Lactoferrin and Lysozyme Inhibit the Proteolytic Activity and Cytopathic Effect of Naegleria fowleri Enzymes.

Naegleria fowleri is a ubiquitous free-living amoeba that causes primary amoebic meningoencephalitis. As a part of the innate immune response at the mucosal level, the proteins lactoferrin (Lf) and lysozyme (Lz) are secreted and eliminate various microorganisms. We demonstrate that N. fowleri survives the individual and combined effects of bovine milk Lf (bLf) and chicken egg Lz (cLz). Moreover, amoebic proliferation was not altered, even at 24 h of co-incubation with each protein. Trophozoites' ultrastructure was evaluated using transmission electron microscopy, and these proteins did not significantly alter their organelles and cytoplasmic membranes. Protease analysis using gelatin-zymograms showed that secreted proteases of N. fowleri were differentially modulated by bLf and cLz at 3, 6, 12, and 24 h. The bLf and cLz combination resulted in the inhibition of N. fowleri-secreted proteases. Additionally, the use of protease inhibitors on bLf-zymograms demonstrated that secreted cysteine proteases participate in the degradation of bLf. Nevertheless, the co-incubation of trophozoites with bLf and/or cLz reduced the cytopathic effect on the MDCK cell line. Our study suggests that bLf and cLz, alone or together, inhibited secreted proteases and reduced the cytopathic effect produced by N. fowleri; however, they do not affect the viability and proliferation of the trophozoites.

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.

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.

The Arp2/3 Inhibitory Protein Arpin Is Required for Intestinal Epithelial Barrier Integrity.

The intestinal epithelial barrier (IEB) depends on stable interepithelial protein complexes such as tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton. During inflammation, the IEB is compromised due to TJ protein internalization and actin remodeling. An important actin regulator is the actin-related protein 2/3 (Arp2/3) complex, which induces actin branching. Activation of Arp2/3 by nucleation-promoting factors is required for the formation of epithelial monolayers, but little is known about the relevance of Arp2/3 inhibition and endogenous Arp2/3 inhibitory proteins for IEB regulation. We found that the recently identified Arp2/3 inhibitory protein arpin was strongly expressed in intestinal epithelial cells. Arpin expression decreased in response to tumor necrosis factor (TNF)α and interferon (IFN)γ treatment, whereas the expression of gadkin and protein interacting with protein C-kinase α-subunit 1 (PICK1), other Arp2/3 inhibitors, remained unchanged. Of note, arpin coprecipitated with the TJ proteins occludin and claudin-1 and the AJ protein E-cadherin. Arpin depletion altered the architecture of both AJ and TJ, increased actin filament content and actomyosin contractility, and significantly increased epithelial permeability, demonstrating that arpin is indeed required for maintaining IEB integrity. During experimental colitis in mice, arpin expression was also decreased. Analyzing colon tissues from ulcerative colitis patients by Western blot, we found different arpin levels with overall no significant changes. However, in acutely inflamed areas, arpin was significantly reduced compared to non-inflamed areas. Importantly, patients receiving mesalazine had significantly higher arpin levels than untreated patients. As arpin depletion (theoretically meaning more active Arp2/3) increased permeability, we wanted to know whether Arp2/3 inhibition would show the opposite. Indeed, the specific Arp2/3 inhibitor CK666 ameliorated TNFα/IFNγ-induced permeability in established Caco-2 monolayers by preventing TJ disruption. CK666 treatment also attenuated colitis development, colon tissue damage, TJ disruption, and permeability in dextran sulphate sodium (DSS)-treated mice. Our results demonstrate that loss of arpin triggers IEB dysfunction during inflammation and that low arpin levels can be considered a novel hallmark of acute inflammation.

Overexpression and extra-mitochondrial localization of the chaperonin Hsp60 in ameloblastoma.

