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.

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.

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.

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.

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.

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.

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.

Hepatoprotective Effects of Nonpolar Extracts from Inflorescences of Thistles Cirsium vulgare and Cirsium ehrenbergii on Acute Liver Damage in Rat.

BACKGROUND: Drugs for the treatment of liver diseases are scarce and not effective enough. Some species of the genus Cirsium possess hepatoprotective activity. There are no studies on the hepatoprotective effects of nonpolar extracts from inflorescences of thistles Cirsium vulgare and Cirsium ehrenbergii, and there are few reports on their chemical composition. OBJECTIVE: The aim is to obtain the hexane extract from inflorescences of both thistles and to identify preliminarily their main chemical component, and to evaluate the hepatoprotective properties of the extracts. MATERIALS AND METHODS: Hexane extracts were obtained using a Soxhlet apparatus. The chemical composition was analyzed using infrared spectra and gas chromatography-mass spectrometry. Two doses (250 and 500 mg/kg, p.o.) of both extracts were administered to assess their hepatoprotective effect on acute carbon tetrachloride (TC)-induced liver damage in rats using biochemical markers of necrosis, cholestasis, functionality, oxidative stress, and histological analysis. RESULTS: Extracts were shown to have a very similar chemical profile. Their major constituent seems to be lupeol acetate. The two doses of both extracts demonstrated comparable hepatoprotective properties because they significantly diminished all the liver injury indicators (P < 0.05) and were corroborated using histopathology. CONCLUSION: This is the first study on the hepatoprotective effects of nonpolar extracts from inflorescences of thistles C. vulgare and C. ehrenbergii. Hexane extracts administration totally prevented the acute TC-induced liver damage. The preliminary chemical analysis strongly suggests the lupeol acetate as their major constituent. Lupeol and its derivatives have been previously reported as antiinflammatory and hepatoprotective agents. SUMMARY: Hexane extracts of both thistles kept normal liver functionality and glycogen store in carbon tetrachloride-induced acute liver damageHexane extracts of both thistles showed anti-necrotic and anti-cholestatic effects, also diminished the lipid peroxidation and nitric oxide levels on the carbon tetrachloride-induced acute liver damageThe two doses of hexane extracts administered (250 and 500 mg/kg) prevented the liver injury in a very similar extentBoth nonpolar extracts are chemically very similar and their main compound seems to be lupeol acetate. Abbreviations used: TC: Carbon tetrachloride; FT-IR: Fourier transform Infrared spectroscopy; GC-MS: Gas chromatography - Mass spectrometry; V: Vehicle; E: Extract; Ecv: Extract of Cirsium vulgare; Ece: Extract of Cirsium ehrenbergii; AP: Alkaline phosphatase; GGTP: γ-Glutamyl transpeptidase; ALT: Alanine aminotransferase; DB: Direct bilirubin; TB: Total bilirubin; LP: Lipid peroxidation; MDA: Malondialdehyde; NO: Nitric oxide; TNF-α: Tumor necrosis factor-α.

Myosin 1F Regulates M1-Polarization by Stimulating Intercellular Adhesion in Macrophages.

Intestinal macrophages are highly mobile cells with extraordinary plasticity and actively contribute to cytokine-mediated epithelial cell damage. The mechanisms triggering macrophage polarization into a proinflammatory phenotype are unknown. Here, we report that during inflammation macrophages enhance its intercellular adhesion properties in order to acquire a M1-phenotype. Using in vitro and in vivo models we demonstrate that intercellular adhesion is mediated by integrin-αVß3 and relies in the presence of the unconventional class I myosin 1F (Myo1F). Intercellular adhesion mediated by αVß3 stimulates M1-like phenotype in macrophages through hyperactivation of STAT1 and STAT3 downstream of ILK/Akt/mTOR signaling. Inhibition of integrin-αVß3, Akt/mTOR, or lack of Myo1F attenuated the commitment of macrophages into a pro-inflammatory phenotype. In a model of colitis, Myo1F deficiency strongly reduces the secretion of proinflammatory cytokines, decreases epithelial damage, ameliorates disease activity, and enhances tissue repair. Together our findings uncover an unknown role for Myo1F as part of the machinery that regulates intercellular adhesion and polarization in macrophages.

Toll-like receptors participate in Naegleria fowleri recognition.

