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In a previous work we described the transcriptional silencing of the amoebapore A (AP-A) gene (Ehap-a) of Entamoeba histolytica strain HM-1:IMSS. The silencing occurred following transfection with a plasmid containing a 5' upstream region (473 bp) of Ehap-a that included a truncated segment (140 bp) of a short interspersed nuclear element (SINE1). Silencing remained in effect even after removal of the plasmid (clone G3). Neither short interfering RNA nor methylated DNA were detected, but the chromatin domain of Ehap-a in the gene-silenced trophozoites was modified. Two other similar genes (Ehap-b and one encoding a Saposin-like protein, SAPLIP 1) also became silenced. In the present work we demonstrate the silencing of a second gene of choice, one that encodes the light subunit of the Gal/GalNAc inhibitable lectin (Ehlgl1) and the other, the cysteine proteinase 5 (EhCP-5). This silencing occurred in G3 trophozoites transfected with a plasmid in which the 473 bp 5' upstream Ehap-a fragment was directly ligated to the second gene. Transcriptional silencing occurred in both the transgene and the chromosomal gene. SINE1 sequences were essential, as was a direct connection between the Ehap-a upstream region and the beginning of the open reading frame of the second gene. Gene silencing did not occur in strain HM-1:IMSS with any of these plasmid constructs. The trophozoites with two silenced genes were virulence-attenuated as were those of clone G3. In addition, trophozoites not expressing Lgl1 and AP-A proteins had a significantly reduced ability to cap the Gal/GalNAc-lectin to the uroid region when incubated with antibodies against the heavy (170 kDa) subunit of the lectin. Lysates of trophozoites lacking cysteine proteinase 5 and AP-A proteins had 30% less cysteine proteinase activity than those of HM-1:IMSS strain or the G3 clone. Silencing of other genes in G3 amoebae could provide a model to study their various functions. In addition, double gene-silenced, virulence-attenuated trophozoites may be an important tool in vaccine development.

Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion.

Entamoeba histolytica is an extracellular tissue parasite causing colitis and occasional liver abscess in humans. E. histolytica-derived secretory products (SPs) contain large amounts of cysteine proteases (CPs), one of the important amoebic virulence factors. Although tissue-residing mast cells play an important role in the mucosal inflammatory response to this pathogen, it is not known whether the SPs induce mast cell activation. In this study, when human mast cells (HMC-1 cells) were stimulated with SPs collected from pathogenic wild-type amoebae, interleukin IL-8 mRNA expression and production were significantly increased compared with cells incubated with medium alone. Inhibition of CP activity in the SPs with heat or the CP inhibitor E64 resulted in significant reduction of IL-8 production. Moreover, SPs obtained from inhibitors of cysteine protease (ICP)-overexpressing amoebae with low CP activity showed weaker stimulatory effects on IL-8 production than the wild-type control. Preincubation of HMC-1 cells with antibodies to human protease-activated receptor 2 (PAR2) did not affect the SP-induced IL-8 production. These results suggest that cysteine proteases in E. histolytica-derived secretory products stimulate mast cells to produce IL-8 via a PAR2-independent mechanism, which contributes to IL-8-mediated tissue inflammatory responses during the early phase of human amoebiasis. Entamoeba histolytica est un parasite extracellulaire des tissus provoquant des colites et occasionnellement des abcès du foie chez l’homme. Les produits de sécrétion dérivés d’E. histolytica (SPs) contiennent de grandes quantités de cystéine-protéases (CPs), l’un des principaux facteurs de virulence amibiens. Bien que les mastocytes tissulaires jouent un rôle important dans la réponse inflammatoire de la muqueuse à ce pathogène, on ne sait pas si les SPs induisent l’activation des mastocytes. Dans cette étude, lorsque des mastocytes humains (cellules HMC-1) ont été stimulés avec des SPs recueillis à partir d’amibes pathogènes de type sauvage, l’expression et la production de l’interleukine IL-8 ont été significativement augmentées par rapport à des cellules incubées avec du milieu seul. L’inhibition de l’activité des CPs dans les SPs avec la chaleur ou avec E64, un inhibiteur de CP, a entraîné une réduction significative de la production d’IL-8. En outre, les SPs obtenus à partir d’amibes surexprimant l’inhibiteur de protéases à cystéine (ICP) à faible activité de CP ont montré des effets stimulants plus faibles sur la production d’IL-8 que le contrôle de type sauvage. La pré-incubation des cellules HMC-1 avec des anticorps contre le récepteur 2 activé par la protéase humaine (PAR2) n’a pas affecté la production d’IL-8 induite par SPs. Ces résultats suggèrent que les cystéine-protéases des produits de sécrétion dérivés d’E. histolytica stimulent les mastocytes pour produire de l’IL-8 par l’intermédiaire d’un mécanisme indépendant de PAR2, ce qui contribue à la réponse inflammatoire tissulaire médiée par IL-8 au cours de la phase précoce de l’amibiase humaine.