OBJECTIVES: Ameloblastoma is an odontogenic neoplasm of the mandible and maxilla with various histological types and subtypes. It has been reported that some ameloblastomas could arise from dentigerous cyst walls; thus, the development of ameloblastoma from dentigerous cysts may be due to differential protein expression. Our aim was to identify a membrane protein that is differentially expressed in ameloblastomas with respect to dentigerous cysts. METHODS: We analyzed the SDS-PAGE profiles of membrane proteins from ameloblastomas and dentigerous cysts. The protein in a band present in the ameloblastoma sample, but apparently absent in the dentigerous cyst sample was identified via mass spectrometry as the chaperonin Hsp60. We used western blotting and immunohistochemistry to analyze its overexpression and localization in ameloblastoma. RESULTS: We found a differential band of 95 kDa in the membrane proteins of ameloblastoma. In this band, the chaperonin Hsp60 was identified, and its overexpression was corroborated using western blotting and immunohistochemistry. Hsp60 was localized in the plasma membrane of all ameloblastoma samples studied; in addition, it was found in the cell nucleus of the plexiform subtype of conventional ameloblastoma. CONCLUSIONS: Our results suggest that Hsp60 may be involved in ameloblastoma development, and could therefore be a potential therapeutic target for ameloblastoma treatment.

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.

Lectins as virulence factors in Entamoeba histolytica and free-living amoebae.

Currently, there is growing interest in the identification and purification of microbial lectins due to their involvement in the pathogenicity mechanisms of pathogens, such as Entamoeba histolytica and free-living amoebae. The Gal/GalNAc lectin from E. histolytica participates in adhesion, cytotoxicity and regulation of immune responses. Furthermore, mannose- and galactose-binding protein have been described in Acanthamoeba castellanii and Balamuthia mandrillaris, respectively and they also contribute to host damage. Finally, in Naegleria fowleri, molecules containing mannose and fucose are implicated in adhesion and cytotoxicity. Considering their relevance in the pathogenesis of the diseases caused by these protozoa, lectins appear to be promising targets in the diagnosis, vaccination and treatment of these infections.

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.

Intestinal amoebiasis: 160 years of its first detection and still remains as a health problem in developing countries.

Amoebiasis is a parasitic disease caused by Entamoeba histolytica (E. histolytica), an extracellular enteric protozoan. This infection mainly affects people from developing countries with limited hygiene conditions, where it is endemic. Infective cysts are transmitted by the fecal-oral route, excysting in the terminal ileum and producing invasive trophozoites (amoebae). E. histolytica mainly lives in the large intestine without causing symptoms; however, possibly as a result of so far unknown signals, the amoebae invade the mucosa and epithelium causing intestinal amoebiasis. E. histolytica possesses different mechanisms of pathogenicity for the adherence to the intestinal epithelium and for degrading extracellular matrix proteins, producing tissue lesions that progress to abscesses and a host acute inflammatory response. Much information has been obtained regarding the virulence factors, metabolism, mechanisms of pathogenicity, and the host immune response against this parasite; in addition, alternative treatments to metronidazole are continually emerging. An accesible and low-cost diagnostic method that can distinguish E. histolytica from the most nonpathogenic amoebae and an effective vaccine are necessary for protecting against amoebiasis. However, research about the disease and its prevention has been a challenge due to the relationship between E. histolytica and the host during the distinct stages of the disease is multifaceted. In this review, we analyze the interaction between the parasite, the human host, and the colon microbiota or pathogenic microorganisms, which together give rise to intestinal amoebiasis.

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.

A comparative study of the membrane proteins from Naegleria species: A 23-kDa protein participates in the virulence of Naegleria fowleri.

