Rabeprazole inhibits several functions of Entamoeba histolytica related with its virulence.

Amoebiasis is a human parasitic disease caused by Entamoeba histolytica. The parasite can invade the large intestine and other organs such as liver; resistance to the host tissue oxygen is a condition for parasite invasion and survival. Thioredoxin reductase of E. histolytica (EhTrxR) is a critical enzyme mainly involved in maintaining reduced the redox system and detoxifying the intracellular oxygen; therefore, it is necessary for E. histolytica survival under both aerobic in vitro and in vivo conditions. In the present work, it is reported that rabeprazole (Rb), a drug widely used to treat heartburn, was able to inhibit the EhTrxR recombinant enzyme. Moreover, Rb affected amoebic proliferation and several functions required for parasite virulence such as cytotoxicity, oxygen reduction to hydrogen peroxide, erythrophagocytosis, proteolysis, and oxygen and complement resistances. In addition, amoebic pre-incubation with sublethal Rb concentration (600 µM) promoted amoebic death during early liver infection in hamsters. Despite the high Rb concentration used to inhibit amoebic virulence, the wide E. histolytica pathogenic-related functions affected by Rb strongly suggest that its molecular structure can be used as scaffold to design new antiamoebic compounds with lower IC50 values.

Functional characterization and subcellular distribution of two recombinant cytosolic HSP70 isoforms from Entamoeba histolytica under normal and stress conditions.

Amoebiasis is a human intestinal disease caused by the parasite Entamoeba histolytica. It has been previously demonstrated that E. histolytica heat shock protein 70 (EhHSP70) plays an important role in amoebic pathogenicity by protecting the parasite from the dangerous effects of oxidative and nitrosative stresses. Despite its relevance, this protein has not yet been characterized. In this study, the EhHSP70 genes were cloned, and the two recombinant EhHSP70 proteins were expressed, purifying and biochemically characterized. Additionally, after being subjected to some host stressors, the intracellular distribution of the proteins in the parasite was documented. Two amoebic HSP70 isoforms, EhHSP70-A and EhHSP70-B, with 637 and 656 amino acids, respectively, were identified. Kinetic parameters of ATP hydrolysis showed low rates, which were in accordance with those of the HSP70 family members. Circular dichroism analysis showed differences in their secondary structures but similarities in their thermal stability. Immunocytochemistry in trophozoites detected EhHSP70 in the nuclei and cytoplasm as well as a slight overexpression when the parasites were subjected to oxidants and heat. The structural differences of amoebic HSP70s with their human counterparts may be used to design specific inhibitors to treat human amoebiasis.

M2 macrophage immunotherapy abolishes glucose intolerance by increasing IL-10 expression and AKT activation.

Aim: Glucose intolerance associates with M1/M2 macrophage unbalance. We thus wanted to examine the effect of M2 macrophage administration on mouse model of glucose intolerance. Materials & methods: C57BL/6 mice fed a high-fat diet (HFD) for 12 weeks and then received thrice 20 mg/kg streptozotocin (HFD-GI). Bone marrow-derived stem cells were collected from donor mice and differentiated/activated into M2 macrophages for intraperitoneal administration into HFD-GI mice. Results: M2 macrophage treatment abolished glucose intolerance independently of obesity. M2 macrophage administration increased IL-10 in visceral adipose tissue and serum, but showed no effect on serum insulin. While nitric oxide synthase-2 and arginase-1 remained unaltered, M2 macrophage treatment restored AKT phosphorylation in visceral adipose tissue. Conclusion: M2 macrophage treatment abolishes glucose intolerance by increasing IL-10 and phosphorylated AKT.

Mechanisms of natural resistance of Balb/c mice to experimental liver amoebiasis.