This study describes the use of in vivo magnetic resonance spectrocopy (MRS) to monitor brain glutamate and lactate levels in a paraoxon (PO) intoxication model. Our results show that the administration of recombinant glutamate-oxaloacetate transaminase (rGOT) in combination with oxaloacetate (OxAc) significantly reduces the brain-accumulated levels of glutamate. Previously we have shown that the treatment causes a rapid decrease of blood glutamate levels and creates a gradient between the brain and blood glutamate levels which leads to the efflux of excess brain glutamate into the blood stream thereby reducing its potential to cause neurological damage. The fact that this treatment significantly decreased the brain glutamate and lactate levels following PO intoxication suggests that it could become a new effective neuroprotective agent.

Recent studies have shown that blood glutamate grabbing is an effective strategy to reduce the excitotoxic effect of extracellular glutamate released during ischemic brain injury. The purpose of the study was to investigate the effect of two of the most efficient blood glutamate grabbers (oxaloacetate and recombinant glutamate oxaloacetate transaminase 1: rGOT1) in a rat model of intracerebral hemorrhage (ICH). Intracerebral hemorrhage was produced by injecting collagenase into the basal ganglia. Three treatment groups were developed: a control group treated with saline, a group treated with oxaloacetate, and a final group treated with human rGOT1. Treatments were given 1 hour after hemorrhage. Hematoma volume (analyzed by magnetic resonance imaging (MRI)), neurologic deficit, and blood glutamate and GOT levels were quantified over a period of 14 days after surgery. The results observed showed that the treatments used induced a significant reduction of blood glutamate levels; however, they did not reduce the hematoma, nor did they improve the neurologic deficit. In the present experimental study, we have shown that this novel therapeutic strategy is not effective in case of ICH pathology. More importantly, these findings suggest that blood glutamate grabbers are a safe treatment modality that can be given in cases of suspected ischemic stroke without previous neuroimaging.

We have previously discovered a unique mechanism of epigenetic transcriptional gene silencing in the Entamoeba histolytica trophozoites of strain HM-1:IMSS that resulted in the persistent downregulation of the amoebapore A (ap-a) gene, and that could be successfully applied to silence other virulence genes (cpA5, lgl1). In order to understand how the silencing is maintained throughout generations, we analysed whether modifications occurred at the chromatin level. Chromatin immunoprecipitation assays were done with antibodies specific to the methylated lysine 4 of E. histolytica histone H3. When the genes were in a transcriptionally silent state, the methylation levels of H3K4 in their coding region were significantly reduced. In contrast, the levels of core histone H3 were consistently higher in the silenced genes. Controlled chromatin digestion with micrococcal nuclease was used to assess changes in nucleosome compaction. We found a significant resistance to digestion in the promoter region of the silenced ap-a and cpA5 genes as compared to the parental strain that expresses those genes. Our data lend further support to the idea that histone modifications and heterochromatin formations are at the basis of the transcriptional silencing of genes in E. histolytica.

Background Allicin in garlic is the primary active compound known to rapidly interact with free thiols. Aims of the study To examine the effect of allicin on gene expression and glutathione cellular level in vascular endothelial cells. Methods Cultured endothelial cells were exposed to allicin; mRNA was prepared and subjected to Micro-array and Real-Time PCR. Glutathione cellular level was determined on cell lysates. Results Micro-array analysis demonstrated allicin-induced up- and down-regulation of 116 and 100 genes, respectively. Up-regulated genes included the phase II detoxifying enzymes thioredoxin reductase 1 and 2, heme oxygenase-1 and glutamate cysteine lygaze modifier subunit, the rate limiting enzyme in glutathione biosynthesis. Endothelial cells exposed to allicin and its derivatives containing glutathione or cysteine residues increased cellular glutathione. Allicin increased the glutathione level in a concentration and time-dependent manner up to 8-fold at a concentration of 10-20 μM after 28 h exposure. Furthermore, allicin derivative-treated cultures demonstrated a 50% decrease in tBuOOH cytotoxicity. Conclusions These results may suggest a putative role for allicin and its derivatives in preventing reactive oxygen species damage by up-regulating the phase II detoxifying enzymes and increasing the cellular glutathione level.

