Exploring the possible role of lysine acetylation on Entamoeba histolytica virulence: a focus on the dynamics of the actin cytoskeleton.

Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

Altered expressions of MMP-2, MMP-9, and TIMP-2 in placentas from women exposed to lead.

Experimental studies have shown that prenatal exposure to lead (Pb) produces morphological changes related to extracellular matrix remodelling. To analyse whether the matrix metalloproteinases (MMPs), particularly MMP-2, MMP-9, and the tissue inhibitor of metalloproteinases-2 (TIMP-2), are associated with morphological alterations found in placentas, the expression of these enzymes was evaluated by immunohistochemical and image analyses in placentas of women with histories of environmental exposure to Pb. The median maternal concentration of Pb in blood was 4.68 µg/dL (x = 5.85 ± 6.48 µg/dL). Significant differences related to the exposure to Pb were not detected in newborn or placenta weight. MMP-2, MMP-9, and TIMP-2 were expressed in the syncytiotrophoblast layer of placental villi. A significant increase in both MMP-2 and MMP-9 was observed in placentas of women with concentrations of Pb in blood ≥4.68 µg/dL (p = 0.01 and 0.03 for MMP-2 and MMP-9, respectively) and decrease in TIMP-2 expression (p = 0.01) resulted in a significant increase in MMP-2/TIMP-2 ratio (p < 0.01). Increased expression of MMPs may be induced to aid in repairing placental tissue damaged by the exposure to Pb and that TIMP-2 decreases its expression to permit tissue repair. Increased expression of MMPs may be important to consider as a mechanism for generating placental abnormalities and in the induction of preterm delivery or abortion.

Decrease of muscarinic cholinergic receptors expression in placenta from rats exposed to methyl parathion.

Placental transfer of methyl parathion (MP), an organophosphate pesticide, could involve effects on cholinergic system. To analyze whether placental cholinergic system is altered by prenatal exposure to MP, expression of muscarinic cholinergic receptors (M1 and M2 subtypes; mAChR) was determined in pregnant rats exposed to MP at 0.0, 1.0, 1.5, and 2.0 mg/kg. An immunohistochemical analysis for M1 and M2 mAChR was performed, and the density of the mAChR signal was measured by image analysis. M1 and M2 mAChR were found in the trophoblast present in the labyrinth, with an 18% predominance of M2 over M1 in the non-exposed group. The expression of M1 and M2 mAChR in placentas exposed to MP showed a decrease when compared with the non-exposed group (P < 0.05); a dose-response effect was not detected. These results demonstrate that prenatal exposure to MP causes changes in the placental expression of mAChR M1 and M2, suggesting that related placental cholinergic functions could be affected.

Placental immaturity and hyperinsulinaemia in full-term newborns.

BACKGROUND: Evidence from large studies suggests that low birthweight is a risk factor for cardiovascular disease and glucose metabolism disorders in adulthood, but the physiological mechanisms involved in intrauterine growth conditioning low birthweight are not completely understood. The objectives of this study were to determine whether placental immaturity (PI), defined as the lower quartile of placental maturity index (PMI), is associated to hyperinsulinaemia at birth and to identify the risk factors associated with PI. MATERIALS AND METHODS: Cross-sectional study conducted at medical research units of two Mexican general hospitals. A total of 272 full-term newborns with gestational age >/= 38 and < 41 weeks were allocated into the corresponding group according to the quartile distribution of PMI. Data from the lower (PMI < 13.3) and higher quartile (PMI >/= 24.3) were compared. The PMI was estimated by dividing the number of epithelial plates by the average thickness of the epithelial plate. Serum measures included cord glucose and insulin levels of the newborns at birth. RESULTS: A total of 74 (27.2%) children had hyperinsulinaemia at birth, of them 47 (63.5%) with PI. The adjusted multiple regression analysis showed a strong association between PI and hyperinsulinaemia at birth [odds ratio (OR) 2.6; CI 95% 1.3-4.3). Additional adjusted analysis showed that both mother's age

Entamoeba histolytica: inflammatory process during amoebic liver abscess formation involves cyclooxygenase-2 expression in macrophages and trophozoites.

It has been demonstrated that expression of cyclooxygenase-2 (COX-2) isoform is induced by Entamoeba histolytica in macrophages and polymorphonuclear cells during amoebic liver abscess (ALA) formation in hamsters. Trophozoites present in the lesion were also positive for COX-2 signal. However, no cross reactivity of the anti-COX-2 antibody with protein extract of cultivated trophozoites was found. To clarify if trophozoites are involved in PGE(2) production during ALA development, COX-2 expression was detected by in situ hybridization and RT-PCR in liver tissue from intrahepatically infected hamsters. COX-2 mRNA was in polymorphonuclear cells since 4h postinfection, and subsequently, local macrophages expressed COX-2 mRNA in a similar way. Additionally, a positive signal for COX-2 mRNA expression was detected in E. histolytica trophozoites, suggesting that, in vivo, parasite COX expression may be an important mechanism to promote inflammation.

