Mineko Shibayama

Kupffer cells inhibition prevents hepatic lipid peroxidation and damage induced by carbon tetrachloride.

The aim of this work was to determine if the action mechanism of gadolinium on CCl(4)-induced liver damage is by preventing lipid peroxidation (that may be induced by Kupffer cells) and its effects on liver carbohydrate metabolism. Four groups of rats were treated with CCl(4), CCl(4)+GdCl(3), GdCl(3), and vehicles. CCl(4) was given orally (0.4 g 100 g(-1) body wt.) and GdCl(3) (0.20 g 100 g(-1) body wt.) was administered i.p. All the animals were killed 24 h after treatment with CCl(4) or vehicle. Glycogen and lipid peroxidation were measured in liver. Alkaline phosphatase, gamma-glutamyl transpeptidase, alanine amino transferase activities and bilirubins were measured in rat serum. A liver histological analysis was performed. CCl(4) induced significant elevations on enzyme activities and bilirubins; GdCl(3) completely prevented this effect. Liver lipid peroxidation increased 2.5-fold by CCl(4) treatment; this effect was also prevented by GdCl(3). Glycogen stores were depleted by acute intoxication with CCl(4). However, GdCl(3) did not prevent this effect. The present study shows that Kupffer cells may be responsible for liver damage induced by carbon tetrachloride and that lipid peroxidation is produced or stimulated by Kupffer cells, since their inhibition with GdCl(3) prevented both lipid peroxidation and CCl(4)-induced liver injury.

Curcumin prevents and reverses cirrhosis induced by bile duct obstruction or CCl4 in rats: role of TGF-β modulation and oxidative stress

Curcumin is a phytophenolic compound, which is highly efficacious for treating several inflammatory diseases. The aim of this study was to evaluate the efficacy of curcumin in preventing or reversing liver cirrhosis. A 4‐week bile duct ligation (BDL) rat model was used to test the ability of curcumin (100 mg/kg, p.o., daily) to prevent cirrhosis. To reverse cirrhosis, CCl4 was administered chronically for 3 months, and then it was withdrawn and curcumin administered for 2 months. Alanine aminotransferase, γ‐glutamyl transpeptidase, liver histopathology, bilirubin, glycogen, reduced and oxidized glutathione, and TGF‐β (mRNA and protein) levels were assessed. Curcumin preserved normal values of markers of liver damage in BDL rats. Fibrosis, assessed by measuring hydroxyproline levels and histopathology, increased nearly fivefold after BDL and this effect was partially but significantly prevented by curcumin. BDL increased transforming growth factor‐beta (TGF‐β) levels (mRNA and proteins), while curcumin partially suppressed this mediator of fibrosis. Curcumin also partially reversed the fibrosis induced by CCl4. Curcumin was effective in preventing and reversing cirrhosis, probably by its ability of reducing TGF‐β expression. These data suggest that curcumin might be an effective antifibrotic and fibrolitic drug in the treatment of chronic hepatic diseases.

Entamoeba histolytica: erythrophagocytosis, collagenolysis, and liver abscess production as virulence markers

High rates of erythrophagocytosis and collagenolysis in vitro have been regarded as indicative of virulence in vivo of Entamoeba histolytica trophozoites. In the present study, the erythrophagocytic index and the collagenolytic activity of 3 axenic lines of E. histolytica, strain HM1:IMSS, were measured. The 3 lines shared the same pathogenic zymodeme but showed clear-cut differences in the extent of liver damage induced in hamsters. A direct correlation between collagenolysis in vitro and the size of liver abscesses produced by each line of E. histolytica trophozoites was found. In contrast, the line with the highest erythrophagocytic index produced small amoebic abscesses in hamsters, whereas the line with a relatively low erythrophagocytic index produced the largest liver lesions. It is concluded that the extent of collagenolytic activity is a better marker of virulence of E. histolytica cultured under axenic conditions than is erythrophagocytosis.

