Epithelial barrier dysfunction is a hallmark of inflammatory bowel diseases; however, the mechanisms underlying such impairment remain incompletely understood. In the present study, a dextran sulfate sodium-induced colitis model was used to investigate how the inflammatory environment damages the intestinal mucosa. The results demonstrated that colitogenic ambiance enhances intestinal epithelial cell death, delays epithelial cell proliferation, and exacerbates mucosal erosion. Unexpectedly, this work identified a previously unrecognized role for Notch signaling in mediating these effects. Specifically, the colitogenic milieu reduces Notch/mechanistic target of rapamycin complex 1 (mTORC1)-mediated intestinal epithelial cell proliferation to promote goblet cell differentiation. Chemical activation of Notch signaling stimulated intestinal epithelial cell proliferation and reduced goblet cell differentiation in the colitic mucosa, further aggravating mucosal damage. Conversely, inhibition of Notch or mTORC1 signaling during mucosal repair reduced intestinal epithelial cell proliferation and enhanced goblet cell differentiation, corroborating the implication of Notch and mTORC1 signaling in both processes. Collectively, these findings uncover a context-dependent role for the Notch-mechanistic target of rapamycin axis in regulating intestinal epithelial cell proliferation and differentiation in the colitic mucosa and suggest that its targeted modulation may hold therapeutic potential in inflammatory bowel diseases.

The genus Naegleria comprises free-living amoebae characterized as amphizoic and ubiquitous microorganisms. Naegleria fowleri is the only species pathogenic to humans, causing primary amebic meningoencephalitis. The 26S proteasome represents the principal catalytic complex responsible for the degradation and recycling of intracellular proteins in eukaryotic cells. This complex consists of the 20S and 19S proteasome subunits, with the latter involved in the recognition and processing of ubiquitinated proteins and their delivery to the degradation site. Although the 26S proteasome has been characterized in various pathogenic protozoa, only the 20S proteasome has been studied within the genus Naegleria. The objective of this study was to demonstrate the presence of 19S subunits in N. fowleri. Bioinformatics analyses were employed to evaluate the presence and homology of non-ATPase subunits (Rpn10, Rpn11, and Rpn13) and ATPase subunits (Rpt2, Rpt3, and Rpt5). Additionally, the presence, localization, and correlation of 19S proteasome proteins with the 20S proteasome were assessed using experimental approaches. The results indicate that N. fowleri possesses proteins corresponding to the 19S proteasome, which, together with the 20S core particle, contribute to the formation of the 26S proteasome.

IntroductionThe oncoprotein CagA is the major virulence factor of Helicobacter pylori and plays a central role in the development of gastric disorders. H. pylori CagA+ infection has been implicated with an increased risk of developing extragastric diseases such as pancreatic cancer; however, the mechanisms remain unclear.MethodsIn this study, BxPC-3 pancreatic epithelial cells were infected with H. pylori bacterial strains CagA+ (26695 and PMSS1) or CagA− (Tx30a).ResultsInfection with CagA+ strains resulted in a significant disruption of epithelial barrier integrity, as demonstrated by transepithelial electrical resistance assays. Western blot and immunofluorescence analyses revealed altered expression and cytoplasmic relocalization of key apical junctional complex proteins. Meanwhile, claudin-4 and occludin levels increased, that of ZO-1 remained unchanged, and those of E-cadherin and β-catenin decreased. Likewise, cytoskeletal rearrangements were observed, particularly the loss of the actin apical ring and mislocalization of actin-binding proteins, such as cortactin and vinculin, at the cell borders. The loss of barrier integrity and cytoskeletal alterations were associated with morphological changes and increased cell motility, as demonstrated by wound healing assays. In addition, infection was accompanied by an increase in IL- 8 secretion.DiscussionThese findings suggest that CagA alters the pancreatic epithelial cells’ functions. Therefore, CagA represents an undescribed risk factor in the pathogenesis of H. pylori-associated pancreatic illness.