Naegleria fowleri is a protozoan that invades the central nervous system and causes primary amoebic meningoencephalitis. It has been reported that N. fowleri induces an important inflammatory response during the infection. In the present study, we evaluated the roles of Toll-like receptors in the recognition of N. fowleri trophozoites by human mucoepithelial cells, analyzing the expression and production of innate immune response mediators. After amoebic interactions with NCI-H292 cells, the expression and production levels of IL-8, TNF-α, IL-1ß, and human beta defensin-2 were evaluated by RT-PCR, ELISA, immunofluorescence, and dot blot assays, respectively. To determine whether the canonical signaling pathways were engaged, we used different inhibitors, namely, IMG-2005 for MyD88 and BAY 11-7085 for the nuclear factor NFkB. Our results showed that the expression and production of the pro-inflammatory cytokines and beta defensin-2 were induced by N. fowleri mainly through the canonical TLR4 pathway in a time-dependent manner.

Naringenin prevents experimental liver fibrosis by blocking TGFß-Smad3 and JNK-Smad3 pathways.

AIM: To study the molecular mechanisms involved in the hepatoprotective effects of naringenin (NAR) on carbon tetrachloride (CCl4)-induced liver fibrosis. METHODS: Thirty-two male Wistar rats (120-150 g) were randomly divided into four groups: (1) a control group (n = 8) that received 0.7% carboxy methyl-cellulose (NAR vehicle) 1 mL/daily p.o.; (2) a CCl4 group (n = 8) that received 400 mg of CCl4/kg body weight i.p. 3 times a week for 8 wk; (3) a CCl4 + NAR (n = 8) group that received 400 mg of CCl4/kg body weight i.p. 3 times a week for 8 wk and 100 mg of NAR/kg body weight daily for 8 wk p.o.; and (4) an NAR group (n = 8) that received 100 mg of NAR/kg body weight daily for 8 wk p.o. After the experimental period, animals were sacrificed under ketamine and xylazine anesthesia. Liver damage markers such as alanine aminotransferase (ALT), alkaline phosphatase (AP), γ-glutamyl transpeptidase (γ-GTP), reduced glutathione (GSH), glycogen content, lipid peroxidation (LPO) and collagen content were measured. The enzymatic activity of glutathione peroxidase (GPx) was assessed. Liver histopathology was performed utilizing Masson's trichrome and hematoxylin-eosin stains. Zymography assays for MMP-9 and MMP-2 were carried out. Hepatic TGF-ß, α-SMA, CTGF, Col-I, MMP-13, NF-κB, IL-1, IL-10, Smad7, Smad3, pSmad3 and pJNK proteins were detected via western blot. RESULTS: NAR administration prevented increases in ALT, AP, γ-GTP, and GPx enzymatic activity; depletion of GSH and glycogen; and increases in LPO and collagen produced by chronic CCl4 intoxication (P < 0.05). Liver histopathology showed a decrease in collagen deposition when rats received NAR in addition to CCl4. Although zymography assays showed that CCl4 produced an increase in MMP-9 and MMP-2 gelatinase activity; interestingly, NAR administration was associated with normal MMP-9 and MMP-2 activity (P < 0.05). The anti-inflammatory, antinecrotic and antifibrotic effects of NAR may be attributed to its ability to prevent NF-κB activation and the subsequent production of IL-1 and IL-10 (P < 0.05). NAR completely prevented the increase in TGF-ß, α-SMA, CTGF, Col-1, and MMP-13 proteins compared with the CCl4-treated group (P < 0.05). NAR prevented Smad3 phosphorylation in the linker region by JNK since this flavonoid blocked this kinase (P < 0.05). CONCLUSION: NAR prevents CCl4 induced liver inflammation, necrosis and fibrosis, due to its antioxidant capacity as a free radical inhibitor and by inhibiting the NF-κB, TGF-ß-Smad3 and JNK-Smad3 pathways.

Entamoeba histolytica: Overexpression of the gal/galnac lectin, ehcp2 and ehcp5 genes in an in vivo model of amebiasis.

