IFN-γ stimulates Paneth cell secretion through necroptosis mTORC1 dependent.

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.

The Helicobacter pylori infection alters the intercellular junctions on the pancreas of gerbils (Meriones unguiculatus).

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, ß-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.

Dietary Fiber Modulates the Release of Gut Bacterial Products Preventing Cognitive Decline in an Alzheimer's Mouse Model.

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.

(-)-Epicatechin protects from amebic liver abscess development in hamster.

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 1x106E. 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-α, IL-1ß and IL-10. All these events observed in animals treated with (-)-epicatechin could contribute to the elimination of trophozoites and liver healing.

Curcumin downregulates Smad pathways and reduces hepatic stellate cells activation in experimental fibrosis.

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 α-SMA mRNA and protein levels were studied. Western blotting by TGF-ß, CTGF, Col-I, MMP-13, NF-κB, 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-κB, IL-1, IL-10, TGF-ß, CTGF, Col-I, MMP-13, and Smad7 protein levels. On the other hand, curcumin decreased JNK and Smad3 phosphorylation. Furthermore, curcumin treatment decreased α-SMA and Smad3 protein and mRNA levels. Curcumin normalized GSH, and NF-κB, JNK-Smad3, and TGF-ß-Smad3 pathways, leading to a decrement in activated hepatic stellate cells, thereby producing its antifibrotic effects.

Amphotericin B induces apoptosis-like programmed cell death in Naegleria fowleri and Naegleria gruberi.

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.

In vitro efficacy of corifungin against Acanthamoeba castellanii trophozoites and cysts.

Painful blinding keratitis and fatal granulomatous amebic encephalitis are caused by the free-living amebae Acanthamoeba spp. Several prescription eye medications are used to treat Acanthamoeba keratitis, but the infection can be difficult to control because of recurrence of infection. For the treatment of encephalitis, no single drug was found useful, and in spite of the use of a combination of multiple drugs, the mortality rate remains high. Therefore, efficient, novel drugs are urgently needed for the treatment of amebic keratitis and granulomatous amebic encephalitis. In this study, we identified corifungin, a water-soluble polyene macrolide, as amebicidal. In vitro, it was effective against both the trophozoites and the cysts. Transmission electron microscopy of Acanthamoeba castellanii incubated with corifungin showed the presence of swollen mitochondria, electron-dense granules, degeneration of cytoplasm architecture, and loss of nuclear chromatin structure. These changes were followed by lysis of amebae. Corifungin also induced the encystment process of A. castellanii. There were alterations in the cyst cell wall followed by lysis of the cysts. Corifungin is a promising therapeutic option for keratitis and granulomatous amebic encephalitis.

Disruption of MDCK cell tight junctions by the free-living amoeba Naegleria fowleri.

Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis. This parasite invades its host by penetrating the olfactory mucosa. However, the mechanism of epithelium penetration is not well understood. In the present study, we evaluated the effect of N. fowleri trophozoites and the non-pathogenic Naegleria gruberi on Madin-Darby canine kidney (MDCK) tight junction proteins, including claudin-1, occludin and ZO-1, as well as on the actin cytoskeleton. Trophozoites from each of the free-living amoeba species were co-cultured with MDCK cells in a 1 : 1 ratio for 1, 3, 6 or 10 h. Light microscopy revealed that N. fowleri caused morphological changes as early as 3 h post-infection in an epithelial MDCK monolayer. Confocal microscopy analysis revealed that after 10 h of co-culture, N. fowleri trophozoites induced epithelial cell damage, which was characterized by changes in the actin apical ring and disruption of the ZO-1 and claudin-1 proteins but not occludin. Western blot assays revealed gradual degradation of ZO-1 and claudin-1 as early as 3 h post-infection. Likewise, there was a drop in transepithelial electrical resistance that resulted in increased epithelial permeability and facilitated the invasion of N. fowleri trophozoites by a paracellular route. In contrast, N. gruberi did not induce alterations in MDCK cells even at 10 h post-infection. Based on these results, we suggest that N. fowleri trophozoites disrupt epithelial monolayers, which could enable their penetration of the olfactory epithelium and subsequent invasion of the central nervous system.

Acanthamoeba castellanii Genotype T4: Inhibition of Proteases Activity and Cytopathic Effect by Bovine Apo-Lactoferrin.

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.

Corifungin, a new drug lead against Naegleria, identified from a high-throughput screen.

Primary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living ameba Naegleria fowleri. The drug of choice in treating PAM is the antifungal antibiotic amphotericin B, but its use is associated with severe adverse effects. Moreover, few patients treated with amphotericin B have survived PAM. Therefore, fast-acting and efficient drugs are urgently needed for the treatment of PAM. To facilitate drug screening for this pathogen, an automated, high-throughput screening methodology was developed and validated for the closely related species Naegleria gruberi. Five kinase inhibitors and an NF-kappaB inhibitor were hits identified in primary screens of three compound libraries. Most importantly for a preclinical drug discovery pipeline, we identified corifungin, a water-soluble polyene macrolide with a higher activity against Naegleria than that of amphotericin B. Transmission electron microscopy of N. fowleri trophozoites incubated with different concentrations of corifungin showed disruption of cytoplasmic and plasma membranes and alterations in mitochondria, followed by complete lysis of amebae. In vivo efficacy of corifungin in a mouse model of PAM was confirmed by an absence of detectable amebae in the brain and 100% survival of mice for 17 days postinfection for a single daily intraperitoneal dose of 9 mg/kg of body weight given for 10 days. The same dose of amphotericin B did not reduce ameba growth, and mouse survival was compromised. Based on these results, the U.S. FDA has approved orphan drug status for corifungin for the treatment of PAM.

