RESUMEN
Here we evaluated the ability of L-theanine in preventing experimental hepatic cirrhosis and investigated the roles of nuclear factor-κB (NF-κB) activation as well as transforming growth factor ß (TGF-ß) and connective tissue growth factor (CTGF) regulation. Experimental hepatic cirrhosis was established by the administration of carbon tetrachloride (CCl4) to rats (0.4 g/kg, intraperitoneally, three times per week, for 8 weeks), and at the same time, adding L-theanine (8.0 mg/kg) to the drinking water. Rats had ad libitum access to water and food throughout the treatment period. CCl4 treatment promoted NF-κB activation and increased the expression of both TGF-ß and CTGF. CCl4 increased the serum activities of alanine aminotransferase and γ-glutamyl transpeptidase and the degree of lipid peroxidation, and it also induced a decrease in the glutathione and glutathione disulfide ratio. L-Theanine prevented increased expression of NF-κB and down-regulated the pro-inflammatory (interleukin (IL)-1ß and IL-6) and profibrotic (TGF-ß and CTGF) cytokines. Furthermore, the levels of messenger RNA encoding these proteins decreased in agreement with the expression levels. L-Theanine promoted the expression of the anti-inflammatory cytokine IL-10 and the fibrolytic enzyme metalloproteinase-13. Liver hydroxyproline contents and histopathological analysis demonstrated the anti-fibrotic effect of l-theanine. In conclusion, L-theanine prevents CCl4-induced experimental hepatic cirrhosis in rats by blocking the main pro-inflammatory and pro-fibrogenic signals.
Asunto(s)
Intoxicación por Tetracloruro de Carbono/patología , Intoxicación por Tetracloruro de Carbono/prevención & control , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Factor de Crecimiento del Tejido Conjuntivo/biosíntesis , Glutamatos/uso terapéutico , Cirrosis Hepática/patología , Cirrosis Hepática/prevención & control , FN-kappa B/biosíntesis , Factor de Crecimiento Transformador beta/biosíntesis , Alanina Transaminasa/sangre , Animales , Antioxidantes/metabolismo , Aspartato Aminotransferasas/sangre , Factor de Crecimiento del Tejido Conjuntivo/efectos de los fármacos , Citocinas/biosíntesis , Regulación hacia Abajo/genética , Peroxidación de Lípido/efectos de los fármacos , Cirrosis Hepática/inducido químicamente , Masculino , Metaloproteinasa 13 de la Matriz/biosíntesis , FN-kappa B/efectos de los fármacos , Ratas , Ratas Wistar , Factor de Crecimiento Transformador beta/efectos de los fármacosRESUMEN
It has been demonstrated that this sulfasalazine (SF) inhibits the nuclear factor κB (NFκB) pathway, which regulates important genes during inflammation and immune answer. The aim of this work was to evaluate the effects of SF on carbon tetrachloride (CCl(4))-induced liver fibrosis. We formed the following experimental groups of rats: controls, damage induced by chronic CCl(4) (0.4 g/kg, intraperitoneally, three times a week for 8 weeks) administration and CCl(4) + SF (100 mg/kg/day, postoperatively for 8 weeks) administration. We determined the activities of alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GTP), cyclooxygenase (COX)-1 and COX-2, lipid peroxidation, glutathione levels, collagen content, expression of transforming growth factor-ß (TGF-ß) and nuclear translocation of NFκB. SF was capable to inhibit the ALT and γ-GTP elevated levels induced with the CCl(4) administration. SF had antioxidant properties, prevented the lipid peroxidation and the imbalance of reduced and oxidized glutathione produced by CCl(4). Importantly, SF blocked the accumulation of collagen in the liver, the expression of TGF-ß, the nuclear translocation of NFκB and the activity of COX-2, all induced with the administration of CCl(4) in the rat. These results show that SF has strong antifibrotic properties because of its antioxidant properties and its ability to prevent nuclear translocation of NFκB and consequently the expression of TGF-ß and the activity of COX-2.