Naegleria fowleri is a free-living amoeba that occurs ubiquitously in soil and water worldwide. As the infective form for humans, N. fowleri trophozoite enters the nasal cavity during recreational activities in contaminated water, crosses the olfactory neuroepithelium, travels through the olfactory nerves and the cribriform plate, and reaches the olfactory bulbs in the central nervous system, where it produces primary amoebic meningoencephalitis (PAM). Characterized by an extensive and robust inflammatory reaction, this acute and fatal disease is mainly attributed to the activities of neutrophils and some macrophages. In the absence of accurate diagnosis and specific treatment, this disease has a mortality rate of 95%–97% and a recovery rate of only 5%. Therefore elucidation of the pathogenic mechanisms and identification of the virulence factors of N. fowleri are critical for the development of novel therapeutic approaches for its ultimate control and eradication.

Immune mediators affect multiple biological functions of intestinal epithelial cells (IECs) and, like Paneth and Paneth-like cells, play an important role in intestinal epithelial homeostasis. IFN-γ a prototypical proinflammatory cytokine disrupts intestinal epithelial homeostasis. However, the mechanism underlying the process remains unknown. In this study, using in vivo and in vitro models we demonstrate that IFN-γ is spontaneously secreted in the small intestine. Furthermore, we observed that this cytokine stimulates mitochondrial activity, ROS production, and Paneth and Paneth-like cell secretion. Paneth and Paneth-like secretion downstream of IFN-γ, as identified here, is mTORC1 and necroptosis-dependent. Thus, our findings revealed that the pleiotropic function of IFN-γ also includes the regulation of Paneth cell function in the homeostatic gut.

Helicobacter pylori is a common resident in the stomach of at least half of the world's population and recent evidence suggest its emergence in other organs such as the pancreas. In this organ, the presence of H. pylori DNA has been reported in cats, although the functional implications remain unknown. In this work, we determined distinct features related to the H. pylori manifestation in pancreas in a rodent model, in order to analyse its functional and structural effect. Gerbils inoculated with H. pylori exhibited the presence of this bacterium, as revealed by the expression of some virulence factors, as CagA and OMPs in stomach and pancreas, and confirmed by urease activity, bacterial culture, PCR and immunofluorescence assays. Non-apparent morphological changes were observed in pancreatic tissue of infected animals; however, delocalization of intercellular junction proteins (claudin-1, claudin-4, occludin, ZO-1, E-cadherin, β-catenin, desmoglein-2 and desmoplakin I/II) and rearrangement of the actin-cytoskeleton were exhibited. This structural damage was consistent with alterations in the distribution of insulin and glucagon, and a systemic inflammation, event demonstrated by elevated IL-8 levels. Overall, these findings indicate that H. pylori can reach the pancreas, possibly affecting its function and contributing to the development of pancreatic diseases.

Arpin was discovered as an inhibitor of the Arp2/3 complex localized at the lamellipodial tip of fibroblasts, where it regulated migration steering. Recently, we showed that arpin stabilizes the epithelial barrier in an Arp2/3-dependent manner. However, the expression and functions of arpin in endothelial cells (EC) have not yet been described. Arpin mRNA and protein are expressed in EC and downregulated by pro-inflammatory cytokines. Arpin depletion in Human Umbilical Vein Endothelial Cells causes the formation of actomyosin stress fibers leading to increased permeability in an Arp2/3-independent manner. Instead, inhibitors of ROCK1 and ZIPK, kinases involved in the generation of stress fibers, normalize the loss-of-arpin effects on actin filaments and permeability. Arpin-deficient mice are viable but show a characteristic vascular phenotype in the lung including edema, microhemorrhage, and vascular congestion, increased F-actin levels, and vascular permeability. Our data show that, apart from being an Arp2/3 inhibitor, arpin is also a regulator of actomyosin contractility and endothelial barrier integrity.

