s)
Mapeo Epitopo/métodos , Epítopos de Linfocito B/metabolismo , Interacciones Huésped-Patógeno/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Neuraminidasa/metabolismo , Infecciones por Orthomyxoviridae/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/metabolismo , Biología Computacional , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Gripe Humana/diagnóstico , México , Modelos Animales , Datos de Secuencia Molecular , Mutación/genética , Neuraminidasa/genética , Neuraminidasa/inmunología , Unión Proteica , Conformación Proteica , Conejos , VacunaciónRESUMEN
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.
Asunto(s)
Transferasas de Carboxilo y Carbamoilo/metabolismo , Entamoeba histolytica/enzimología , Regulación Enzimológica de la Expresión Génica/fisiología , Proteínas Protozoarias/metabolismo , Animales , Transferasas de Carboxilo y Carbamoilo/genética , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Genoma de Protozoos , Proteínas Protozoarias/genéticaRESUMEN
During amebic invasion, neutrophils are a key component in either protecting against invading trophozoites or contributing to tissue damage. Upon degranulating or being lysed, neutrophils release toxic substances that can kill amebas as well as damage host tissue. In a previous study we identified a protein from nonspecifically stimulated peritoneal exudates of hamster that has peroxidase and marked amebicidal activity. In the current study we analyzed the in vitro amebicidal effect of purified hamster myeloperoxidase (MPO). The results demonstrate that MPO must bind directly to the surface of Entamoeba histolytica trophozoites in order to carry out amebicidal activity by using the H(2) O(2) produced by the amebas themselves. Myeloperoxidase-incubated amebas showed important morphological and ultrastructural alterations that increased with incubation time. Changes included an increase of vacuoles in the cytoplasm, a decrease of glycogen, alterations of nuclear morphology and disturbances in the plasma membrane culminating in complete ameba destruction.
Asunto(s)
Antiprotozoarios/farmacología , Entamoeba histolytica/efectos de los fármacos , Neutrófilos/enzimología , Peroxidasa/farmacología , Trofozoítos/efectos de los fármacos , Animales , Antiprotozoarios/aislamiento & purificación , Antiprotozoarios/metabolismo , Supervivencia Celular , Cricetinae , Entamoeba histolytica/citología , Masculino , Mesocricetus , Peroxidasa/aislamiento & purificación , Peroxidasa/metabolismo , Unión Proteica , Trofozoítos/citologíaRESUMEN
We previously reported that intranasal administration of Cry1Ac protoxin alone or in combination with amoebic lysates increases protection against Naegleria fowleri meningoencephalitis in mice. Those results suggested that both antibody responses and innate immune mechanisms may be participating in the protective effects observed. The present study was aimed to investigate whether the STAT6-induced Th2 immune response is essential for the resistance to N. fowleri infection, conferred by immunization with amoebic lysates plus Cry1Ac. STAT6-deficient (STAT6-/-) and wild-type (STAT6+/+) BALB/c mice were immunized by the intranasal route with a combination of N. fowleri lysates plus Cry1Ac, and subsequently challenged with lethal doses of N. fowleri trophozoites. STAT6+/+ mice displayed 100% protection, while no protection was observed in STAT6-/- mice. Significantly higher titres of Th2-associated IgG1 as well as interleukin-4 (IL-4) were found in STAT6+/+ mice, whereas in STAT6-/- mice significantly more IL-12 and IFN-gamma as well as significantly higher titres of Th1-associated IgG2a were detected. Thus, whereas protected STAT6+/+-immunized mice elicited a Th-2 type inclined immune response that produced predominantly humoral immunity, unprotected STAT6-/- mice exhibited a polarized Th1 type cellular response. These findings suggest that the STAT6-signalling pathway is critical for defence against N. fowleri infection.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Proteínas Bacterianas/administración & dosificación , Infecciones Protozoarias del Sistema Nervioso Central/prevención & control , Endotoxinas/administración & dosificación , Proteínas Hemolisinas/administración & dosificación , Naegleria fowleri/inmunología , Vacunas Antiprotozoos/administración & dosificación , Factor de Transcripción STAT6/inmunología , Células Th2/inmunología , Administración Intranasal , Animales , Toxinas de Bacillus thuringiensis , Evaluación Preclínica de Medicamentos , Inmunización , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteínas Recombinantes/administración & dosificaciónRESUMEN
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.