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Antioxidantes/uso terapéutico , Cirrosis Hepática Experimental/tratamiento farmacológico , Cirrosis Hepática/prevención & control , Sulfasalazina/uso terapéutico , Alanina Transaminasa/sangre , Animales , Antiinflamatorios no Esteroideos/farmacología , Antioxidantes/farmacología , Tetracloruro de Carbono , Colágeno/metabolismo , Ciclooxigenasa 2/metabolismo , Glutatión/metabolismo , Disulfuro de Glutatión/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/metabolismo , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/metabolismo , Masculino , FN-kappa B/metabolismo , Ratas , Ratas Wistar , Sulfasalazina/farmacología , Factor de Crecimiento Transformador beta/metabolismo , gamma-Glutamiltransferasa/sangreRESUMEN
Amoebic liver abscess (ALA) is the most important extraintestinal complication of Entamoeba histolytica infection. Amoebic liver abscess development causes severe destruction of the liver tissue concomitant with a strong inflammatory reaction. We analyse the in situ expression of TNF-α, IFN-γ, IL-1ß, 1L-8 and IL-10 at different stages of ALA development in a susceptible animal model. Results showed that after inoculation, neutrophils (PMN) and some macrophages infiltrated the liver and were positive for TNF-α and IFN-γ at the acute phase of amoeba infection. The presence of these cytokines was transient and decreased as tissue damage progressed. In contrast, IL-1ß and IL-8 were detected mainly in neutrophils and macrophages from the periods of acute infection to subacute and chronic infection and decreased when granulomas were formed. The IL-10 was expressed in PMN and mononuclear cells and only during a short period at the onset of acute infection. The qRT-PCR of mRNA revealed a relationship with the expression of the cytokines in cells found in the ALA. Furthermore, our data suggest that IL-10 does not regulate local production of these cytokines. Our results indicate that an exacerbated inflammatory milieu is established and contributes to liver tissue damage and probably supports the survival of the parasites.
Asunto(s)
Citocinas , Entamoeba histolytica/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/inmunología , Inflamación/inmunología , Absceso Hepático Amebiano/inmunología , Absceso Hepático Amebiano/metabolismo , Hígado/inmunología , Hígado/metabolismo , Macrófagos/inmunología , Neutrófilos/inmunología , ARN Mensajero/análisis , Animales , Cricetinae , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Entamoeba histolytica/metabolismo , Inmunohistoquímica , Inflamación/metabolismo , Hígado/parasitología , Hígado/ultraestructura , Absceso Hepático Amebiano/parasitología , Macrófagos/metabolismo , Masculino , Neutrófilos/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
We have recently proposed revival of the name Entamoeba nuttalli Castellani, 1908 for a virulent amoeba (P19-061405 strain) isolated from a rhesus monkey (Macaca mulatta) and located phylogenetically between E. histolytica and E. dispar. In this study, E. nuttalli was isolated from feces of captive Japanese macaques (M. fuscata) in an open-air corral in Japan. The sequence of the 18S rRNA gene in the isolates differed from the P19-061405 strain in 2 nucleotide positions, but was identical to the EHMfas1 strain isolated previously from a cynomolgus monkey (M. fascicularis). One of the E. nuttalli isolates from Japanese macaques, named the NASA6 strain, was axenized and cloned. In isoenzyme analysis, the mobilities of hexokinase and phosphate glucose isomerase in the NASA6 strain were identical to those in the P19-061405 and EHMfas1 strains, but the mobility of phosphoglucomutase was different. These results were supported by gene analyses of these enzymes. Inoculation of NASA6 strain trophozoites into the liver of hamsters led to formation of an amoebic liver abscess. The liver lesions were characterized by extensive necrosis associated with inflammatory reactions. These results demonstrate that the NASA6 strain is potentially virulent and that E. nuttalli should be recognized as a common parasite in macaques.