Entamoeba histolytica calreticulin: an endoplasmic reticulum protein expressed by trophozoites into experimentally induced amoebic liver abscesses.

Entamoeba histolytica calreticulin (EhCRT) is remarkably immunogenic in humans (90-100% of invasive amoebiasis patients). Nevertheless, the study of calreticulin in this protozoan is still in its early stages. The exact location, biological functions, and its role in pathogenesis are yet to be fully understood. The aim of the present work is to determine the location of EhCRT in virulent trophozoites in vivo and the expression of the Ehcrt gene during the development of experimentally induced amoebic liver abscesses (ALA) in hamsters. Antibodies against recombinant EhCRT were used for the immunolocalization of EhCRT in trophozoites through confocal microscopy; immunohistochemical assays were also performed on tissue sections of ALAs at different times after intrahepatic inoculation. The expression of the Ehcrt gene during the development of ALA was estimated through both in situ RT-PCR and real-time RT-PCR. Confocal assays of virulent trophozoites showed a distribution of EhCRT in the cytoplasmic vesicles of different sizes. Apparently, EhCRT is not exported into the hepatic tissue. Real-time RT-PCR demonstrated an over-expression of the Ehcrt gene at 30 min after trophozoite inoculation, reaching a peak at 1-2 h; thereafter, the expression fell sharply to its original levels. These results demonstrate for the first time in an in vivo model of ALA, the expression of Ehcrt gene in E. histolytica trophozoites and add evidence that support CRT as a resident protein of the ER in E. histolytica species. The in vivo experiments suggest that CRT may play an important role during the early stages of the host-parasite relationship, when the parasite is adapting to a new environment, although the protein seems to be constitutively synthesized. Moreover, trophozoites apparently do not export EhCRT into the hepatic tissue in ALA.

The Dichloromethane Fraction of Croton sonorae, A Plant Used in Sonoran Traditional Medicine, Affect Entamoeba histolytica Erythrophagocytosis and Gene Expression.

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.

Differences between Naegleria fowleri and Naegleria gruberi in expression of mannose and fucose glycoconjugates.

Naegleria fowleri is the etiologic agent of primary amoebic meningoencephalitis, a rapidly fatal parasitic disease of humans. The adherence of Naegleria trophozoites to the host cell is one of the most important steps in the establishment and invasiveness of this infectious disease. Currently, little is known about the surface molecules that may participate in the interaction of N. fowleri with their target cells. In the present study, we investigated the composition of glycoconjugates present on the surface of trophozoites of the pathogenic N. fowleri and the nonpathogenic Naegleria gruberi. With the use of biotinylated lectins in western blot and flow cytometric analysis, we showed that N. fowleri trophozoites present high levels of surface glycoconjugates that contain alpha-D-mannose, alpha-D-glucose, and terminal alpha-L-fucose residues. A significant difference in the expression of these glycoconjugates was observed between N. fowleri and the nonpathogenic N. gruberi. Furthermore, we suggest that glycoconjugates that contain D-mannose and L-fucose residues participate in the adhesion of N. fowleri and subsequent damage to MDCK cells.

Naegleria fowleri and Naegleria gruberi 20S proteasome: identification and characterization.

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.

The MPO system participates actively in the formation of an oxidative environment produced by neutrophils and activates the antioxidant mechanism of Naegleria fowleri.

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.

Acanthamoeba mauritaniensis genotype T4D: An environmental isolate displays pathogenic behavior.

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.

Naringenin attenuates the progression of liver fibrosis via inactivation of hepatic stellate cells and profibrogenic pathways.

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-ß, CTGF, Col-I, MMP-13, NF-κB, IL-1ß, IL-10, Smad7, pSmad3 and pJNK protein levels were determined by western blotting. In addition, α-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-κB, IL-1ß, IL-10, TGF-ß, CTGF, Col-I, MMP-13 and Smad7 protein levels. Moreover, naringenin decreased JNK activation and Smad3 phosphorylation in the linker region. Finally, α-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-ß-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.

In Vitro Efficacy of Ebselen and BAY 11-7082 Against Naegleria fowleri.

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.

Characterization of Naegleria fowleri strains isolated from human cases of primary amoebic meningoencephalitis in Mexico.

The protozoon Naegleria fowleri (N. fowleri) is a free-living amoeba that produces primary amoebic meningoencephalitis (PAM), which is an acute and frequently fatal infection of the central nervous system. We characterized the strains of N. fowleri isolated from the cerebrospinal fluid (CSF) of two cases presented in northwestern Mexico. The strains were isolated and cultured in 2% bactocasitone medium. Enflagellation assays, ultrastructural analysis, protein and protease electrophoresis patterns, and PCR were performed as confirmatory tests. Virulence tests were done using in Balb/c mice. Light microscopy analysis of brain tissue showed amoebae with abundant inflammatory reaction and extensive necrotic and hemorrhagic areas. The enflagellation assay was positive and the electron microscopy showed trophozoites with morphologic features typical of the genus. Protein and protease profiles of the isolated strains were identical to the reference strain. Finally, a 1500-bp PCR product was found in all three strains. Based on all the analyses performed, we concluded that the etiologic agent of both PAM cases was N. fowleri. The need for better epidemiological information and educational programs about basic clinical and pathological aspects of free-living amoebae provided by the health authorities are emphasized.

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis.

Inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and ω3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.