The plasma membrane is essential in the pathogenicity of several microorganisms. However, to date, there are few studies related to the plasma membrane proteins in Naegleria fowleri; this amoeba produces a fatal disease called primary amoebic meningoencephalitis. In the present study, we analyzed the electrophoretic pattern of the membrane proteins of N. fowleri and compared it with the nonpathogenic N. lovaniensis and N. gruberi. We detected a 23-kDa protein (Nf23) present at a higher level in N. fowleri than in the nonpathogenic amoebae. The mass spectrometry analysis showed that the Nf23 protein has a sequence of 229 amino acids that corresponds to a membrane protein. The mRNA level of nf23 was overexpressed 4-fold and 40,000-fold in N. fowleri compared with N. lovaniensis and N. gruberi, respectively. Moreover, we found a 5-fold overexpression of nf23 in N. fowleri trophozoites recovered from mouse brains compared with trophozoites axenically cultivated. In addition, the cytopathic effect on Madin-Darby Canine Kidney cells coincubated with N. fowleri diminished in the presence of antibodies against Nf23; nevertheless, the nonpathogenic amoebae did not produce damage to the monolayer cells. These results suggest that the plasma membrane protein Nf23 is probably involved in the virulence of N. fowleri.

Increased heart fibrosis and acute infection in a murine Chagas disease model associated with organophosphorus pesticide metabolite exposure.

Some reports suggest that exposure to organophosphorus (OP) pesticides increases the incidence of infections. Ethylated dialkylphosphates (EtDAPs) are metabolites of OP pesticides widely distributed with immunomodulatory potential. Chagas disease is produced by Trypanosoma cruzi parasites, and resolution of this infection requires the activation of inflammatory macrophages (MΦ), which results in cardiac fibrosis. Some reports indicate that EtDAPs increase the amount of the anti-inflammatory alternatively activated MΦ (M2; CD206+F4/80+). Therefore, we analyzed the course of T. cruzi infection, MΦ profiles from peritoneal exudate cells (PECs), inflammatory cell infiltration and fibrosis in the heart of BALB/c mice exposed to diethyldithiophosphate (DEDTP), diethylthiophosphate (DETP) or diethylphosphate (DEP, 0.01 g/kg), common DAPs produced by OP pesticides, 24 h before infection with T. cruzi. We found that DEDTP increased the parasite burden in blood by 99% at the peak of the infection and enhanced the myocardial damage due to an increase in infiltrated inflammatory cells (induced by DEDTP or DETP) and fibrosis (induced by EtDAPs). In the PECs, exposure to EtDAPs increased the proportion of the MΦ subpopulations of M2a, M2b and M2d, which are associated with tissue repair. These results indicate that exposure to EtDAPs can exacerbate the acute phase of a parasitic infection and increase the long-term damage to the heart.

Syncytiotrophoblast of Placentae from Women with Zika Virus Infection Has Altered Tight Junction Protein Expression and Increased Paracellular Permeability.

The cytotrophoblast of human placenta transitions into an outer multinucleated syncytiotrophoblast (STB) layer that covers chorionic villi which are in contact with maternal blood in the intervillous space. During pregnancy, the Zika virus (ZIKV) poses a serious prenatal threat. STB cells are resistant to ZIKV infections, yet placental cells within the mesenchyme of chorionic villi are targets of ZIKV infection. We seek to determine whether ZIKV can open the paracellular pathway of STB cells. This route is regulated by tight junctions (TJs) which are present in the uppermost portion of the lateral membranes of STB cells. We analyzed the paracellular permeability and expression of E-cadherin, occludin, JAMs -B and -C, claudins -1, -3, -4, -5 and -7, and ZO-1, and ZO-2 in the STB of placentae from ZIKV-infected and non-infected women. In ZIKV-infected placentae, the pattern of expression of TJ proteins was preserved, but the amount of claudin-4 diminished. Placentae from ZIKV-infected women were permeable to ruthenium red, and had chorionic villi with a higher mean diameter and Hofbauer hyperplasia. Finally, ZIKV added to the basolateral surface of a trophoblast cell line reduced the transepithelial electrical resistance. These results suggest that ZIKV can open the paracellular pathway of STB cells.