Acanthamoeba castellanii genotype T4 is a clinically significant free-living amoeba that causes granulomatous amoebic encephalitis and amoebic keratitis in human beings. During the initial stages of infection, trophozoites interact with various host immune responses, such as lactoferrin (Lf), in the corneal epithelium, nasal mucosa, and blood. Lf plays an important role in the elimination of pathogenic microorganisms, and evasion of the innate immune response is crucial in the colonization process. In this study, we describe the resistance of A. castellanii to the microbicidal effect of bovine apo-lactoferrin (apo-bLf) at different concentrations (25, 50, 100, and 500 µM). Acanthamoeba castellanii trophozoites incubated with apo-bLf at 500 µM for 12 h maintained 98% viability. Interestingly, despite this lack of effect on viability, our results showed that the apo-bLf inhibited the cytopathic effect of A. castellanii in MDCK cells culture, and analysis of amoebic proteases by zymography showed significant inhibition of cysteine and serine proteases by interaction with the apo-bLf. From these results, we conclude that bovine apo-Lf influences the activity of A. castellanii secretion proteases, which in turn decreases amoebic cytopathic activity.

Fiber intake is associated with a lower risk for Alzheimer´s disease (AD) in older adults. Intake of plant-based diets rich in soluble fiber promotes the production of short-chain fatty acids (SCFAs: butyrate, acetate, propionate) by gut bacteria. Butyrate administration has antiinflammatory actions, but propionate promotes neuroinflammation. In AD patients, gut microbiota dysbiosis is a common feature even in the prodromal stages of the disease. It is unclear whether the neuroprotective effects of fiber intake rely on gut microbiota modifications and specific actions of SCFAs in brain cells. Here, we show that restoration of the gut microbiota dysbiosis through the intake of soluble fiber resulted in lower propionate and higher butyrate production, reduced astrocyte activation and improved cognitive function in 6-month-old male APP/PS1 mice. The neuroprotective effects were lost in antibiotic-treated mice. Moreover, propionate promoted higher glycolysis and mitochondrial respiration in astrocytes, while butyrate induced a more quiescent metabolism. Therefore, fiber intake neuroprotective action depends on the modulation of butyrate/propionate production by gut bacteria. Our data further support and provide a mechanism to explain the beneficial effects of dietary interventions rich in soluble fiber to prevent dementia and AD. Fiber intake restored the concentration of propionate and butyrate by modulating the composition of gut microbiota in male transgenic (Tg) mice with Alzheimer´s disease. Gut dysbiosis was associated with intestinal damage and high propionate levels in control diet fed-Tg mice. Fiber-rich diet restored intestinal integrity and promoted the abundance of butyrate-producing bacteria. Butyrate concentration was associated with better cognitive performance in fiber-fed Tg mice. A fiber-rich diet may prevent the development of a dysbiotic microbiome and the related cognitive dysfunction in people at risk of developing Alzheimer´s disease.

In this work the effect of (-)-epicatechin on the development of amebic liver abscess in hamsters was evaluated. (-)-epicatechin is a flavonoid present in plants that possesses various biological properties, including its activity against some protozoal parasites; however its antiamebic activity in a living model had not been evaluated. Syrian golden hamsters were intrahepatically inoculated with 1x10(6) E. histolytica trophozoites, three days after inoculation they received nine intraperitoneal doses of (-)-epicatechin (10 mg/100 g) every 48 h. Animals without treatments and treated with metronidazole were included as controls. Macroscopic characteristics of the hepatic abscess, histopathological analysis of the tissue and the levels of inflammatory cytokines were determined. (-)-epicatechin produced a decrease in liver abscess progression being observed only 9.49% of damage compared to 84% shown by untreated animals. During treatment with (-)-epicatechin hepatic tissue showed signs of liver repair and absence of amoebae. Additionally, (-)-epicatechin produced a modulating effect on inflammatory cytokines TNF-alpha, IL-1 beta and IL-10. All these events observed in animals treated with (-)-epicatechin could contribute to the elimination of trophozoites and liver healing.