Entamoeba histolytica is the parasite responsible for human amoebiasis. The analysis of the natural resistance mechanisms of some rodents to amoebic liver abscess (ALA) may reveal alternative pathogenicity mechanisms to those previously discovered in the experimental model of ALA in hamsters. In this work the natural resistance of BALB/c mice to ALA was explored by performing: (i) in vivo chemotaxis analysis with a specifically designed chamber; (ii) in vitro amoebic survival in fresh and decomplemented serum; (iii) histological temporal course analysis of ALA development in mice with different treatments (hypocomplementemic, hyperimmune and treated with iNOS and NADPH oxidase inhibitors) and (iv) mouse liver amoebic infection by both in situ implantation of ALA from hamsters and inoculation of parasites into the peritoneal cavity. The results show that E. histolytica clearance from the mouse liver is related to a low chemotactic activity of complement, which results in poor inflammatory response and parasite inability to cause tissue damage. Also, the absence of amoebic tropism for the mouse liver is correlated with resistance to experimental liver amoebiasis.

Complement is a rat natural resistance factor to amoebic liver infection.

Amoebiasis is a parasitic disease caused by Entamoeba histolytica This illness is prevalent in poor countries causing 100,000 deaths worldwide. Knowledge of the natural resistance mechanisms of rats to amoebic liver abscess (ALA) development may help to discover new pathogenic factors and to design novel therapeutic strategies against amoebiasis. In this work, histologic analyses suggested that the complement system may play a central role in rat natural resistance to ALA. E. histolytica trophozoites disappeared from rat liver within 6 h post-infection with minimal or no inflammatory infiltrate. In vitro findings indicate that rat complement was lethal for the parasite. Furthermore, hamsters became resistant to ALA by intravenous administration of fresh rat serum before infection. The amoebicidal potency of rat complement was 10 times higher than hamster complement and was not related to their respective CH50 levels. The alternative pathway of complement plays a central role in its toxicity to E. histolytica since trypan blue, which is a C3b receptor inhibitor, blocks its amoebicidal activity. These results suggest that amoebic membrane affinity, high for C3b and/or low for Factor H, in comparison with the hamster ones, may result in higher deposition of membrane complex attack on parasite surface and death.

The oxygen reduction pathway and heat shock stress response are both required for Entamoeba histolytica pathogenicity.

Several species belonging to the genus Entamoeba can colonize the mouth or the human gut; however, only Entamoeba histolytica is pathogenic to the host, causing the disease amoebiasis. This illness is responsible for one hundred thousand human deaths per year worldwide, affecting mainly underdeveloped countries. Throughout its entire life cycle and invasion of human tissues, the parasite is constantly subjected to stress conditions. Under in vitro culture, this microaerophilic parasite can tolerate up to 5 % oxygen concentrations; however, during tissue invasion the parasite has to cope with the higher oxygen content found in well-perfused tissues (4-14 %) and with reactive oxygen and nitrogen species derived from both host and parasite. In this work, the role of the amoebic oxygen reduction pathway (ORP) and heat shock response (HSP) are analyzed in relation to E. histolytica pathogenicity. The data suggest that in contrast with non-pathogenic E. dispar, the higher level of ORP and HSPs displayed by E. histolytica enables its survival in tissues by diminishing and detoxifying intracellular oxidants and repairing damaged proteins to allow metabolic fluxes, replication and immune evasion.

Severity of non-alcoholic fatty liver disease is associated with high systemic levels of tumor necrosis factor alpha and low serum interleukin 10 in morbidly obese patients.