Silencing Survivin Splice Variant 2B Leads to Antitumor Activity in Taxane-Resistant Ovarian Cancer

Purpose: To study the role of survivin and its splice variants in taxane-resistant ovarian cancer. Experimental Design: We assessed the mRNA levels of survivin splice variants in ovarian cancer cell lines and ovarian tumor samples. siRNAs targeting survivin were designed to silence all survivin splice variants (T-siRNA) or survivin 2B (2B-siRNA) in vitro and orthotopic murine models of ovarian cancer. The mechanism of cell death was studied in taxane-resistant ovarian cancer cells and in tumor sections obtained from different mouse tumors. Results: Taxane-resistant ovarian cancer cells express higher survivin mRNA levels than their taxane-sensitive counterparts. Survivin 2B expression was significantly higher in taxane-resistant compared with -sensitive cells. Silencing survivin 2B induced growth inhibitory effects similar to silencing total survivin in vitro. In addition, survivin 2B-siRNA incorporated into DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) nanoliposomes resulted in significant reduction in tumor growth (P < 0.05) in orthotopic murine models of ovarian cancer, and these effects were similar to T-siRNA-DOPC. The antitumor effects were further enhanced in combination with docetaxel chemotherapy (P < 0.01). Finally, we found a significant association between survivin 2B expression and progression-free survival in 117 epithelial ovarian cancers obtained at primary debulking surgery. Conclusions: These data identify survivin 2B as an important target in ovarian cancer and provide a translational path forward for developing new therapies against this target. Clin Cancer Res; 17(11); 3716–26. ©2011 AACR.

Human Amebiasis: Breaking the Paradigm?

For over 30 years it has been established that the Entamoeba histolytica protozoan included two biologically and genetically different species, one with a pathogenic phenotype called E. histolytica and the other with a non-pathogenic phenotype called Entamoeba dispar. Both of these amoebae species can infect humans. E. histolytica has been considered as a potential pathogen that can cause serious damage to the large intestine (colitis, dysentery) and other extraintestinal organs, mainly the liver (amebic liver abscess), whereas E. dispar is a species that interacts with humans in a commensal relationship, causing no symptoms or any tissue damage. This paradigm, however, should be reconsidered or re-evaluated. In the present work, we report the detection and genotyping of E. dispar sequences of DNA obtained from patients with amebic liver abscesses, including the genotyping of an isolate obtained from a Brazilian patient with a clinical diagnosis of intestinal amebiasis that was previously characterized as an E. dispar species. The genetic diversity and phylogenetic analysis performed by our group has shown the existence of several different genotypes of E. dispar that can be associated to, or be potentiality responsible for intestinal or liver tissue damage, similar to that observed with E. histolytica.

Ultrastructural study of the midgut and hindgut in eight species of the genus Dendroctonus Erichson (Coleoptera: Scolytidae)

Abstract Chemical communication mediated by pheromones is a crucial aspect in the life cycle of beetles in the genus Dendroctonus. This communication plays an important role in mate location and in the colonization of host conifers. The study of the alimentary canal of these species is of importance not only because this organ is involved in the processes of digestion, detoxification, nutrient absorption, and transport, but also in the production of semiochemical compounds, such as pheromones. To better understand these functions and where they occur, the ultrastructural differences between the anterior and posterior midgut and the hindgut and their different cellular types were characterized. Adult specimens of both sexes from eight species were dissected and the alimentary canal was removed. It was sectioned into three parts: anterior midgut, posterior midgut, and hindgut, and analyzed by transmission electron microscopy. Results show that the epithelial tissue of the midgut possesses ultrastructural characteristics that permit differentiation of the anterior and posterior midgut. There are no ultrastructural differences within sexes of the same species, but differences exist among species. The ultrastructural characteristics of the hindgut do not differ between sexes or among species, but they do differ from those of the midgut.

Characterization of brain inflammation during primary amoebic meningoencephalitis

Naegleria fowleri is a free-living amoeba and the etiologic agent of primary amoebic meningoencephalitis (PAM). Trophozoites reach the brain by penetrating the olfactory epithelium, and invasion of the olfactory bulbs results in an intense inflammatory reaction. The contribution of the inflammatory response to brain damage in experimental PAM has not been delineated. Using both optical and electron microscopy, we analyzed the morphologic changes in the brain parenchyma due to inflammation during experimental PAM. Several N. fowleri trophozoites were observed in the olfactory bulbs 72 h post-inoculation, and the number of amoebae increased rapidly over the next 24 h. Eosinophils and neutrophils surrounding the amoebae were then noted at later times during infection. Electron microscopic examination of the increased numbers of neutrophils and the interactions with trophozoites indicated an active attempt to eliminate the amoebae. The extent of inflammation increased over time, with a predominant neutrophil response indicating important signs of damage and necrosis of the parenchyma. These data suggest a probable role of inflammation in tissue damage. To test the former hypothesis, we used CD38-/- knockout mice with deficiencies in chemotaxis to compare the rate of mortality with the parental strain, C57BL/6J. The results showed that inflammation and mortality were delayed in the knockout mice. Based on these results, we suggest that the host inflammatory response and polymorphonuclear cell lysis contribute to a great extent to the central nervous system tissue damage.

