Correction: Differential expression of pathogenic genes of Entamoeba histolytica vs E. dispar in a model of infection using human liver tissue explants.

[This corrects the article DOI: 10.1371/journal.pone.0181962.].

Differential expression of pathogenic genes of Entamoeba histolytica vs E. dispar in a model of infection using human liver tissue explants.

We sought to establish an ex vivo model for examining the interaction of E. histolytica with human tissue, using precision-cut liver slices (PCLS) from donated organs. E. histolytica- or E. dispar-infected PCLS were analyzed at different post-infection times (0, 1, 3, 24 and 48 h) to evaluate the relation between tissue damage and the expression of genes associated with three factors: a) parasite survival (peroxiredoxin, superoxide dismutase and 70 kDa heat shock protein), b) parasite virulence (EhGal/GalNAc lectin, amoebapore, cysteine proteases and calreticulin), and c) the host inflammatory response (various cytokines). Unlike E. dispar (non-pathogenic), E. histolytica produced some damage to the structure of hepatic parenchyma. Overall, greater expression of virulence genes existed in E. histolytica-infected versus E. dispar-infected tissue. Accordingly, there was an increased expression of EhGal/GalNAc lectin, Ehap-a and Ehcp-5, Ehcp-2, ehcp-1 genes with E. histolytica, and a decreased or lack of expression of Ehcp-2, and Ehap-a genes with E. dispar. E. histolytica-infected tissue also exhibited an elevated expression of genes linked to survival, principally peroxiredoxin, superoxide dismutase and Ehhsp-70. Moreover, E. histolytica-infected tissue showed an overexpression of some genes encoding for pro-inflammatory interleukins (ILs), such as il-8, ifn-γ and tnf-α. Contrarily, E. dispar-infected tissue displayed higher levels of il-10, the gene for the corresponding anti-inflammatory cytokine. Additionally, other genes were investigated that are important in the host-parasite relationship, including those encoding for the 20 kDa heat shock protein (HSP-20), the AIG-1 protein, and immune dominant variable surface antigen, as well as for proteins apparently involved in mechanisms for the protection of the trophozoites in different environments (e.g., thioredoxin-reductase, oxido-reductase, and 9 hypothetical proteins). Some of the hypothetical proteins evidenced interesting overexpression rates, however we should wait to their characterization. This finding suggest that the present model could be advantageous for exploring the complex interaction between trophozoites and hepatocytes during the development of ALA, particularly in the initial stages of infection.

Differential expression of E-type prostanoid receptors 2 and 4 in microglia stimulated with lipopolysaccharide.

BACKGROUND: Cyclooxygenase-2 (COX-2) is induced under inflammatory conditions, and prostaglandin E2 (PGE2) is one of the products of COX activity. PGE2 has pleiotropic actions depending on the activation of specific E-type prostanoid EP1-4 receptors. We investigated the involvement of PGE2 and EP receptors in glial activation in response to an inflammatory challenge induced by LPS. METHODS: Cultures of mouse microglia or astroglia cells were treated with LPS in the presence or absence of COX-2 inhibitors, and the production of PGE2 was measured by ELISA. Cells were treated with PGE2, and the effect on LPS-induced expression of TNF-α messenger RNA (mRNA) and protein was studied in the presence or absence of drug antagonists of the four EP receptors. EP receptor expression and the effects of EP2 and EP4 agonists and antagonists were studied at different time points after LPS. RESULTS: PGE2 production after LPS was COX-2-dependent. PGE2 reduced the glial production of TNF-α after LPS. Microglia expressed higher levels of EP4 and EP2 mRNA than astroglia. Activation of EP4 or EP2 receptors with selective drug agonists attenuated LPS-induced TNF-α in microglia. However, only antagonizing EP4 prevented the PGE2 effect demonstrating that EP4 was the main target of PGE2 in naïve microglia. Moreover, the relative expression of EP receptors changed during the course of classical microglial activation since EP4 expression was strongly depressed while EP2 increased 24 h after LPS and was detected in nuclear/peri-nuclear locations. EP2 regulated the expression of iNOS, NADPH oxidase-2, and vascular endothelial growth factor. NADPH oxidase-2 and iNOS activities require the oxidation of NADPH, and the pentose phosphate pathway is a main source of NADPH. LPS increased the mRNA expression of the rate-limiting enzyme of the pentose pathway glucose-6-phosphate dehydrogenase, and EP2 activity was involved in this effect. CONCLUSIONS: These results show that while selective activation of EP4 or EP2 exerts anti-inflammatory actions, EP4 is the main target of PGE2 in naïve microglia. The level of EP receptor expression changes from naïve to primed microglia where the COX-2/PGE2/EP2 axis modulates important adaptive metabolic changes.