We have previously demonstrated that inhibition of expression of amoebapore A (AP-A) by antisense RNA caused a marked decrease in the virulence of the parasite. A four-fold over-expression of AP-A was obtained with plasmid (pA7) which has theap-a gene under the control of geneEhgLE-3-RP-L21. The virulence of the transfected trophozoites, however, was also decreased. Excess of AP-A protein was found in the cytosol and a significant amount was released into the surrounding media. Transfection of the parasite with a plasmid (psAP-1) in which theap-a gene was introduced with its own regulatory sequences, caused a total suppression of the transcription and translation of both the genomic and episomalap-a genes. The silenced transfectant was not virulent at all. These results demonstrate that important factors need to be expressed at the correct cellular location and that the parasite has additional internal control mechanisms such as transcriptional gene silencing which can prevent excess amounts of gene expression.[PUBLICATION ABSTRACT]

Hypertension often coexists with hyperlipidemia, insulin resistance, and glucose intolerance in metabolic syndrome. Allylmercaptocaptopril is a conjugate of the angiotensin-converting enzyme inhibitor captopril with allicin, an active principle in garlic with multiple beneficial actions on metabolic-syndrome abnormalities. We sought to test the hypothesis that the conjugation of allicin to captopril may confer additional therapeutic actions in metabolic disease. We compared allylmercaptocaptopril (53.5 mg/kg/day orally for 60 days) to an equimolar dose of captopril (40 mg/kg/day) in the spontaneously hypertensive, obese rat (SHROB) model. Allylmercaptocaptopril prevented progressive weight gain, without a detectable effect on food intake. Both captopril and allylmercaptocaptopril lowered blood pressure, but allylmercaptocaptopril was more effective. Allylmercaptocaptopril, but not captopril, improved cardiac hypertrophy, as indicated by heart weight and ventricular-wall thickness. Allylmercaptocaptopril improved, whereas captopril impaired, oral glucose tolerance after a fast. Triglycerides were decreased by both captopril and allylmercaptocaptopril. Total cholesterol and non-HDL cholesterol were reduced by captopril but not by allylmercaptocaptopril. The SHROB rats developed severe glomerulosclerosis and renal failure. Allylmercaptocaptopril showed significant nephro-protection, as indicated by reductions in urinary protein loss, urinary protein-to-creatinine ratio, and plasma creatinine. Captopril showed the same trends and also prevented the decline of creatinine clearance. Finally, both allylmercaptocaptopril and captopril reduced the basal level of lipolysis in isolated abdominal adipocytes, and restored the response to catecholamine stimulation. Both captopril and allylmercaptocaptopril are effective in attenuating multiple abnormalities of metabolic syndrome. Allylmercaptocaptopril may have additional effectiveness on improving glucose tolerance, further lowering blood pressure, reducing cardiac hypertrophy, preventing weight gain, and protecting against renal disease.

Commercially available garlic preparations in the form of garlic oil, garlic powder and pills are widely used for certain therapeutic purposes, including lowering blood pressure and improving lipid profile. Despite the impressive effects of garlic most studies are limited by lack of controlled methods and suitable double-blinding, and by the use of preparations with unknown amounts and chemical identification of the active ingredient. Allicin, a synthetic preparation of an active constituent of garlic, was found to lower blood pressure, insulin, and triglycerides levels in fructose-fed rats. Thus, it was considered important to assess its effect on the weight of the animals. Male Sprague-Dawley rats weighing 240 to 250 g were fed a fructose-enriched diet consisting of 21% protein, 5% fat, 60% carbohydrate, 0.49% sodium and 0.49% potassium for 5 weeks, which produced hyperinsulinemia, hypertension, and hypertriglyceridemia. Group I (controls) rats were fed a diet enriched by fructose only; group II was given allicin the last 2 weeks, and group III was given allicin the first 3 weeks. The three groups consumed the same amount of food. Weight was measured at the beginning of the experiment and after 3 and 5 weeks on the diet. Weight in the control group rose from 249.4 ± 10.04 g to 274.5 ± 15.5 g after 3 weeks and to 306.9 ± 22.2 g after 5 weeks. Weight in group II rose from baseline 259.1 ± 12.1 g to 306.9 ± 22.2 g after 3 weeks on fructose alone, and declined slightly to 282.4 ± 17.4 g after 2 weeks of allicin ( P < .02). In group III, in which the protocol was reversed, the baseline weight of 260.4 ± 13.25 g rose only to 269.8 ±15.3 g ( P < .431) after 3 weeks on fructose and allicin. The control group that was fed a diet enriched by fructose alone continued to gain weight, whereas the groups fed allicin did not. The difficulty of preventing weight gain after reaching the nadir of weight loss underscores the practical value of allicin for weight control.