Morbid obesity has been shown to increase the risk to develop hepatic steatosis, also referred to as non-alcoholic fatty liver disease (NAFLD). Emerging evidence suggests that the severity of NAFLD may associate with increased serum levels of inflammatory markers as well as decreased concentration of mediators with anti-inflammatory actions, such as tumor necrosis factor alpha (TNF-α) and interleukin (IL) 10, respectively. We thus examined the serum levels of TNF-α and IL-10 in 102 morbidly obese women and men (body mass index > 40 kg/m(2)), exhibiting different grades of NAFLD. Blood glucose, glycated hemoglobin, insulin, the homeostatic model assessment of insulin resistance (HOMA-IR), total cholesterol, triglycerides, high- and low-density lipoproteins, parameters of liver function, TNF-α, and IL-10 were measured in each subject. The stage of NAFLD was estimated by abdominal ultrasound imaging. In comparison with morbidly obese subjects without steatosis, morbidly obese patients with NAFLD showed increased age (39.23 ± 9.80 years), HOMA-IR (6.74 ± 1.62), total cholesterol (219.7 ± 9.58 mg/dl), aspartate aminotransferase (36.25 ± 3.24 UI/l), gamma-glutamyl transpeptidase (37.12 ± 3.41 UI/l), and TNF-α (37.41 ± 1.72 pg/ml) as well as decreased serum levels of IL-10 (61.05 ± 2.43 pg/ml). Interestingly, the systemic levels of TNF-α increased, while IL-10 decreased in accordance with the severity of NAFLD, which supports a role for systemic inflammatory mediators in promoting steatosis progression. Further clinical prospective studies need to be addressed to elucidate the role of TNF-α and IL-10 in the development of NAFLD while also establishing their clinical utility in the assessment of morbidly obese patients at higher risk to develop severe steatosis.

Maintenance of intracellular hypoxia and adequate heat shock response are essential requirements for pathogenicity and virulence of Entamoeba histolytica.

Adhesion to cells, cytotoxicity and proteolysis are functions required for virulence and pathogenicity of Entamoeba histolytica. However, there was no correlation between these in vitro functions and the early elimination of non-pathogenic E. dispar and non-virulent E. histolytica (nvEh) in experimental amoebic liver abscesses developed in hamsters. Thus, additional functions may be involved in amoebic pathogenicity and virulence. In the present study, an integral experimental assessment, including innovative technologies for analyses of amoebal pathophysiology, cell biology, biochemistry and transcriptomics, was carried out to elucidate whether other cellular processes are involved in amoebal pathogenicity and virulence. In comparison with virulent E. histolytica, the data indicated that the main reasons for the early clearance of nvEh from hamster liver are decreased intracellular H2 O2 detoxification rate and deficient heat shock protein expression, whereas for E. dispar, it is a relatively lower capacity for O2 reduction. Therefore, maintenance of an intracellular hypoxic environment combined with the induction of an adequate parasite response to oxidative stress are essential requirements for Entamoeba survival in the liver, and therefore for pathogenicity.

Bone lineage proteins in the entheses of the midfoot in patients with spondyloarthritis.

OBJECTIVE: Patients with juvenile-onset spondyloarthritis (SpA) may develop ankylosis of the midfoot resembling the spinal changes seen in patients with ankylosing spondylitis (AS). The study of the histopathology of the feet of patients with tarsitis could help us understand the pathogenesis of bone formation in affected structures in the SpA. The objective of our study was to describe the histopathologic characteristics of the midfoot in patients with tarsitis associated with SpA. METHODS: We obtained synovial sheaths, entheses, and bone samples from 20 patients with SpA with midfoot pain/tenderness and swelling. Tissue samples underwent H&E staining; immunohistochemistry for CD3, CD4, CD8, CD68, and CD20 cell identification; and immunofluorescence for bone lineage proteins, including osteocalcin, osteopontin, parathyroid hormone-related protein, bone sialoprotein, and alkaline phosphatase. RESULTS: Slight edema and hyalinization were found in some tendon sheaths, and few inflammatory cells were detected in the entheses. In bones, we found some changes suggesting osteoproliferation, including endochondral and intramembranous ossification, but no inflammatory cells. In entheses showing bone proliferation, we detected osteocalcin and osteopontin in cells with a fibroblast-mesenchymal phenotype, suggesting the induction of entheseal cells toward an osteoblast phenotype. CONCLUSION: Osteoproliferation and abnormal expression of bone lineage proteins, but no inflammatory infiltration, characterize midfoot involvement in patients with SpA. In this sense, tarsitis (or ankylosing tarsitis) resembles the involvement of the spine in patients with AS. Ossification may be in part explained by the differentiation of mesenchymal entheseal cells toward the osteoblastic lineage.