Entamoeba histolytica and Entamoeba dispar infection in Mexican school children: genotyping and phylogenetic relationship.

BACKGROUND: This study aimed to determine the frequency of Entamoeba histolytica and Entamoeba dispar infection in school children in the community of Tlaltizapan, in order to understand the dynamics of infection within the school and family spheres of this population. Amoebiasis is an unsolved public health problem and an endemic disease in Mexico. The incidence rate varies depending on the state; the most affected states show the highest numbers of new cases of amoebiasis per year. Previously, we reported the molecular frequency of infection with E. histolytica and/or E. dispar in other rural communities of the state of Morelos. METHODS: Children from 3 schools were studied to estimate the frequency of intestinal parasites through microscopic examination of fresh stool samples. The number of studied individuals were 309 school children. The molecular characterization of E. histolytica or E. dispar was carried out by Polymerase Chain Reaction (PCR) using species-specific primers to amplify short tandem repeats (STR) in non-coding sequences associated with the tRNA gene; the amplified fragments were sequenced and analyzed. RESULTS: Eight different genotypes were obtained from E. dispar isolates with the molecular marker NKD3-D5. None of the cases in which the species E. histolytica was detected developed symptoms attributable to an invasive process of disease. Moreover, the parasitized condition appeared to have no significant impact on the development or nutritional status of affected children. Genotype 1, which corresponds to the reference strain E. dispar SAW760, considered a non-pathogenic amoeba, was the most prevalent. CONCLUSIONS: The comparison of the genotypes of Entamoeba species did not show a correlation between children and their relatives. In this community, the species Entamoeba dispar genotype 1 was the most widespread. Based on the indicators of growth, development and nutrition status, the studied community seems to be reasonably adapted to constant exposure to intestinal parasites, since there were no evidences of a serious impact of the parasitized condition on the children's health.

IL-10 regulates adult neurogenesis by modulating ERK and STAT3 activity.

The adult subventricular zone (SVZ) contains Nestin+ progenitors that differentiate mainly into neuroblasts. Our previous data showed that interleukin-10 (IL-10) regulates SVZ adult neurogenesis by up-regulating the expression of pro-neural genes and modulating cell cycle exit. Here we addressed the specific mechanism through which IL-10 carries out its signaling on SVZ progenitors. We found that, in vitro and in vivo, IL-10 targets Nestin+ progenitors and activates the phosphorylation of ERK and STAT3. The action of IL-10 on Nestin+ progenitors is reversed by treatment with a MEK/ERK inhibitor, thus restoring neurogenesis to normal levels. Silencing STAT3 expression by lentiviral vectors also impaired neurogenesis by blocking the effects of IL-10. Our findings unveil ERK and STAT3 as effectors of IL-10 in adult SVZ neurogenesis.

Entamoeba histolytica and E. dispar Calreticulin: inhibition of classical complement pathway and differences in the level of expression in amoebic liver abscess.