Intestinal parasites are a global problem, mainly in developing countries. Obtaining information about plants and compounds that can combat gastrointestinal disorders and gastrointestinal symptoms is a fundamental first step in designing new treatment strategies. In this study, we analyzed the antiamoebic activity of the aerial part of Croton sonorae. The dichloromethane fraction of C. sonorae (CsDCMfx) contained flavonoids, terpenes, alkaloids, and glycosides. The ultrastructural morphology of the amoebae treated for 72 h with CsDCMfx was completely abnormal. CsDCMfx reduced erythrophagocytosis of trophozoites and the expression of genes involved in erythrocyte adhesion (gal/galnac lectin) and actin cytoskeleton rearrangement in the phagocytosis pathway (rho1 gtpase and formin1). Interestingly, CsDCMfx decreased the expression of genes involved in Entamoeba histolytica trophozoite pathogenesis, such as cysteine proteases (cp1, cp4, and cp5), sod, pfor, and enolase. These results showed that C. sonorae is a potential source of antiamoebic compounds.

Acanthamoeba spp. are free-living amoebae with a worldwide distribution. These amoebae can cause granulomatous amoebic encephalitis and amoebic keratitis in humans. Proteases are considered virulence factors in pathogenic Acanthamoeba. The objective of this study was to evaluate the behavior of Acanthamoeba mauritaniensis, a nonpathogenic amoeba. We analyzed the cytopathic effect of A. mauritaniensis on RCE1(5 T5) and MDCK cells and compared it to that of Acanthamoeba castellanii. A partial biochemical characterization of proteases was performed in total crude extracts (TCE) and conditioned medium (CM). Finally, we evaluated the effect of proteases on tight junction (TJ) proteins and the transepithelial electrical resistance of MDCK cells. The results showed that this amoeba can induce substantial damage to RCE1(5T5) and MDCK cells. Moreover, the zymograms and Azocoll assays of amoebic TCE and CM revealed different protease activities, with serine proteases being the most active. Furthermore, A. mauritaniensis induced the alteration and degradation of MDCK cell TJ proteins with serine proteases. After genotyping this amoeba, we determined that it is an isolate of Acanthamoeba genotype T4D. From these data, we suggest that A. mauritaniensis genotype T4D behaves similarly to the A. castellanii strain.

The Naegleria are ubiquitous free-living amoebae and are characterized by the presence of three phases in their biological cycle: trophozoite, cyst and flagellate. Of this genus, only Naegleria fowleri has been reported as pathogenic to humans. The proteasome is a multi-catalytic complex and is considered to be the most important structure responsible for the degradation of intracellular proteins. This structure is related to the maintenance of cellular homeostasis and, in pathogenic microorganisms, to the modulation of their virulence. Until now, the proteasome and its function have not been described for the Naegleria genus. In the current study, using bioinformatic analysis, protein sequences homologous to those reported for the subunits of the 20S proteasome in other organisms were found, and virtual modelling was used to determine their three-dimensional structure. The presence of structural and catalytic subunits of the 20S proteasome was detected by Western and dot blot assays. Its localization was observed by immunofluorescence microscopy to be mainly in the cytoplasm, and a leading role of the chymotrypsin-like catalytic activity was determined using fluorogenic peptidase assays and specific proteasome inhibitors. Finally, the role of the 20S proteasome in the proliferation and differentiation of Naegleria genus trophozoites was demonstrated.

