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 β (TGF-β) and connective tissue growth factor (CTGF) regulation. Experimental hepatic cirrhosis was established by the administration of carbon tetrachloride (CCl 4 ) 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. CCl 4 treatment promoted NF-κB activation and increased the expression of both TGF-β and CTGF. CCl 4 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)-1β and IL-6) and profibrotic (TGF-β 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 CCl 4 -induced experimental hepatic cirrhosis in rats by blocking the main pro-inflammatory and pro-fibrogenic signals.

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-β (TGF-β) 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-β, 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-β and the activity of COX-2.

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-1β, 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-1β 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.

SUMMARY We have recently proposed revival of the name Entamoeba nuttalliCastellani, 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.

Sporothrix schenckii is a pathogenic dimorphic fungus for humans; this mycopathogen is a causative agent of sporotrichosis, a fungal skin / subcutaneous disease-associated lymphatic system. Sporotrichosis is classified as an emerging mycosis. The main event during the establishment, colonization and invasion of host tissues by pathogenic microorganisms is adherence to host cell. Currently unknown biological basis responsible of physiopathology of sporotrichosis. The purpose of this study was elucidating the interaction processes between yeast cells of S. schenckii with epithelial tissue. Using a cultured epithelial monolayer was carried out the interaction with yeast cells S. schenckii. We determinate the adhesion kinetics and morphological changes by S. schenckii toward epithelium at epifluorescence microscopy level. The results indicate that the adherence of yeasts cells to the epithelium occurs in a time-dependent. It was also noted that a long interaction time (48 h occurred cytopathic effect on the epithelial monolayer. The interaction of pathogen, promoted arrangement of the epithelial microtubule cytoskeleton in a time-dependent. Furthermore, the ultrastructure showed changes in the cell surface, which occurs in loss of cellular contacts with the integrity of the monolayer and consequently cell polarity. Surprisingly at 48 h of interaction was observed internalization of yeast by the epithelium. This research can be useful to understand the dynamic aspects of sporotrichosis.

Amoebic liver abscess (ALA) constitutes the most important and common extraintestinal complication of Entamoeba histolytica infection. Information regarding the role of cell-mediated immunity to confer protection against amoebiasis is still contradictory. Several in vitro and in vivo studies have shown that amoebic antigens induce the production of different cytokines: IIFN gamma, TNF alpha, IL-2, Il-4, IL-10; however, the local and systemic roles of these molecules in the pathogenesis of invasive amoebiasis is poorly understood. We analyzed the in situ presence of TNF alpha, IFN gamma and IL-10 during the evolution of ALA in hamsters. Results showed inflammatory cells labeled with TNF alpha, IFN gamma and IL-10 mainly during the acute phase of the ALA development. Presence of these cytokines was temporarily and decreased with the progress of the amoebic damage. Nevertheless, local production of cytokines appears to be non-sufficient to evade or arrest the development of ALA.

Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS-PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to inhibit crude extract protease activity on Madin-Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.

ABSTRACT Acanthamoeba castellani and Acanthamoeba polyphaga are free-living amebae that cause keratitis and granulomatous encephalitis in humans. We have analyzed the early morphological and electrophysiological changes occurring during the in vitro interaction of cultured amebae with intact or physically damaged corneas obtained from hamsters. Both species of Acanthamoeba produced similar cytopathic changes, as seen by light microscopy and scanning electron microscopy. After adhesion to the epithelial surface, trophozoites formed clumps and migrated toward the cell borders, causing the separation of adjacent cells at 1 h of coculture. At later stages (2 to 4 h), some amebae were found under desquamating epithelial cells whereas others were seen associated with damaged cells or forming amebostome-like structures to ingest detached epithelial cells. Control corneas incubated in culture medium conditioned with amebae showed a cytoplasmic vacuolization and blurring of the epithelial-stromal junction. The early stages of corneal epithelial damage caused by amebae were also analyzed by measuring the transepithelial resistance changes in corneas mounted in Ussing chambers. Both species of Acanthamoeba caused a rapid decrease in electrical resistance. The present observations demonstrate that under in vitro conditions, Acanthamoeba trophozoites rapidly cause significant damage to the corneal epithelium. Furthermore, in our experimental model, previous physical damage to the corneas was not a prerequisite for the development of amebic corneal ulcerations.

BACKGROUND:The aim of the present study was to determine if the inflammation and/or immunosuppression induced by Entamoeba histolytica may contribute to amebic invasion.MATERIAL/METHODS:Dexamethasone was administered three days before and three days after inoculation of hamsters with E. histolytica. Seven days alter inoculation the animals were sacrificed and the sizes of their amebic liver abscesses were determined. The number of neutrophils, macrophages, T and B cells in the peritoneum as well as the production of nitric oxide and the susceptibility to Listeria monocytogenes infection was also determined.RESULTS:Dexamethasone treatment significantly reduced the number of T lymphocytes in thymus and spleen. The number of neutrophils, macrophages and T lymphocytes in the peritoneal exudate was also reduced as well as the production of nitric oxide and the microbicidal activity against Listeria monocytogenes. However, in the animals treated with a high dose of dexamethasone the size of the liver abscesses was significantly smaller than in the untreated animals.CONCLUSIONS:The results suggest that macrophage and T cell-mediated immunity is not relevant as a protective mechanism because tissue invasion by E. histolytica was reduced in immunosuppresed animals. On the contrary, the inflammatory process may contribute to the invasion and liver damage.