The role of calreticulin (CRT) in host-parasite interactions has recently become an important area of research. Information about the functions of calreticulin and its relevance to the physiology of Entamoeba parasites is limited. The present work demonstrates that CRT of both pathogenic E. histolytica and nonpathogenic E. dispar species specifically interacted with human C1q inhibiting the activation of the classical complement pathway. Using recombinant EhCRT protein, we demonstrate that CRT interaction site and human C1q is located at the N-terminal region of EhCRT. The immunofluorescence and confocal microscopy experiments show that CRT and human C1q colocalize in the cytoplasmic vesicles and near to the surface membrane of previously permeabilized trophozoites or are incubated with normal human serum which is known to destroy trophozoites. In the presence of peripheral mononuclear blood cells, the distribution of EhCRT and C1q is clearly over the surface membrane of trophozoites. Nevertheless, the level of expression of CRT in situ in lesions of amoebic liver abscess (ALA) in the hamster model is different in both Entamoeba species; this molecule is expressed in higher levels in E. histolytica than in E. dispar. This result suggests that EhCRT may modulate some functions during the early moments of the host-parasite relationship.

Case report: Cutaneous amebiasis: the importance of molecular diagnosis of an emerging parasitic disease.

Cutaneous amebiasis is the least common clinical form of human amebiasis in Mexico, sexual amebiasis was only occasionally observed before the late 1980s. However, in the last few decades, most of the documented cases of cutaneous amebiasis from around the world are sexually transmitted. We present two cases of sexually transmitted genital amebiasis. The molecular characterization of the Entamoeba species in the affected tissues underlines the importance of an etiological diagnosis using specific and sensitive techniques that avoid the rapid destruction of tissues and the irreversible sequelae to the anatomy and function of the affected organs. In addition, for those interested in the study of the human-amoebic disease relationship and its epidemiology, the detection of a new, mixed infection in an invasive case of amebiasis reveals new perspectives in the study of the extraordinarily complex host-parasite relationship in amebiasis.

Induction of COX-2 enzyme and down-regulation of COX-1 expression by lipopolysaccharide (LPS) control prostaglandin E2 production in astrocytes.

Pathological conditions and pro-inflammatory stimuli in the brain induce cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism mediating the production of prostanoids that, among other actions, have strong vasoactive properties. Although low basal cerebral COX-2 expression has been reported, COX-2 is strongly induced by pro-inflammatory challenges, whereas COX-1 is constitutively expressed. However, the contribution of these enzymes in prostanoid formation varies depending on the stimuli and cell type. Astrocyte feet surround cerebral microvessels and release molecules that can trigger vascular responses. Here, we investigate the regulation of COX-2 induction and its role in prostanoid generation after a pro-inflammatory challenge with the bacterial lipopolysaccharide (LPS) in astroglia. Intracerebral administration of LPS in rodents induced strong COX-2 expression mainly in astroglia and microglia, whereas COX-1 expression was predominant in microglia and did not increase. In cultured astrocytes, LPS strongly induced COX-2 and microsomal prostaglandin-E(2) (PGE(2)) synthase-1, mediated by the MyD88-dependent NFκB pathway and influenced by mitogen-activated protein kinase pathways. Studies in COX-deficient cells and using COX inhibitors demonstrated that COX-2 mediated the high production of PGE(2) and, to a lesser extent, other prostanoids after LPS. In contrast, LPS down-regulated COX-1 in an MyD88-dependent fashion, and COX-1 deficiency increased PGE(2) production after LPS. The results show that astrocytes respond to LPS by a COX-2-dependent production of prostanoids, mainly vasoactive PGE(2), and suggest that the coordinated down-regulation of COX-1 facilitates PGE(2) production after TLR-4 activation. These effects might induce cerebral blood flow responses to brain inflammation.

Astrocyte TLR4 activation induces a proinflammatory environment through the interplay between MyD88-dependent NFκB signaling, MAPK, and Jak1/Stat1 pathways.