Asunto(s)
Entamoeba/clasificación , Entamoeba/patogenicidad , Entamebiasis , Macaca/parasitología , Enfermedades de los Monos , Animales , Cricetinae , Entamoeba/genética , Entamoeba/aislamiento & purificación , Entamebiasis/epidemiología , Entamebiasis/parasitología , Entamebiasis/patología , Heces/parasitología , Isoenzimas/análisis , Japón , Hígado/parasitología , Hígado/patología , Absceso Hepático Amebiano/parasitología , Absceso Hepático Amebiano/patología , Macaca/clasificación , Enfermedades de los Monos/epidemiología , Enfermedades de los Monos/parasitología , Enfermedades de los Monos/patología , ARN Ribosómico 18S/genética , Análisis de Secuencia de ADN , Especificidad de la Especie , VirulenciaRESUMEN
In the life cycle of Entamoeba species, the cyst and all the processes associated to it have been poorly studied. Entamoeba invadens, a serpent's parasite, has been commonly accepted as a model for the study of encystation and excystation. Here we analyzed through scanning and transmission electron microscopy the in vitro morphological differentiation of both processes. During encystation, the formation of an irregular net of fibrillar material on the surface of precysts was observed. In thin sections of cryofixed and cryosubstituted specimens, abundant vacuoles containing a microfibrillar material of similar appearance to the structural components of the cyst wall were found in the cytoplasm. Assays with a calcofluor probe on cryosections of encysting trophozoites and precysts showed the presence of fluorescent circular cytoplasmic structures. In the cyst stage, the fluorescence was located on the surface. During excystation, the detachment of the metacyst from the cyst wall was observed through scanning electron microscopy. Metacysts endocyting amorphous material which may correspond to cyst wall residues were commonly found. By transmission electron microscopy the formation of a crescent-shaped space between the plasma membrane and the cyst wall was observed. Abundant small electrondense bodies were found in the cytoplasm. Many of them were in close apposition to the plasma membrane and frequently some of them were seen projecting towards this newly formed space. Our results suggest that the microfibrillar content of the vacuoles corresponds to the cyst wall material, that the electrondense bodies may be involved in the excystation process, and that part of the cyst wall residues may be endocyted by the parasite.
Asunto(s)
Entamoeba/ultraestructura , Microscopía Electrónica de Rastreo/métodos , Animales , Entamoeba/fisiologíaRESUMEN
Studies in mice have not rendered conclusive data on cell and humoral factors to support the resistance of this rodent to Entamoeba histolytica infection. In Balb/c and C3H/HeJ mice inoculated with live or fixed trophozoites, we studied the evolution of the hepatic lesion, the kinetics of inflammatory cells, and the participation of some humoral factors in the development of the hepatic amoebic lesion. From the first hour, amoebae were surrounded by neutrophils containing inducible nitric oxide synthase (iNOS); macrophages also expressing iNOS appeared lately, whereas NK cells were not part of the inflammatory infiltrates. On the fourth day, neutrophils, macrophages, T and B lymphocytes, plasma cells, and some NK cells limited the lesions and anti-amoeba antibodies appeared when most parasites had been eliminated. Therefore, the resistance of the mice to E. histolytica probably lies in non-specific immune responses, among which the activation of neutrophils and the production of nitric oxide (NO) may be important amoebicide factors.
Asunto(s)
Entamoeba histolytica/inmunología , Entamebiasis/inmunología , Neutrófilos/inmunología , Óxido Nítrico/metabolismo , Animales , Entamoeba histolytica/patogenicidad , Entamebiasis/parasitología , Entamebiasis/patología , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Inmunohistoquímica , Hígado/parasitología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Necrosis , Neutrófilos/enzimología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo IIRESUMEN
The protozoan parasite Entamoeba histolytica is the etiological agent of human amebiasis. The pathology of the disease starts with the cytolysis of the host target cells by amoebae. It is initiated by the adhesion of trophozoites to the host cells, through surface lectin via specific receptors. These adherence lectins have been demonstrated to be highly conserved, and can be recognised by serum antibodies from patients with invasive amebiasis. Some of these molecules have been used as antigens in serologic studies, which has been very helpful in the diagnosis of invasive intestinal amebiasis. However, false-positive serologic reactivity can occur using E. histolytica extracts and purified antigens. Additional problems are because the extracts display a great enzymatic activity. Several diagnostic methods, using different molecules and techniques, have been described. However, the problem still remains since these tests are not