Dynamic changes in hepatic DNA methylation during the development of nonalcoholic fatty liver disease induced by a high-sugar diet.

DNA methylation is an important epigenetic mechanism of gene expression control. The present study aimed to evaluate the temporal effect of isocaloric high-sugar diet (HSD) intake on the development of nonalcoholic fatty liver disease (NAFLD) and the role of DNA methylation in this event. Newly weaned Wistar rats were divided into eight groups and fed a standard chow diet or an HSD ad libitum for 4 weeks, 8 weeks, 15 weeks, and 18 weeks. After the experimental periods, the animals were euthanized and their livers were removed for histological analysis, gene expression of maintenance methylase (Dnmt1), de novo methylases (Dnmt3a and Dnmt3b), demethylases (Tet2 and Tet3) of DNA, and global DNA methylation. HSD intake led to the gradual development of NAFLD. HSD intake for 18 weeks was associated with downregulation of Dnmt1 expression and global DNA hypomethylation; these results were negatively correlated with more severe steatosis scores observed in these animals. The HSD consumption for 18 weeks was also associated with a decrease in Dnmt3a and Tet2 expression. Interestingly, the expression of de novo methyltransferase Dnmt3b was reduced by HSD during all experimental periods. Together, these results indicate that the downregulation of de novo DNA methylation, Dnmt3b, induced by HSD is the primary factor in the development of NAFLD. On the other hand, disease progression is associated with downregulation of maintenance DNA methylation and global DNA hypomethylation. These results suggest a link between the dynamic changes in hepatic DNA methylation and the development of NAFLD induced by an HSD intake.

Benznidazole Treatment: Time- and Dose-Dependence Varies with the Trypanosoma cruzi Strain.

As the development of new drugs for Chagas disease is not a priority due to its neglected disease status, an option for increasing treatment adherence is to explore alternative treatment regimens, which may decrease the incidence of side effects. Therefore, we evaluated the efficacy of different therapeutic schemes with benznidazole (BNZ) on the acute and chronic phases of the disease, using mice infected with strains that have different BNZ susceptibilities. Our results show that the groups of animals infected by VL-10 strain, when treated in the chronic phase with a lower dose of BNZ for a longer period of time (40 mg/kg/day for 40 days) presented better treatment efficacy than with the standard protocol (100 mg/kg/day for 20 days) although the best result in the treatment of the animals infected by the VL-10 strain was with100 mg/kg/day for 40 days. In the acute infection by the Y and VL-10 strains of T. cruzi, the treatment with a standard dose, but with a longer time of treatment (100 mg/kg/day for 40 days) presented the best results. Given these data, our results indicate that for BNZ, the theory of dose and time proportionality does not apply to the phases of infection.

A High-Fat Diet Exacerbates the Course of Experimental Trypanosoma cruzi Infection That Can Be Mitigated by Treatment with Simvastatin.

The protozoan Trypanosoma cruzi is responsible for triggering a damage immune response in the host cardiovascular system. This parasite has a high affinity for host lipoproteins and uses the low-density lipoprotein (LDL) receptor for its invasion. Assuming that the presence of LDL cholesterol in tissues could facilitate T. cruzi proliferation, dietary composition may affect the parasite-host relationship. Therefore, the aim of this study was to evaluate myocarditis in T. cruzi-infected C57BL/6 mice-acute phase-fed a high-fat diet and treated with simvastatin, a lipid-lowering medication. Animals (n = 10) were infected with 5 × 103 cells of the VL-10 strain of T. cruzi and treated or untreated daily with 20 mg/kg simvastatin, starting 24 h after infection and fed with a normolipidic or high-fat diet. Also, uninfected mice, treated or not with simvastatin and fed with normolipidic or high-fat diet, were evaluated as control groups. Analyses to measure the production of chemokine (C-C motif) ligand 2 (CCL2), interferon- (IFN-) γ, interleukin- (IL-) 10, and tumor necrosis factor (TNF); total hepatic lipid dosage; cholesterol; and fractions, as well as histopathological analysis, were performed on day 30 using cardiac and fat tissues. Our results showed that the high-fat diet increased (i) parasite replication, (ii) fat accumulation in the liver, (iii) total cholesterol and LDL levels, and (iv) the host inflammatory state through the production of the cytokine TNF. However, simvastatin only reduced the production of CCL2 but not that of other inflammatory mediators or biochemical parameters. Together, our data suggest that the high-fat diet may have worsened the biochemical parameters of the uninfected and T. cruzi-infected animals, as well as favored the survival of circulating parasites.