Asunto(s)
Amebiasis/inmunología , Proteínas Bacterianas/inmunología , Toxinas Bacterianas/inmunología , Endotoxinas/inmunología , Proteínas Hemolisinas/inmunología , Inmunización , Inmunoglobulina A Secretora/análisis , Meningoencefalitis/inmunología , Naegleria fowleri/inmunología , Mucosa Olfatoria/inmunología , Adyuvantes Inmunológicos , Administración Intranasal , Amebiasis/parasitología , Animales , Anticuerpos Antiprotozoarios/análisis , Antígenos de Protozoos/inmunología , Toxinas de Bacillus thuringiensis , Masculino , Meningoencefalitis/parasitología , Metaplasia , Ratones , Ratones Endogámicos BALB C , Mucosa Nasal/inmunología , Mucosa Nasal/parasitología , Proteínas Recombinantes/inmunologíaRESUMEN
Nasal-associated lymphoid tissue (NALT) and nasal passages (NP) are considered as inductive and effector sites, respectively. The differences among lymphocyte populations of these nasal compartments have not been clearly established. The aim of this work was to contribute to the characterization of NALT and NP lymphocytes in mice. We isolated lymphocytes from both compartments, determined the frequencies of B220(+) cells as well as CD8(+), CD4(+) T cells; and analysed the expression of CD69 and CD25. Besides we analysed the proportion of T cells producing IL-2, IL-4, IL-5, IL-10, IFN-gamma and TNF-alpha. We found differences between NALT and NP. Two populations of B cells, B220+(hi) and B220+(low) were clearly distinguished only in NP, but not in NALT. Both (hi) and (low) B220(+) cells expressed CD19, but only a fraction of the B220+(low) population, expressed the plasma cell marker CD138(+). More B than T lymphocytes, as well as higher frequencies of CD4(+) than CD8(+) T cells were found in both compartments. A small fraction of NK cells (CD3(-)DX5(+)) along with a significant proportion of double negative CD4(-)CD8(-)CD3(+)DX5(-) T cells was detected in both nasal tissues. Furthermore, as expected for a mucosal effector site, NP contained major proportions of B220(+), T CD4(+) and T CD8(+) cells expressing CD25 and CD69 in comparison to NALT. Likewise, the proportion of T cells spontaneously producing IL-2, IFN-gamma, and IL-4, was higher in NP than in NALT. These data provide further evidence indicating that distinctive phenotypic and functional features exist in the lymphocyte populations residing at NALT and NP.
Asunto(s)
Inmunidad Mucosa , Subgrupos Linfocitarios/inmunología , Linfocitos/inmunología , Cavidad Nasal/inmunología , Mucosa Nasal/citología , Animales , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos de Diferenciación de Linfocitos T/metabolismo , Citocinas/biosíntesis , Citocinas/inmunología , Citometría de Flujo , Lectinas Tipo C , Activación de Linfocitos/inmunología , Subgrupos Linfocitarios/metabolismo , Linfocitos/metabolismo , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Masculino , Ratones , Mucosa Nasal/inmunología , FenotipoRESUMEN
The objective of this study was to evaluate the secretory IgA (SIgA) antibody response to Naegleria fowleri (Nf) in individuals living in a parasite endemic area. Saliva and serum samples were obtained from both healthy subjects and patients suffering from a respiratory illness (chronic bronchitis or rhinitis) and were analyzed by immunoblot assay. SIgA from the patients' samples recognized more intensely a greater number of Nf proteins than did SIgA from the healthy control group. The proteins more frequently recognized were those with a molecular weight of 171, 107, 102, 62, 50, 46, and 10 kDa. Some IgA antibodies recognized proteins from Nf and Entamoeba histolytica (Eh) of similar molecular weight. These results suggest that some of those antibodies could have been elicited by a previous intestinal infection with Eh. Through the common mucosal immune system the IgA B-cells activated by Eh antigens can be disseminated to all the mucosae, including the nasal mucosa. SIgA antibodies recognizing Nf proteins, induced either by specific immunization or by cross-reaction, could participate in the resistance to the infection, probably by inhibiting the adherence of Nf trophozoites to the nasal mucosa.