Influence of serological factors and BMI on the blood pressure/hematocrit association in healthy young men and women.

The association between mean arterial blood pressure (MAP) and hematocrit (Hct) as a surrogate for blood viscosity was investigated in a young (average 20.0±2.3 years), healthy population of 174 men and 442 women. Health status was assessed by clinical examination and serological evaluation. Individuals with severe anemia or hemoconcentration, prior traumas or major surgical intervention, smokers, and pregnant or lactating women were excluded from the study. The MAP/Hct association was positive and significant (P=0.04) for women and negative, albeit not significantly so, for men. The MAP/Hct association was also evaluated in subgroups of the same population with a progressive step-by-step exclusion of: individuals with cholesterol >200 mg/dL; triglycerides >200 mg/dL; body mass index >25 kg/m(2); and glucose >100 mg/dL. This consecutively reduced the strength of the positive MAP/Hct association in women, which became negative - although not significantly so - when all anomalously high factors were excluded. The same trend was found in men. Our study indicates that previously reported positive trends in the relationship between the MAP and Hct in the population are not present in a young, healthy population of men or women that excludes individuals with the confounding factors of above normal serological values and BMI.

Neuroprotective effect of silymarin in a MPTP mouse model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease secondary to the loss of dopaminergic neurons in the substantia nigra. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces in mice and primates histopathological changes similar to PD in humans. A common feature of PD and MPTP models is neuronal death and dopamine depletion. Silymarin is a complex of flavonolignans derived from the seeds of the plant Silybum marianum and has mainly antioxidant, anti-inflammatory, cytoprotective and neuroprotective effects. In order to explore whether silymarin has a neuroprotective effects in a mouse model of PD we determined the concentration of striatal dopamine by HPLC, the number of apoptotic cells by in situ Tunel assay and the number of tyrosine hydroxylase positive neurons by immunohistochemistry in substantia nigra of vehicle-treated, silymarin-treated, MPTP-intoxicated and MPTP-silymarin treated C57BL/6J male mice. MPTP (30 mg/kg) and silymarin doses (25, 50, 100, 200, 250, 300 or 400mg/kg) were administered intraperitoneally once daily for five consecutive days. Silymarin treatment showed a non-monotonic dose-response curve and only 50 and 100mg/kg doses preserved dopamine levels (62% and 69%, respectively) after MPTP intoxication. Additionally, 100mg/kg silymarin treatment significantly diminished the number of apoptotic cells and preserved dopaminergic neurons in the substantia nigra of MPTP-intoxicated mice. These results show the neuroprotective properties of 100mg/kg silymarin and may be of interest in the treatment of PD.

Amibiasis: mecanismos moleculares de la patogenicidad de Entamoeba histolytica / Amebiasis: Molecular mechanisms of Entamoeba histolytica pathogenicity

La amibiasis es un padecimiento que afecta al 10% de la población mundial, y puede tener un comportamiento muy diverso, tanto en el intestino como en diversos órganos (hígado, pulmones, cerebro, piel). Se conoce su ciclo biológico, los síntomas y signos de su penetración al organismo, así como su diagnóstico y tratamiento, pero aún hay controversias sobre los mecanismos moleculares de la patogenicidad de la E. Histolítica, para lo cual se ha utilizado en particular el absceso hepático experimental en Hamsters (AHAH). Durante mucho tiempo se sostuvo que la patogenicidad de E. Histolítica se debía a su capacidad para destruir tejidos, pero encontramos que la E. Histolítica virulenta, per se es incapaz de causar daño al hígado del hámster leucopénico. Este estudio se dedicó a estudiar los mecanismos de virulencia de la amiba mediante la comparación funcional y molecular entre E. Histolítica virulenta y E. Histolítica no virulenta. Encontramos que la virulencia de este parásito no se puede explicar solamente por la actividad de sus moléculas citotóxicas (adhesinas, fosfolipasas y ameboporos) o proteolíticas (proteasas), y los hallazgos sugieren que cuando las amibas virulentas arriban al hígado del hámster y se encuentran una concentración tóxica de oxígeno, éste las sensibiliza a la lisis por el complemento, el peróxido de hidrógeno y el ácido hipocloroso. Las consecuencias de estos hallazgos pueden abrir nuevas perspectivas para el diseño de terapias alternativas para el tratamiento de este padecimiento.