The Schistosomiasis SpleenOME: Unveiling the Proteomic Landscape of Splenomegaly Using Label-Free Mass Spectrometry.

Schistosomiasis is a neglected parasitic disease that affects millions of people worldwide and is caused by helminth parasites from the genus Schistosoma. When caused by S. mansoni, it is associated with the development of a hepatosplenic disease caused by an intense immune response to the important antigenic contribution of adult worms and to the presence of eggs trapped in liver tissue. Although the importance of the spleen for the establishment of immune pathology is widely accepted, it has received little attention in terms of the molecular mechanisms operating in response to the infection. Here, we interrogated the spleen proteome using a label-free shotgun approach for the potential discovery of molecular mechanisms associated to the peak of the acute phase of inflammation and the development of splenomegaly in the murine model. Over fifteen hundred proteins were identified in both infected and control individuals and 325 of those proteins were differentially expressed. Two hundred and forty-two proteins were found upregulated in infected individuals while 83 were downregulated. Functional enrichment analyses for differentially expressed proteins showed that most of them were categorized within pathways of innate and adaptive immunity, DNA replication, vesicle transport and catabolic metabolism. There was an important contribution of granulocyte proteins and antigen processing and presentation pathways were augmented, with the increased expression of MHC class II molecules but the negative regulation of cysteine and serine proteases. Several proteins related to RNA processing were upregulated, including splicing factors. We also found indications of metabolic reprogramming in spleen cells with downregulation of proteins related to mitochondrial metabolism. Ex-vivo imunophenotyping of spleen cells allowed us to attribute the higher abundance of MHC II detected by mass spectrometry to increased number of macrophages (F4/80+/MHC II+ cells) in the infected condition. We believe these findings add novel insights for the understanding of the immune mechanisms associated with the establishment of schistosomiasis and the processes of immune modulation implied in the host-parasite interactions.

Understanding global changes of the liver proteome during murine schistosomiasis using a label-free shotgun approach.

Schistosomiasis is an endemic disease affecting over 207 million people worldwide caused by helminth parasites of the genus Schistosoma. In Brazil the disease is responsible for the loss of up to 800 lives annually, resulting from the desabilitating effects of this chronic condition. In this study, we infected Balb/c mice with Schistosoma mansoni and analysed global changes in the proteomic profile of soluble liver proteins. Our shotgun analyses revealed predominance of up-regulation of proteins at 5weeks of infection, coinciding with the onset of egg laying, and a remarkable down-regulation of liver constituents at 7weeks, when severe tissue damage is installed. Representatives of glycolytic enzymes and stress response (in particular at the endoplasmic reticulum) were among the most differentially expressed molecules found in the infected liver. Collectively, our data contribute over 70 molecules not previously reported to be found at altered levels in murine schistosomiasis to further exploration of their potential as biomarkers of the disease. Moreover, understanding their intricate interaction using bioinformatics approach can potentially bring clarity to unknown mechanisms linked to the establishment of this condition in the vertebrate host. SIGNIFICANCE: To our knowledge, this study refers to the first shotgun proteomic analysis to provide an inventory of the global changes in the liver soluble proteome caused by Schistosoma mansoni in the Balb/c model. It also innovates by yielding data on quantification of the identified molecules as a manner to clarify and give insights into the underlying mechanisms for establishment of Schistosomiasis, a neglected tropical disease with historical prevalence in Brazil.

The crowding effect in Ancylostoma ceylanicum: density-dependent effects on an experimental model of infection.

This study compared the course of Ancylostoma ceylanicum infection in hamsters infected with different inocula and the consequences for the host and helminth populations. The average of adult worms recovered, according to the number of third stage larva used, were 28.0, 24.8, 24.6, and 24.8% to inocula size of 25 L3, 75 L3, 125 L3, and 250 L3, respectively. The size of the inoculum did not affect the establishment, survival, or fecundity of adult helminths. Reductions in the red blood cell and hemoglobin levels in the infected group were inversely proportional to the number of white blood cells. Moreover, differential cell counting revealed a positive correlation between the worm load and leucocyte numbers. The humoral response against excretion-secretion antigens was more robust and sensitive compared with the response against crude extract, with no direct linear correlation with the number of worms. The effect of the population density was more evident in females.