capable of differentiating between amoebic liver abscess (ALA) and intestinal amebiasis.Here, the research has been addressed to the 66-kDa antigen, which is a part of the outer membrane proteins from the E. histolytica strain HM1-IMSS trophozoites. First of all, we characterized the 66-kDa antigen in order to prove the relevance. We found that the 66-kDa antigen is a part of the plasma membranes and is distributed rather homogeneously on the cell surface of trophozoites. Apparently, the 66-kDa antigen is a glycoprotein. Using a monoclonal antibody (MAb), we found 25% of inhibition in the erythrophagocytosis by the trophozoites. Starting form one monoclonal antibody, we prepared an anti-idiotype (anti-Id) antibody reagent, with the purpose of searching for the different expressions of the idiotype between the sera from ALA and the intestinal amebiasis patients. Moreover, we produced the antibody Ab3 that is capable of recognising the 66-kDa antigen; it means that the Ab2 displays the internal image of the antigen. We found that 91.6% of the serum from ALA patients displayed the expression of the Id. In contrast, 15.7% of the E. histolytica asymtomatic cyst carriers displayed the Id expression, 6.6% of the patients with another parasite infection, and 11% of the negative controls (serum from umbilical cords of newborn babies). Our results showed that the expression of the Id could be differentiated among the AHA patients from the other groups with a 91.6% sensibility and 88.3% specificity.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Antígenos de Protozoos/inmunología , Entamoeba histolytica/inmunología , Entamoeba histolytica/aislamiento & purificación , Entamebiasis/diagnóstico , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Antígenos de Protozoos/análisis , Entamebiasis/inmunología , Humanos , Inmunoensayo , Ratones , Ratones Endogámicos BALB C , Sensibilidad y EspecificidadRESUMEN
We studied the early in situ interactions of live and fixed Entamoeba histolytica trophozoites with hamster hepatic parenchymal and inflammatory cells using immunoperoxidase and immunoelectronmicroscopy. Close contact between trophozoites and endothelial cells and the diffusion of amoebic molecules from trophozoites towards nearby endothelial cells and distant hepatocytes were observed. The inflammatory cells around the amoebae and the remnants of parenchymal cells and hepatocytes located close to the lesion had a positive stain for amoebic molecules. In the amoebae, at the ultrastructural level, molecules were attached to the membranes and inside the vesicles. These molecules were apparently released into the space formed between the parasite and the endothelial cells. The endothelial cells and the nearby and distant hepatocytes captured amoebic molecules, and later they became necrotic. Contrarily, when fixed amoebae were inoculated, amoebic molecules were captured by endothelial cells and polymorphonuclear (PMN) leukocytes, but neither suffered any damage. In this work, we are presenting evidence clearly showing that some molecules of the amoeba can diffuse away long distances causing cytotoxic effects and even necrosis on hepatic cells of hamster liver without the need of the trophozoite being in close contact with the target cells. They also may promote lytic or proinflammatory effects by inducing the secretion of enzymes or cytokines in other nonparenchymal cells, like PMN leukocytes and endothelial cells. Our results suggest that the accepted mechanisms of cytotoxicity by amoebae are not exclusively restricted to the following sequence: adhesion, phagocytosis, and necrosis.
Asunto(s)
Entamoeba histolytica/fisiología , Entamebiasis/patología , Parasitosis Hepáticas/patología , Animales , Cricetinae , Entamoeba histolytica/inmunología , Entamebiasis/fisiopatología , Hepatitis/parasitología , Hepatitis/patología , Hepatocitos/patología , Hepatocitos/ultraestructura , Interacciones Huésped-Parásitos , Leucocitos/fisiología , Leucocitos/ultraestructura , Parasitosis Hepáticas/parasitología , Masculino , Microscopía InmunoelectrónicaRESUMEN
The objective of the current study was to analyze the hepatotoxic effect caused by cypermethrin (CYP) in rats, and to evaluate the possible protective effect of the antioxidant alpha-tocopherol (alpha-T). Fifty male Wistar rats were given daily i.p. doses of 300 mg/kg per day of CYP during 7 days. Half of them were administered three previous doses of 100 mg/kg per day of alpha-T, followed by seven subsequent oral doses of 40 mg/kg per day of alpha-T. The levels of biochemical indicators and histological liver damage were determined, as well as DCVA in urine. CYP altered the lipid metabolism. Such alterations were inhibited 32% by alpha-T, except for LDL. Alterations in AST were modulated in 29%. In the histology, alpha-T reduced mitochondria damage, and swelling of the endoplasmic reticulum of the liver cells. The results suggest that alpha-T can modify CYP metabolism, changing the lipidic profile and the histological analysis.