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.

Homoectoine Protects Against Colitis by Preventing a Claudin Switch in Epithelial Tight Junctions.

BACKGROUND: Inflammatory bowel diseases (IBD) are multifactorial disorders affecting millions of people worldwide with alarmingly increasing incidences every year. Dysfunction of the intestinal epithelial barrier is associated with IBD pathogenesis, and therapies include anti-inflammatory drugs that enhance intestinal barrier function. However, these drugs often have adverse side effects thus warranting the search for alternatives. Compatible solutes such as bacterial ectoines stabilize cell membranes and proteins. AIM: To unravel whether ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) and homoectoine (4,5,6,7-tetrahydro-2-methyl-1H-(1,3)-diazepine-4-carboxylic acid), a synthetic derivative of ectoine, have beneficial effects during dextran sulfate sodium (DSS)-induced colitis in mice. METHODS/RESULTS: We found that the disease activity index was significantly reduced by both ectoines. DSS-induced edema formation, epithelial permeability, leukocyte recruitment and tissue damage were reduced by ectoine and homoectoine, with the latter having stronger effects. Interestingly, the claudin switch usually observed during colitis (decreased expression of claudin-1 and increased expression of the leaky claudin-2) was completely prevented by homoectoine, whereas ectoine only reduced claudin-2 expression. Concomitantly, only homoectoine ameliorated the drop in transepithelial electrical resistance induced by IFN-γ and TNF-α in Caco-2 cells. Both ectoines inhibited loss of ZO-1 and occludin and prevented IFN-γ/TNF-α-induced increased paracellular flux of 4 kDa FITC-dextran in vitro. Moreover, both ectoines reduced expression of pro-inflammatory cytokines and oxidative stress during colitis. CONCLUSION: While both ectoine and homoectoine have protective effects on the epithelial barrier during inflammation, only homoectoine completely prevented the inflammatory claudin switch in tight junctions. Thus, homoectoine may serve as diet supplement in IBD patients to reach or extend remission.

The organophosphate pesticide methamidophos opens the blood-testis barrier and covalently binds to ZO-2 in mice.

Methamidophos (MET) is an organophosphate (OP) pesticide widely used in agriculture in developing countries. MET causes adverse effects in male reproductive function in humans and experimental animals, but the underlying mechanisms remain largely unknown. We explored the effect of MET on mice testes (5 mg/kg/day/4 days), finding that this pesticide opens the blood-testis barrier and perturbs spermatogenesis, generating the appearance of immature germ cells in the epididymis. In the seminiferous tubules, MET treatment changed the level of expression or modified the stage-specific localization of tight junction (TJ) proteins ZO-1, ZO-2, occludin, and claudin-3. In contrast, claudin-11 was barely altered. MET also modified the shape of claudin-11, and ZO-2 at the cell border, from a zigzag to a more linear pattern. In addition, MET diminished the expression of ZO-2 in spermatids present in seminiferous tubules, induced the phosphorylation of ZO-2 and occludin in testes and reduced the interaction between these proteins assessed by co-immunoprecipitation. MET formed covalent bonds with ZO-2 in serine, tyrosine and lysine residues. The covalent modifications formed on ZO-2 at putative phosphorylation sites might interfere with ZO-2 interaction with regulatory molecules and other TJ proteins. MET bonds formed at ZO-2 ubiquitination sites likely interfere with ZO-2 degradation and TJ sealing, based on results obtained in cultured epithelial cells transfected with ZO-2 mutated at a MET target lysine residue. Our results shed light on MET male reproductive toxicity and are important to improve regulations regarding the use of OP pesticides and to protect the health of agricultural workers.

Flavonoids as a Natural Treatment Against Entamoeba histolytica.