Amoebiasis is a disease that affects 10 % of the world population, and it may have a different behavior when attacks bowels, liver, lungs, brain, etc. Its biological cycle is well known, as well as its symptoms and signs of its penetration into those organs, its diagnosis and treatment, but it is still a controversy on the molecular mechanism of its pathogenesis; to study them it, the experimental hepatic abscess in hamsters has been employed. For years it was considered that the pathogenicity of E. Histolítica was due to its capacity to destroy tissues, but we found that virulent E. Histoliticaperse is unable to produce liver damage in leucopenic hamster; we therefore studied the mechanisms of virulence of the amoeba by functional and molecular comparison between virulent and non virulent E. Histolitica. We found that the parasit virulence cannot be explained only by the activity of citotoxic or proteolytic molecules (adhesines, phospholypases and amebopores, or proteases), and the findings suggest that when amoebas arrives to the hamster liver and find a toxic concentration of oxygen, this sensibilizes them to lysis by complement, hydrogen peroxide and hypoclorose acid. The consequences of those findings may open new perspectives for the design of new therapies for the treatment of this disease.

Cellular senescence in livers from children with end stage liver disease.

BACKGROUND: Senescent cells occur in adults with cirrhotic livers independent of the etiology. AIM: Investigate the presence rate of cellular senescence and expression of cell cycle check points in livers from children with end stage disease. METHODOLOGY/PRINCIPAL FINDINGS: Livers of five children aged three years or less undergoing liver transplantation due to tyrosinemia (n = 1), biliary atresia (n = 2), or fulminant hepatitis (n = 2) were analyzed for senescence associated beta-galactosidase (SA-betagal) activity and p16INK4a, p21cip1 and p53. All livers displayed positive cellular staining for SA-betagal in the canals of Hering and interlobular biliary ducts. In the presence of cirrhosis (3/5 cases) SA-betagal was found at the cholangioles and hepatocytes surrounding the regenerative nodules. Children with fulminant hepatic failure without cirrhosis had significant ductular transformation with intense SA-betagal activity. No SA-betagal activity was evident in the fibrous septa. Staining for p53 had a similar distribution to that observed for SA-betagal. Staining for p16(INK4a) and p21(cip1) was positive in the explanted liver of the patient with tyrosinemia, in the hepatocytes, the canals of Hering, cholangioles and interlobular bile ducts. In the livers with fulminant hepatitis, p21(cip1) staining occurred in the areas of ductular transformation and in the interlobular bile ducts. CONCLUSIONS/SIGNIFICANCE: Cellular senescence in livers of children with end stage disease is associated with damage rather than corresponding to an age dependent phenomenon. Further studies are needed to support the hypothesis that these senescence markers correlate with disease progression.

Molecular nature of virulence in Entamoeba histolytica.