Asunto(s)
Antioxidantes/farmacología , Insecticidas/toxicidad , Hígado/efectos de los fármacos , Piretrinas/toxicidad , alfa-Tocoferol/farmacología , Animales , Lípidos/sangre , Hígado/patología , Hígado/fisiología , Masculino , Piretrinas/metabolismo , Ratas , Ratas WistarRESUMEN
The aim of this work was to determine if the action mechanism of gadolinium on CCl(4)-induced liver damage is by preventing lipid peroxidation (that may be induced by Kupffer cells) and its effects on liver carbohydrate metabolism. Four groups of rats were treated with CCl(4), CCl(4)+GdCl(3), GdCl(3), and vehicles. CCl(4) was given orally (0.4 g 100 g(-1) body wt.) and GdCl(3) (0.20 g 100 g(-1) body wt.) was administered i.p. All the animals were killed 24 h after treatment with CCl(4) or vehicle. Glycogen and lipid peroxidation were measured in liver. Alkaline phosphatase, gamma-glutamyl transpeptidase, alanine amino transferase activities and bilirubins were measured in rat serum. A liver histological analysis was performed. CCl(4) induced significant elevations on enzyme activities and bilirubins; GdCl(3) completely prevented this effect. Liver lipid peroxidation increased 2.5-fold by CCl(4) treatment; this effect was also prevented by GdCl(3). Glycogen stores were depleted by acute intoxication with CCl(4). However, GdCl(3) did not prevent this effect. The present study shows that Kupffer cells may be responsible for liver damage induced by carbon tetrachloride and that lipid peroxidation is produced or stimulated by Kupffer cells, since their inhibition with GdCl(3) prevented both lipid peroxidation and CCl(4)-induced liver injury.
Asunto(s)
Gadolinio/farmacología , Glucógeno/antagonistas & inhibidores , Macrófagos del Hígado/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Fallo Hepático/metabolismo , Hígado/metabolismo , Alanina Transaminasa/sangre , Alanina Transaminasa/efectos de los fármacos , Fosfatasa Alcalina/sangre , Fosfatasa Alcalina/efectos de los fármacos , Animales , Bilirrubina/agonistas , Bilirrubina/sangre , Tetracloruro de Carbono , Membrana Celular/enzimología , Hígado/patología , Fallo Hepático/inducido químicamente , Masculino , Ratas , Ratas Wistar , gamma-Glutamiltransferasa/sangre , gamma-Glutamiltransferasa/efectos de los fármacosRESUMEN
Antibodies from IgA and IgM classes that recognize Naegleria fowleri (Nf) proteins were detected by the ELISA assay in serum and saliva from three groups of people: (i) subjects with upper respiratory tract infections (URTI) living in the parasite-endemic area, (ii) healthy persons from the same area, and (iii) healthy persons from a parasite-nonendemic area. In serum and in saliva the titers of IgA antibodies to Naegleria fowleri in the group of patients with URTI was significantly higher than that of the healthy group in the parasite-endemic area; also the titers of IgM antibodies in serum were significantly higher in patients. On the contrary, in saliva the antibodies were higher in healthy people from the parasite-endemic area. In all cases the subjects from the parasite-nonendemic area had lower antibody titers in serum and saliva.
Asunto(s)
Amebiasis/inmunología , Anticuerpos Antiprotozoarios/análisis , Isotipos de Inmunoglobulinas/análisis , Naegleria fowleri/inmunología , Infecciones del Sistema Respiratorio/inmunología , Saliva/inmunología , Adulto , Animales , Anticuerpos Antiprotozoarios/sangre , Humanos , Inmunoglobulina A/análisis , Inmunoglobulina M/análisisRESUMEN
Entamoeba histolytica trophozoites were inoculated into the liver of hamsters and serum nitrate/nitrite levels [expressed as nitric oxide (NO) production] were determined at different times during amebic liver abscess (ALA) development. We also tested the effects of NO synthase (NOS) inhibitors such as N(G)-nitro-L-arginine methyl ester (L-NAME), aminoguanidine, and dexamethasone during ALA production. Since NOS activity has been correlated with expression of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) in tissues, we performed histochemistry studies to determine the activity of the latter in livers infected with E. histolytica trophozoites. Production of NO in serum was directly proportional to the size of ALAs, and NOS inhibitors caused low levels of NO and smaller ALAs. Our data suggest that NO does not have any lytic effect on E. histolytica trophozoites and is therefore incapable of providing protection against the amebic liver infection. In addition, NADPHd activity was detected histochemically in hepatocytes and inflammatory cells associated with focal necrosis containing trophozoites. The positive reactivity observed in these parasites may be attributable to a close biochemical similarity of NADPHd to the NADPH:flavin oxidoreductase described in E. histolytica by other investigators.