N-acetylcysteine prevents carbon tetrachloride-induced liver cirrhosis: role of liver transforming growth factor-beta and oxidative stress

Objectives N-acetylcysteine (NAC) is an antioxidant, a precursor of reduced glutathione, and an inhibitor of the profibrotic cytokine liver transforming growth factor-beta (TGF-&bgr;). Carbon tetrachloride (CCl4) cirrhosis is characterized by oxidative stress and fibrosis. Therefore, the aim of this work was to study the effect of NAC on experimental cirrhosis. Methods CCl4 was chronically administered for 8 weeks along with 300 mg/kg of NAC orally once a day. Alkaline phosphatase, alanine aminotransferase, and &ggr;-glutamyltranspeptidase were measured in plasma. Hydroxyproline, glycogen, lipid peroxidation, glutathione were determined in liver samples by colorimetric methods. TGF-&bgr; was evaluated by western blotting, and a histopathological analysis was performed. Results Serum markers of liver damage increased by CCl4 intoxication (P<0.05), whereas cotreatment with NAC prevented these increases (P<0.05); glycogen was depleted in the cirrhotic group (P<0.05), but preserved by NAC (P<0.05). Lipid peroxidation increased and glutathione decreased by the administration of CCl4 (P<0.05), again NAC prevented both effects (P<0.05). Importantly, collagen increased by about seven-fold in the CCl4 group (P<0.05); administration of NAC preserved the normal levels of collagen (P<0.05). Biochemical determinations were corroborated by hematoxylin and eosin, and trichromic stains. Western blots revealed a four-fold increase in TGF-&bgr; in the group receiving CCl4, NAC cotreatment abolished TGF-&bgr; signal (P<0.05). Conclusion Our results strongly suggest that NAC prevents experimental cirrhosis by two mechanisms: by preventing oxidative stress and by downregulating the profibrogenic cytokine TGF-&bgr;. As NAC is currently used in humans intoxicated with paracetamol, it can be tested in fibrotic or cirrhotic patients under controlled trials.

A Biochemical Comparison of Proteases from Pathogenic Naegleria fowleri and Non-Pathogenic Naegleria gruberi

ABSTRACT. Naegleria fowleri is the etiologic agent of primary amoebic meningoencephalitis (PAM). Proteases have been suggested to be involved in tissue invasion and destruction during infection. We analyzed and compared the complete protease profiles of total crude extract and conditioned medium of both pathogenic N. fowleri and non‐pathogenic Naegleria gruberi trophozoites. Using SDS‐PAGE, we found differences in the number and molecular weight of proteolytic bands between the two strains. The proteases showed optimal activity at pH 7.0 and 35 °C for both strains. Inhibition assays showed that the main proteolytic activity in both strains is due to cysteine proteases although serine proteases were also detected. Both N. fowleri and N. gruberi have a variety of different protease activities at different pH levels and temperatures. These proteases may allow the amoebae to acquire nutrients from different sources, including those from the host. Although, the role of the amoebic proteases in the pathogenesis of PAM is not clearly defined, it seems that proteases and other molecules of the parasite as well as those from the host, could be participating in the damage to the human central nervous system.

Antifibrotic and fibrolytic properties of celecoxib in liver damage induced by carbon tetrachloride in the rat

Background: Transforming growth factor‐β (TGF‐β) plays a pivotal role in liver fibrosis, because it activates hepatic stellate cells, stimulating extracellular matrix deposition. Cyclooxygenase‐2 (COX‐2) has been associated with TGF‐β because its inhibition decreases TGF‐β expression and collagen production in some cultured cell types.