Introduction and Objectives: Curcumin, a polyphenol, is a natural compound that has been widely studied as a hepatoprotector; however, only a few studies have examined its ability to reduce fibrosis in previously established cirrhosis. The objective of this study was to investigate whether curcumin could reduce carbon tetrachloride (CCl4)-induced fibrosis and if so, to determine the action mechanisms involved in the reduction process. Materials and Methods: CCl4 was administered to male Wistar rats (400 mg/kg, three times a week, i. p.) for 12 weeks; curcumin (100 mg/kg body weight twice per day, p. o.) was administered from week 9-12 of CCl4 treatment. Biochemical markers of hepatic injury and oxidative stress were evaluated. Hematoxylin and eosin, Masson's trichrome stains, transmission electron microscopy; immunohistochemistry, and zymography assays were carried out. Moreover, Smad3 and alpha-SMA mRNA and protein levels were studied. Western blotting by TGF-alpha, CTGF, Col-I, MMP-13, NF-kappa B, IL-1, IL-10, Smad7, pSmad3, and pJNK proteins was developed. Results and Conclusions: Curcumin reduced liver damage, oxidative stress, fibrosis, and restored normal activity of MMP-9 and MMP-2. Besides, curcumin restored NF-kappa B, IL-1, IL-10, TGF-beta, CTGF, Col-I, MMP-13, and Smad7 protein levels. On the other hand, curcumin decreased JNK and Smad3 phosphorylation. Furthermore, curcumin treatment decreased alpha-SMA and Smad3 protein and mRNA levels. Curcumin normalized GSH, and NF-kappa B, JNK-Smad3, and TGF-beta-Smad3 pathways, leading to a decrement in activated hepatic stellate cells, thereby producing its antifibrotic effects. (C) 2020 Fundacion Clinica Medica Sur, A.C. Published by Elsevier Espana, S.L.U.

Naegleria fowleri produces a fatal disease called primary amebic meningoencephalitis (PAM), which is characterized by an extensive inflammatory reaction in the CNS. It is known that the immune response is orchestrated mainly by neutrophils, which activate several defense mechanisms in the host, including phagocytosis, the release of different enzymes such as myeloperoxidase (MPO), and the production of neutrophil extracellular traps. However, the mechanisms by which amoebas evade the neutrophil response are still unknown. In this study, we analyzed the ability of N. fowleri to respond to the stress exerted by MPO. Interestingly, after the interaction of trophozoites with neutrophils, the amoeba viability was not altered; however, ultrastructural changes were observed. To analyze the influence of MPO against N. fowleri and its participation in free radical production, we evaluated its enzymatic activity, expression, and localization with and without the specific 4-aminobenzoic acid hydrazide inhibitor. The production of oxidizing molecules is the principal mechanism used by neutrophils to eliminate pathogens. In this context, we demonstrated an increase in the production of NO, superoxide anion, and reactive oxygen species; in addition, the overexpression of several antioxidant enzymes present in the trophozoites was quantified. The findings strongly suggest that N. fowleri possesses antioxidant machinery that is activated in response to an oxidative environment, allowing it to evade the neutrophil-mediated immune response, which may contribute to the establishment of PAM.

There is no effective treatment for hepatic fibrosis. Previously, we demonstrated that naringenin possesses the ability to prevent experimental chronic liver damage. Therefore, the objective of this work was to investigate whether naringenin could reverse carbon tetrachloride (CCl4)-induced fibrosis in rats and, if so, to search for the mechanisms involved. CCl4 was given to male Wistar rats (400 mg/kg, three times per week, i. p.) for 12 weeks; naringenin (100 mg/kg twice per day, p. o.) was administered from weeks 9-12 of the CCl4 treatment. Liver damage and oxidative stress markers were measured. Masson's trichrome, hematoxylin-eosin staining and immunohistochemistry were performed. Zymography assays for MMP-9 and MMP-2 were carried out. TGF-beta, CTGF, Col-I, MMP-13, NF-kappa B, IL-1 beta, IL-10, Smad7, pSmad3 and pJNK protein levels were determined by western blotting. In addition, alpha-SMA and Smad3 protein and mRNA levels were studied. Naringenin reversed liver damage, biochemical and oxidative stress marker elevation, and fibrosis and restored normal MMP-9 and MMP-2 activity. The flavonoid also preserved NF-kappa B, IL-1 beta, IL-10, TGF-beta, CTGF, Col-I, MMP-13 and Smad7 protein levels. Moreover, naringenin decreased JNK activation and Smad3 phosphorylation in the linker region. Finally, alpha-SMA and Smad3 protein and mRNA levels were reduced by naringenin administration. The results of this study demonstrate that naringenin blocks oxidative stress, inflammation and the TGF-beta-Smad3 and JNK-Smad3 pathways, thereby carrying out its antifibrotic effects and making it a good candidate to treat human fibrosis, as previously demonstrated in toxicological and clinical studies.