The parasite Entamoeba histolytica causes intestinal amebiasis and amebic liver abscess as its main extraintestinal manifestation. To study the in vivo events related to inflammation and the interactions between hosts and parasites during amebiasis, we designed a novel model of host-parasite interactions using cellulose membrane dialysis bags containing E. histolytica trophozoites. A bag is placed into the hamster peritoneal cavity, as has been reported in previous studies of programmed cell death (PCD) in E. histolytica trophozoites. To determine if virulence factors such as cysteine proteinases (EhCP2 and EhCP5) and Gal/GalNAc lectin could be involved in the host-parasite interaction using this model, we examined the relative expression of the ehcp2 and ehcp5 genes and the carbohydrate recognition domain (crd) of Gal/GalNAc lectin using real-time quantitative PCR (qRT-PCR). All analyzed genes were over-expressed 0.5h after the initiation of the host-parasite interaction and were then progressively down-regulated. However, Gal/GalNAc lectin had the greatest increase in gene expression 1.5h after host-parasite interaction; Gal/GalNAc lectin had a 250-fold increase with respect to the axenically grown trophozoites, which over-express Gal/GalNAc lectin in in vivo models. These results support the important role of these molecules in the initiation of cell damage by E. histolytica.

Double staining of ß-galactosidase with fibrosis and cancer markers reveals the chronological appearance of senescence in liver carcinogenesis induced by diethylnitrosamine.

Cellular senescence is characterized by irreversible cell arrest and is associated with the development of chronic diseases, including cancer. Here, we investigated the induction of cellular senescence during liver carcinogenesis. Liver cancer was induced in Fischer 344 rats with a weekly intraperitoneal injection of diethylnitrosamine (50mg/kg body weight) for 16 weeks. Double-detection of ß-galactosidase with Ki67 for cell proliferation; a-SMA and Pdgfrb for cell specificity; p53, p21, p16, and cyclin D1, CDK2, and CDK4 for senescence-associated molecular pathways and γ-glutamyltranspeptidase (GGT) for hepatocarcinogenesis was assessed to determine the association of these markers with cellular senescence. DNA damage was measured through senescence-associated heterochromatin foci (SAHF) detection. Progressive cellular senescence was observed in both fibrotic septa and hepatocytes from week 10 to 18. The maximum peak of positive senescent and fibrotic cells was observed at week 16 and decreased at week 18, but cell proliferation remained high. Whereas the increased p16 expression and SAHF were concomitant with that of ß-galactosidase, those of p53 and p21 were barely detected. Furthermore, ß-galactosidase positive myofibroblast-like cells were mainly surrounding GGT-positive tumors. Our findings showed that in hepatocarcinogenesis by diethylnitrosamine, cellular senescence is associated with p16 pathway activation and is mainly localized in myofibroblast-like cells.

CD40 Ligand Deficient C57BL/6 Mouse Is a Potential Surrogate Model of Human X-Linked Hyper IgM (X-HIGM) Syndrome for Characterizing Immune Responses against Pathogens.

Individuals with X-HIGM syndrome fail to express functional CD40 ligand; consequently they cannot mount effective protective antibody responses against pathogenic bacteria. We evaluated, compared, and characterized the humoral immune response of wild type (WT) and C57-CD40L deficient (C57-CD40L(-/-)) mice infected with Citrobacter rodentium. Basal serum isotype levels were similar for IgM and IgG3 among mice, while total IgG and IgG2b concentrations were significantly lower in C57-CD40L(-/-) mice compared with WT. Essentially IgG1 and IgG2c levels were detectable only in WT mice. C57-CD40L(-/-) animals, orally inoculated with 2 × 10(9) CFU, presented several clinical manifestations since the second week of infection and eventually died. In contrast at this time point no clinical manifestations were observed among C57-CD40L(-/-) mice infected with 1 × 10(7) CFU. Infection was subclinical in WT mice inoculated with either bacterial dose. The serum samples from infected mice (1 × 10(7) CFU), collected at day 14 after infection, had similar C. rodentium-specific IgM titres. Although C57-CD40L(-/-) animals had lower IgG and IgG2b titres than WT mice, C57-CD40L(-/-) mice sera displayed complement-mediated bactericidal activity against C. rodentium. C. rodentium-infected C57-CD40L(-/-) mice are capable of producing antibodies that are protective. C57-CD40L(-/-) mouse is a useful surrogate model of X-HIGM syndrome for studying immune responses elicited against pathogens.