Molecular and biological characterization of Trypanosoma cruzi strains isolated from children from Jequitinhonha Valley, State of Minas Gerais, Brazil

Introduction The biological diversity of Trypanosoma cruzi strains plays an important role in the clinical and epidemiological features of Chagas disease. Methods Eight T. cruzi strains isolated from children living in a Chagas disease vector-controlled area of Jequitinhonha Valley, State of Minas Gerais, Brazil, were genetically and biologically characterized. Results The characterizations demonstrated that all of the strains belonged to T. cruzi II, and showed high infectivity and a variable mean maximum peak of parasitemia. Six strains displayed low parasitemia, and two displayed moderate parasitemia. Later peaks of parasitemia and a predominance of intermediate and large trypomastigotes in all T. cruzi strains were observed. The mean pre-patent period was relatively short (4.2±0.25 to 13.7±3.08 days), whereas the patent period ranged from 3.3±1.08 to 34.5±3.52 days. Mortality was observed only in animals infected with strain 806 (62.5%). Histopathological analysis of the heart showed that strains 501 and 806 caused inflammation, but fibrosis was observed only in animals infected with strain 806. Conclusions The results indicate the presence of an association between the biological behavior in mice and the genetic characteristics of the parasites. The study also confirmed general data from Brazil where T. cruzi II lineage is the most prevalent in the domiciliary cycle and generally has low virulence, with some strains capable of inducing inflammatory processes and fibrosis. .

Prednisolone and cyclosporine A: effects on an experimental model of ancylostomiasis.

Corticosteroids and cyclosporine A (CsA) are important clinical immunosuppressive drugs used in the maintenance of organ transplants and in suppressing undesired autoimmune or allergic immune responses. To study the effect of CsA and prednisolone on the course of an Ancylostoma ceylanicum infection, hamsters were treated with commercially available prednisolone or CsA. For both drugs, half the recommended dose was sufficient to inhibit the proliferation of more than 70% of hamster lymph node cells. There was no difference in the recovery of adult worms; however, animals treated with prednisolone presented with low egg counts in the feces. Infection with A. ceylanicum resulted in an increase in specific antibodies against adult worm antigens, but hamsters treated with either drug presented with lower IgG titers. We observed that A. ceylanicum infection caused peripheral cellular immune suppression, which is characterized by a reduction in the total white cell count, neutropenia and lymphopenia. We also observed a lymphoplasmacytic pattern and few eosinophils in the mucosal inflammatory infiltrate for all the animals. The animals treated with prednisolone showed changes in the architecture of the intestine, including the loss of the mucosa, intense congestion and inflammation. In spleen, we observed hyperplasia of white pulp in all infected animals; in addition, there was a loss of tissue architecture in the animals treated with prednisolone. In conclusion, this work shows that an A. ceylanicum infection leads to acute peripheral cellular immune suppression in hamsters but not humoral immune suppression and that CsA treatment does not interfere with the process of infection. However, prednisolone treatment causes intestinal injury, what could hamper the parasite attachment to the intestinal wall, and as a result affects copulation and, consequently, decreases the number of eggs eliminated in the feces. Moreover, the possibility that the drug can also be exerting an effect on female fertility should be considered.

Hookworm products ameliorate dextran sodium sulfate-induced colitis in BALB/c mice.

BACKGROUND: Several lines of evidence have shown that helminthiasis can significantly reduce disease severity in animal models of intestinal inflammation, airway inflammation/hyperreactivity, diabetes, and multiple sclerosis. Identification and characterization of helminth-derived immunomodulatory molecules that contribute to anticolitis effects could lead to new therapeutic approaches in inflammatory bowel diseases (IBDs) without the need for helminth infection. We evaluated the therapeutic potential of adult human hookworm, Ancylostoma ceylanicum, crude (Aw) and excreted/secreted (ES) products on dextran sulfate sodium (DSS)-induced colitis in BALB/c mice. METHODS: Colitis was induced by 5% DSS oral administration for 7 days. Clinical disease severity was monitored daily during concomitant intraperitoneal treatment with helminth-derived products. Additionally, several pathways of immunological modulation induced by A. ceylanicum products (MPO, EPO, Th1, Th2, and Th17 cytokine responses) in the inflamed intestinal microenvironment were assessed. Finally, the histopathological profile of the colon was characterized. RESULTS: Hookworm products are able to modulate the potent proinflammatory response induced by DSS, mainly through the downregulation of Th1 and Th17 cytokines. These proteins also reduce clinical and colonic microscopic inflammation scores as well as EPO and MPO activity. CONCLUSIONS: Ancylostoma ceylanicum Aw and ES mediators have an important therapeutic potential in experimental colitis in mice, which may provide a more socially acceptable form of therapy for patients with IBDs as opposed to using living worms. Our results support the urgency of further isolation and recombinant expression of active hookworm products responsible for the beneficial effects on colitis.