For many years virulence of pathogenic Entamoeba histolytica has been attributed to the capacity of the parasite to destroy tissues through the expression and/or secretion of various molecules. Such view is supported mainly by in vitro experimentation, whereas data obtained by using animal models of the disease have clearly demonstrated that the host's inflammatory response is primarily responsible for tissue damage. This review analyzes the content and/or activity of some of the presumed toxic amebic molecules present in amebic strains with different degrees of virulence compared to various parasite in vitro functions that are supposed to correlate with in vivo virulence. The analysis suggests that amebic virulence is primarily determined by the parasite's capacity to adapt and survive the aerobic conditions present in animal tissues. This initial episode in the host-parasite relationship is an absolute requirement for the further development of tissue lesions, which result from the concerted action of many molecules derived from both, the host and the parasite.

Entamoeba histolytica: oxygen resistance and virulence.

Entamoeba histolytica virulence has been attributed to several amoebic molecules such as adhesins, amoebapores and cysteine proteinases, but supporting evidence is either partial or indirect. In this work we compared several in vitro and in vivo features of both virulent E. histolytica (vEh) and non-virulent E. histolytica (nvEh) axenic HM-1 IMSS strains, such as complement resistance, proteinase activity, haemolytic, phagocytic and cytotoxic capacities, survival in mice caecum, and susceptibility to O(2). The only difference observed was a higher in vitro susceptibility of nvEh to O(2). The molecular mechanism of that difference was analyzed in both groups of amoebae after high O(2) exposure. vEh O(2) resistance correlated with: (i) higher O(2) reduction (O(2)(-) and H(2)O(2) production); (ii) increased H(2)O(2) resistance and thiol peroxidase activity, and (iii) reversible pyruvate: ferredoxin oxidoreductase (PFOR) inhibition. Despite the high level of carbonylated proteins in nvEh after O(2) exposure, membrane oxidation by reactive oxygen species was not observed. These results suggest that the virulent phenotype of E. histolytica is related to the greater ability to reduce O(2) and H(2)O(2) as well as PFOR reactivation, whereas nvEh undergoes irreversible PFOR inhibition resulting in metabolic failure and amoebic death.

Entamoeba histolytica: apoptosis induced in vitro by nitric oxide species.

Apoptosis has been described in some parasites like Leishmania, Trypanosoma, and Trichomonas. This phenomenon has not been observed yet in Entamoeba histolytica. This work analyzed the in vitro effect of sodium nitroprusside, sodium nitrite and sodium nitrate (NOs) on E. histolytica apoptosis. Parasites incubated for 1h with NOs revealed apoptosis 6h later (95% viability), demonstrated by YOPRO-1, TUNEL, DNA fragmentation and low ATP levels. The caspase inhibitor Z-VAD-FMK inhibited total intracellular cysteine protease activity (CPA) but had no effect on apoptosis. When treated with NOs some amebic functions like complement resistance and hemolytic activity decreased but CPA and erythrophagocytosis remained unchanged. After treatment in vitro with NOs, parasite death was almost complete at 24h; but when injected into hamster livers they disappeared in less than 6h. These results show that apoptosis is induced in vitro by NOs in E. histolytica and renders them incapable of surviving in hamster's livers.

Hepatocyte production of modulators of extracellular liver matrix in normal and cirrhotic rat liver.

In the present study, we found collagenolytic and gelatinolytic activity in the supernatants of hepatocyte cultures from rats with experimental CCl(4)-induced liver cirrhosis, in levels significantly higher than in comparable supernatants of hepatocyte cultures from normal rats. In addition, we clearly detected the messenger ribonucleic acids (mRNA) of four matrix metalloproteinases (MMP-2, MMP-3, MMP-10, and MMP-13) and of two tissue inhibitors of matrix metalloproteinases (TIMP-1 and TIMP-2) in hepatocytes from both normal and cirrhotic rats by RT-PCR and by in situ hybridization. Finally, we demonstrated MMP-2, MMP-3, and MMP-13 and TIMP-1 and TIMP-2 proteins in the same hepatocyte preparations by immunostaining. We conclude that rat hepatocytes produce the major enzymes and inhibitors involved in liver ECM modulation and therefore suggests that they might participate actively in the pathophysiology of liver cirrhosis in rats.