Over the past 20 years, gastrointestinal infections in developing countries have been a serious health problem and are the second leading cause of morbidity among all age groups. Among pathogenic protozoans that cause diarrheal disease, the parasite Entamoeba histolytica produces amebic colitis as well as the most frequent extra-intestinal lesion, an amebic liver abscess (ALA). Usually, intestinal amebiasis and ALA are treated with synthetic chemical compounds (iodoquinol, paromomycin, diloxanide furoate, and nitroimidazoles). Metronidazole is the most common treatment for amebiasis. Although the efficacy of nitroimidazoles in killing amebas is known, the potential resistance of E. histolytica to this treatment is a concern. In addition, controversial studies have reported that metronidazole could induce mutagenic effects and cerebral toxicity. Therefore, natural and safe alternative drugs against this parasite are needed. Flavonoids are natural polyphenolic compounds. Flavonoids depend on malonyl-CoA and phenylalanine to be synthesized. Several flavonoids have anti-oxidant and anti-microbial properties. Since the 1990s, several works have focused on the identification and purification of different flavonoids with amebicidal effects, such as, -(-)epicatechin, kaempferol, and quercetin. In this review, we investigated the effects of flavonoids that have potential amebicidal activity and that can be used as complementary and/or specific therapeutic strategies against E. histolytica trophozoites. Interestingly, it was found that these natural compounds can induce morphological changes in the amebas, such as chromatin condensation and cytoskeletal protein re-organization, as well as the upregulation and downregulation of fructose-1,6-bisphosphate aldolase, glyceraldehyde-phosphate dehydrogenase, and pyruvate:ferredoxin oxidoreductase (enzymes of the glycolytic pathway). Although the specific molecular targets, bioavailability, route of administration, and doses of some of these natural compounds need to be determined, flavonoids represent a very promising and innocuous strategy that should be considered for use against E. histolytica in the era of microbial drug resistance.

Dataset of cathepsin L-like CP inhibition of Naegleria fowleri and Acanthamoeba castellanii by ppTvCP4r from Trichomonas vaginalis.

The recombinant TvCP4 prepro region (ppTvCP4r) acts as an exogenous inhibitor of cathepsin L-like CPs from Trichomonas vaginalis (Cárdenas-Guerra et al., 2015 [1]). Here, we present the dataset of the trichomonad ppTvCP4r inhibitory effect against the CP proteolytic activities from other microorganisms, such as Naegleria fowleri and Acanthamoeba castellanii free-living amoeba. The proteolytic activity inhibition of total crude extracts (TCEs) of N. fowleri and A. castellanii was determined and recorded using a fluorogenic substrate specific for cathepsin L CPs without or with a ppTvCP4r treatment at different concentrations and pH.

An In Vitro Model of the Blood-Brain Barrier: Naegleria fowleri Affects the Tight Junction Proteins and Activates the Microvascular Endothelial Cells.

Naegleria fowleri causes a fatal disease known as primary amoebic meningoencephalitis. This condition is characterized by an acute inflammation that originates from the free passage of peripheral blood cells to the central nervous system through the alteration of the blood-brain barrier. In this work, we established models of the infection in rats and in a primary culture of endothelial cells from rat brains with the aim of evaluating the activation and the alterations of these cells by N. fowleri. We proved that the rat develops the infection similar to the mouse model. We also found that amoebic cysteine proteases produced by the trophozoites and the conditioned medium induced cytopathic effect in the endothelial cells. In addition, N. fowleri can decrease the transendothelial electrical resistance by triggering the destabilization of the tight junction proteins claudin-5, occludin, and ZO-1 in a time-dependent manner. Furthermore, N. fowleri induced the expression of VCAM-1 and ICAM-1 and the production of IL-8, IL-1ß, TNF-α, and IL-6 as well as nitric oxide. We conclude that N. fowleri damaged the blood-brain barrier model by disrupting the intercellular junctions and induced the presence of inflammatory mediators by allowing the access of inflammatory cells to the olfactory bulbs.