There is increasing evidence that astrocytes play important roles in immune regulation in the brain. Astrocytes express toll-like receptors (TLR) and build up responses to innate immune triggers by releasing proinflammatory molecules. We investigate signaling pathways and released molecules after astrocyte TLR4 activation. Purified rodent brain astrocyte cultures were treated with the TLR4 activator bacterial lipopolysaccharide (LPS). Tools used to interfere with this system include small interference RNA, inhibitory drugs, and MyD88 or Stat1 deficient mice. LPS induced early activation of the transcription factor NFκB, through the MyD88 adaptor, and expression of TNF-α, VCAM-1, IL-15, and IL-27. LPS also induced delayed Jak1/Stat1 activation, which was MyD88-independent but was not mediated by IFN-ß. Jak1/Stat1 activation induced the expression of negative cytokine regulator SOCS-1 and CXCL10 chemokine (IP-10). Mitogen-activated protein kinases (MAPK) were also involved in TLR4 signaling in a MyD88-independent fashion. p38 exerted a strong influence on LPS-induced gene expression by regulating the phosphorylation of Stat1 and the transcriptional activity of NFκB, while JNK regulated the Jak1/Stat1 pathway, and ERK1/2 controlled the expression of Egr-1 and influenced MyD88-dependent MMP-9 expression. Interplay between these signals was evidenced by the increased induction of MMP-9 in Stat1-deficient cells challenged with LPS, suggesting that Stat1 negatively regulates the expression of MMP-9 induced by LPS. Therefore, astrocytes are responsive to TLR4 activation by inducing a complex set of cell-dependent molecular reactions mediated by NFκB, MAPK and Jak1/Stat1 signaling pathways. Here we identified cross-talking signals generating a proinflammatory environment that will modulate the response of surrounding cells.

Nitric oxide mediates NMDA-induced persistent inhibition of protein synthesis through dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 and eukaryotic initiation factor 4G proteolysis.

Cerebral ischaemia causes long-lasting protein synthesis inhibition that is believed to contribute to brain damage. Energy depletion promotes translation inhibition during ischaemia, and the phosphorylation of eIF (eukaryotic initiation factor) 2alpha is involved in the translation inhibition induced by early ischaemia/reperfusion. However, the molecular mechanisms underlying prolonged translation down-regulation remain elusive. NMDA (N-methyl-D-aspartate) excitotoxicity is also involved in ischaemic damage, as exposure to NMDA impairs translation and promotes the synthesis of NO (nitric oxide), which can also inhibit translation. In the present study, we investigated whether NO was involved in NMDA-induced protein synthesis inhibition in neurons and studied the underlying molecular mechanisms. NMDA and the NO donor DEA/NO (diethylamine-nitric oxide sodium complex) both inhibited protein synthesis and this effect persisted after a 30 min exposure. Treatments with NMDA or NO promoted calpain-dependent eIF4G cleavage and 4E-BP1 (eIF4E-binding protein 1) dephosphorylation and also abolished the formation of eIF4E-eIF4G complexes; however, they did not induce eIF2alpha phosphorylation. Although NOS (NO synthase) inhibitors did not prevent protein synthesis inhibition during 30 min of NMDA exposure, they did abrogate the persistent inhibition of translation observed after NMDA removal. NOS inhibitors also prevented NMDA-induced eIF4G degradation, 4E-BP1 dephosphorylation, decreased eIF4E-eIF4G-binding and cell death. Although the calpain inhibitor calpeptin blocked NMDA-induced eIF4G degradation, it did not prevent 4E-BP1 dephosphorylation, which precludes eIF4E availability, and thus translation inhibition was maintained. The present study suggests that eIF4G integrity and hyperphosphorylated 4E-BP1 are needed to ensure appropriate translation in neurons. In conclusion, our data show that NO mediates NMDA-induced persistent translation inhibition and suggest that deficient eIF4F activity contributes to this process.