Epithelial cells lining the intestinal mucosa constitute a selective-semipermeable barrier acting as first line of defense in the organism. The number of those cells remains constant during physiological conditions, but disruption of epithelial cell homeostasis has been observed in several pathologies. During colitis, epithelial cell proliferation decreases and cell death augments. The mechanism responsible for these changes remains unknown. Here, we show that the pro-inflammatory cytokine IFNγ contributes to the inhibition of epithelial cell proliferation in intestinal epithelial cells (IECs) by inducing the activation of mTORC1. Activation of mTORC1 in response to IFNγ was detected in IECs present along the crypt axis and in colonic macrophages. mTORC1 inhibition enhances cell proliferation, increases DNA damage in IEC. In macrophages, mTORC1 inhibition strongly reduces the expression of pro-inflammatory markers. As a consequence, mTORC1 inhibition exacerbated disease activity, increased mucosal damage, enhanced ulceration, augmented cell infiltration, decreased survival and stimulated tumor formation in a model of colorectal cancer CRC associated to colitis. Thus, our findings suggest that mTORC1 signaling downstream of IFNγ prevents epithelial DNA damage and cancer development during colitis.

Primary amebic meningoencephalitis (PAM) is a fatal infection caused by the free-living ameba Naegleria fowleri, popularly known as the “brain-eating ameba.” The drugs of choice in treating PAM are the antifungal amphotericin B and an antileishmanial miltefosine, but these are not FDA-approved for this indication and use of amphotericin B is associated with severe adverse effects. Moreover, very few patients treated with the combination therapy have survived PAM. Therefore, development of efficient drugs is a critical unmet need to avert future deaths of children. Since N. fowleri causes extensive inflammation in the brain it is important to select compounds that can enter brain to kill ameba. In this study, we identified two central nervous system (CNS) active compounds, ebselen and BAY 11-7082 as amebicidal with EC50 of 6.2 and 1.6 μM, respectively. The closely related BAY 11-7085 was also found active against N. fowleri with EC50 similar to BAY 11-7082. We synthesized a soluble ebselen analog, which had amebicidal activity similar to ebselen. Transmission electron microscopy of N. fowleri trophozoites incubated for 48 h with EC50 concentration of ebselen showed alteration in the cytoplasmic membrane, loss of the nuclear membrane, and appearance of electron-dense granules. Incubation of N. fowleri trophozoites with EC50 concentrations of BAY 11-7082 and BAY 11-7085 for 48 h showed the presence of large lipid droplets in the cytoplasm, disruption of cytoplasmic and nuclear membranes and appearance of several vesicles and chromatin residues. Blood-brain barrier permeable amebicidal compounds have potential as new drug leads for Naegleria infection.

Naegleria fowleri and Naegleria gruberi belong to the free-living amoebae group. It is widely known that the non-pathogenic species N. gruberi is usually employed as a model to describe molecular pathways in this genus, mainly because its genome has been recently described. However, N. fowleri is an aetiological agent of primary amoebic meningoencephalitis, an acute and fatal disease. Currently, the most widely used drug for its treatment is amphotericin B (AmB). It was previously reported that AmB has an amoebicidal effect in both N. fowleri and N. gruberi trophozoites by inducing morphological changes that resemble programmed cell death (PCD). PCD is a mechanism that activates morphological, biochemical and genetic changes. However, PCD has not yet been characterized in the genus Naegleria. The aim of the present work was to evaluate the typical markers to describe PCD in both amoebae. These results showed that treated trophozoites displayed several parameters of apoptosis-like PCD in both species. We observed ultrastructural changes, an increase in reactive oxygen species, phosphatidylserine externalization and a decrease in intracellular potassium, while DNA degradation was evaluated using the TUNEL assay and agarose gels, and all of these parameters are related to PCD. Finally, we analysed the expression of apoptosis-related genes, such as sir2 and atg8, in N. gruberi. Taken together, our results showed that AmB induces the morphological, biochemical and genetic changes of apoptosis-like PCD in the genus Naegleria.