Numerous sperm functions including the acrosome reaction (AR) are associated with Ca2+ influx through voltage-gated Ca2+ (CaV) channels. Although the electrophysiological characterization of Ca2+ currents in mature sperm has proven difficult, functional studies have revealed the presence of low-threshold (CaV3) channels in spermatogenic cells. However, the molecular identity of these proteins remains undefined. Here, we identified by reverse transcription polymerase chain reaction the expression of CaV3.3 mRNA in mouse male germ cells, an isoform not previously described in these cells. Immunoconfocal microscopy revealed the presence of the three CaV3 channel isoforms in mouse spermatogenic cells. In mature mouse sperm only CaV3.1 and CaV3.2 were detected in the head, suggesting its participation in the AR. CaV3.1 and CaV3.3 were found in the principal and the midpiece of the flagella. All CaV3 channels are also present in human sperm, but only to a minor extent in the head. These findings were corroborated by immunogold transmission electron microscopy. Tail localization of CaV3 channels suggested they may participate in motility, however, mibefradil and gossypol concentrations that inhibit CaV3 channels did not significantly affect human sperm motility. Only higher mibefradil doses that can block high-threshold (HVA) CaV channels caused small but significant motility alterations. Antibodies to HVA channels detected CaV1.3 and CaV2.3 in human sperm flagella.

Chemical communication mediated by pheromones is a crucial aspect in the life cycle of beetles in the genus Dendroctonus. This communication plays an important role in mate location and in the colonization of host conifers. The study of the alimentary canal of these species is of importance not only because this organ is involved in the processes of digestion, detoxification, nutrient absorption, and transport, but also in the production of semiochemical compounds, such as pheromones. To better understand these functions and where they occur, the ultrastructural differences between the anterior and posterior midgut and the hindgut and their different cellular types were characterized. Adult specimens of both sexes from eight species were dissected and the alimentary canal was removed. It was sectioned into three parts: anterior midgut, posterior midgut, and hindgut, and analyzed by transmission electron microscopy. Results show that the epithelial tissue of the midgut possesses ultrastructural characteristics that permit differentiation of the anterior and posterior midgut. There are no ultrastructural differences within sexes of the same species, but differences exist among species. The ultrastructural characteristics of the hindgut do not differ between sexes or among species, but they do differ from those of the midgut.

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.

Objective Thalidomide has anti-inflammatory, anti-tumour necrosis factor-alpha and anti-collagen activities. Cirrhosis is characterized by inflammation and fibrosis. Thus, thalidomide was evaluated in an experimental model of liver cirrhosis. Methods Male Wistar rats were used. Group 1 (n = 8) received mineral oil i.p. (control); group 2 (n = 15) received CCl4 i.p. for 8 weeks to induce cirrhosis; group 3 (n = 15) consisted of rats receiving CCl4 plus thalidomide (200 mg/kg/12 h); animals in group 4 (n = 8) received thalidomide only. Alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (γ-GTP) and alkaline phosphatase (ALP) were measured in serum, while collagen (hydroxyproline), glycogen and lipid peroxidation were determined in liver samples. A liver histopathological analysis was performed by using Gomori's trichromic staining. Results Intoxication with CCl4 induced 33.3% mortality, while thalidomide co-treatment reduced it to 13.3%. The serum activities of ALT, γ-GTP and ALP increased 3, 2 and 4-fold by CCl4 treatment; thalidomide completely prevented elevation of these enzymes. In the liver, lipid peroxidation increased about 20-fold and glycogen was abolished in CCl4 cirrhotic rats; thalidomide completely prevented the former and partially (P < 0.05) the latter. CCl4 treated rats revealed a loss of normal architecture and nodules of hepatocytes surrounded by thick bands of collagen. Thalidomide + CCl4 treated rats showed minor histological alterations and thinner bands of collagen. The anti-fibrotic effect estimated by hydroxyproline was partial but significant (P < 0.05). Conclusion Thalidomide prevented necrosis, cholestasis and fibrosis induced by CCl4. Its mechanism of action may be related to its anti-inflammatory, anti-tumour necrosis factor-alpha and anti-fibrotic activities reported previously.

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.

To study the role of neutrophils in the innate resistance to Entamoeba histolytica intestinal infection in mice, animals were treated with anti-neutrophil monoclonal antibodies prior to intracecal parasite inoculation and the resulting lesions were compared with normal mice that had been equally infected. In contrast to our previous finding that neutrophils are critical in eliminating E. histolytica infection in the liver, we show here that neutrophils are not absolutely required to eliminate E. histolytica infection from the intestine. Although the neutrophils are not critical for resolution of the E. histolytica infection, neutrophils do appear to provide some measure of protection as the intestinal amoeba burden was higher at early timepoints after infection in the neutropenic animals. In addition, we found that while both the normal and the neutrophil-depleted mice developed ulcerative lesions in the colon, the neutropenic mice had an increased frequency of granulomas that formed around the amoeba. Thus, our findings appear to be the first evidence showing that granulomatous inflammation can occur after intestinal infection in mice using axenically cultured amoeba.

Potassium (K + ) channels are believed to regulate mammalian sperm acquisition of fertilising capacity. However, the molecular identity of these proteins in sperm has not been elucidated. In this report, using immunoconfocal and electron microscopy we show that a minimum of four different classes of K + channels (Kv1.1, Kv1.2, Kv3.1 and GIRK1) are present and regionally distributed over the surface of mouse epididymal sperm. In addition, the use of reverse transcription polymerase chain reaction on RNA from mouse spermatogenic cells allowed the amplification of multiple transcripts corresponding to the channels identified by immunocytochemistry. Consistent with this, whole-cell patch-clamp recordings showed the expression of at least two different outwardly rectifying K+ currents in spermatogenic cells.

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.

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.