In vitro assessment of the genotoxicity of ethyl paraoxon in newborns and adults.

This in vitro experiment measured the genotoxic effects of ethyl paraoxon, the active metabolite of ethyl parathion. To assess genotoxicity, we used the micronuclei (MN) technique by blocking cytokinesis, and the 'comet' assay. We cultured peripheral blood samples from healthy adults and umbilical cord blood samples from four clinically healthy newborns to identify the frequency of MN. After 48 hours, we added the following ethyl paraoxon concentrations to the cultures: 0.0, 0.075, 0.100, 0.160, and 0.200 microg/mL. For the comet assay, following Singh's technique, we treated the blood samples for 2 hours with similar doses of the metabolite. The comet assay results, at a concentration of 0.075 microg/mL, showed that ethyl paraoxon causes a greater DNA migration that followed a dose-response pattern, a greater intensity being observed in lymphocytes from newborns. A comparison of the treatment and control groups indicated that only the 0.200 microg/mL concentration produced a slight increase in MN. In conclusion, our study identified primary DNA damage due to ethyl paraoxon, with a major effect on newborn lymphocytes, as well as an effect on the frequency of MN in the study groups at high concentrations only.

Entamoeba histolytica: induction of cyclooxygenase-2 expression during amoebic liver abscess formation in hamsters (Mesocricetus auratus).

Experimental amoebic liver abscess in hamsters curses with an increase in both, systemic levels of prostaglandin E2 (PGE(2)) and local cyclooxygenase activity in liver microsomes. The cellular source of PGE(2) and the isoform of cyclooxygenase responsible are not completely evidenced. Cyclooxygenase-2 (COX-2) protein and gene expression were demonstrated on macrophages and polymorphonuclear cells as a result of Entamoeba histolytica infection in hamsters at 2, 4, and 7 days postinfection by immunohistochemistry and RT-PCR. E. histolytica trophozoites located in the lesion showed a strong positive signal for COX-2, however the enzyme was not detected in cultured trophozoites by Western blot. Our results indicate that the increment in PGE(2) is the result of COX-2 activity from cells of the reticuloendothelial system and reinforce the possibility that PGE(2) production by enzyme induction in macrophages may be a mechanism by which E. histolytica modulates the host immune response in this parasitic infection.

Liver function tests during amoebic liver abscess formation in indomethacin-treated hamsters.

Establishment of Entamoeba histolytica infection is facilitated through macrophage effector disruption by a prostaglandin E2 (PGE2)-mediated mechanism. Infection severity may be measured by weight of abscess formed. Indomethacin (Indo) treatment of infected hamsters reduced abscess weight by 30% at 7 days post-infection presumably by inhibition of PGE2. To explain reductions in abscess development by Indo treatment, we determined liver functionality in Indo-treated or untreated animals, either healthy or infected. Determinations of serum glutamic oxaloacetic (SGOT) and glutamic pyruvic (SGPT) transaminases, serum alkaline phosphatase (SAP), total serum protein (TSP), and bilirubinemia were done. SGOT, SGPT, and SAP activities showed a significant increase in their values by 600% at seven days post-infection in infected animals in both conditions; nonstatistical differences were found between animals treated or not. This increase did not correlate with the percentage of damage. Infected nontreated hamsters showed TSP levels 30% below normal group (P < 0.05). Infected Indo-treated hamsters had no significant differences compared to normal values. Infected nontreated animals showed an increase in bilirubin, particularly in indirect bilirubin, whereas infected Indo-treated hamsters showed total bilirubin values lower than normals (P < 0.05), with a decrease in direct bilirubin levels. Our results demonstrated that E. histolytica infection in hamsters produces similar abnormalities in liver function as it does in humans, and that the beneficial effect of Indo treatment on amoebic abscess development is not related with an improvement of liver functionality.

Role of prostaglandin E2 on amoebic liver abscess formation in hamsters.

Entamoeba histolytica can modulate macrophage functions and cytokine production by a prostaglandin E2 (PGE2) mechanism. To study the participation of PGE2 on amoebic liver abscess formation, we tested the effect of the PG synthesis inhibitor indomethacin (INDO) on abscess development in hamsters infected intrahepatically with E. histolytica trophozoites. Male infected animals had higher levels of plasma PGE2 (5.7 +/- 0.7 pg/ml pre-infection; 26.0 +/- 2.0 pg/ml 7 days postinfection; p < 0.001). INDO prevented this increase, so that infected-treated and control non-infected animals had similar levels of plasma PGE2. INDO reduced liver and abscess weight by 18% and 30% respectively (p < 0.05). Cyclooxygenase (COX) activity determination by thin layer chromatography using (1-14C) arachidonic acid (AA) showed that liver microsomes from infected animals produced more PGE2 than controls. COX activity was considerably inhibited in infected INDO-treated animals. Our data suggest that E. histolytica can stimulate the hepatic production of PGE2 which contributes to pathogenesis of amoebic abscesses through generation and support of the inflammation. The partial effect of INDO treatment suggests that additional factors are involved.