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

Peroxynitrite and Peroxiredoxin in the Pathogenesis of Experimental Amebic Liver Abscess

The molecular mechanisms by which Entamoeba histolytica causes amebic liver abscess (ALA) are still not fully understood. Amebic mechanisms of adherence and cytotoxic activity are pivotal for amebic survival but apparently do not directly cause liver abscess. Abundant evidence indicates that chronic inflammation (resulting from an inadequate immune response) is probably the main cause of ALA. Reports referring to inflammatory mechanisms of liver damage mention a repertoire of toxic molecules by the immune response (especially nitric oxide and reactive oxygen intermediates) and cytotoxic substances released by neutrophils and macrophages after being lysed by amoebas (e.g., defensins, complement, and proteases). Nevertheless, recent evidence downplays these mechanisms in abscess formation and emphasizes the importance of peroxynitrite (ONOO−). It seems that the defense mechanism of amoebas against ONOO−, namely, the amebic thioredoxin system (including peroxiredoxin), is superior to that of mammals. The aim of the present text is to define the importance of ONOO− as the main agent of liver abscess formation during amebic invasion, and to explain the superior capacity of amoebas to defend themselves against this toxic agent through the peroxiredoxin and thioredoxin system

Biodistribution and Tumor Uptake of ⁶⁷Ga-Nimotuzumab in a Malignant Pleural Mesothelioma Xenograft

Malignant pleural mesothelioma (MPM) is the most common tumor of the pulmonary pleura. It is a rare and aggressive malignancy, generally associated with continuous occupational exposure to asbestos. Only a multimodal-approach to treatment, based on surgical resection, chemotherapy and/or radiation, has shown some benefits. However, the survival rate remains low. Nimotuzumab (h-R3), an anti-EGFR (epidermal growth factor receptor) humanized antibody, is proposed as a promising agent for the treatment of MPM. The aim of this research was to implement a procedure for nimotuzumab radiolabeling to evaluate its biodistribution and affinity for EGF (epidermal growth factor) receptors present in a mesothelioma xenograft. Nimotuzumab was radiolabeled with 67Ga; radiolabel efficiency, radiochemical purity, serum stability, and biodistribution were evaluated. Biodistribution and tumor uptake imaging studies by microSPECT/CT in mesothelioma xenografts revealed constant nimotuzumab uptake at the tumor site during the first 48 h after drug administration. In vivo studies using MPM xenografts showed a significant uptake of this radioimmunoconjugate, which illustrates its potential as a biomarker that could promote its theranostic use in patients with MPM

A review of the proposed role of neutrophils in rodent amebic liver abscess models

Host invasion by Entamoeba histolytica, the pathogenic agent of amebiasis, can lead to the development of amebic liver abscess (ALA). Due to the difficulty of exploring host and amebic factors involved in the pathogenesis of ALA in humans, most studies have been conducted with animal models (e.g., mice, gerbils, and hamsters). Histopathological findings reveal that the chronic phase of ALA in humans corresponds to lytic or liquefactive necrosis, whereas in rodent models there is granulomatous inflammation. However, the use of animal models has provided important information on molecules and mechanisms of the host/parasite interaction. Hence, the present review discusses the possible role of neutrophils in the effector immune response in ALA in rodents. Properly activated neutrophils are probably successful in eliminating amebas through oxidative and non-oxidative mechanisms, including neutrophil degranulation, the generation of free radicals (O2−, H2O2, HOCl) and peroxynitrite, the activation of NADPH-oxidase and myeloperoxidase (MPO) enzymes, and the formation of neutrophil extracellular traps (NETs). On the other hand, if amebas are not eliminated in the early stages of infection, they trigger a prolonged and exaggerated inflammatory response that apparently causes ALAs. Genetic differences in animals and humans are likely to be key to a successful host immune response

A review of the proposed role of neutrophils in rodent amebic liver abscess models

Host invasion by Entamoeba histolytica, the pathogenic agent of amebiasis, can lead to the development of amebic liver abscess (ALA). Due to the difficulty of exploring host and amebic factors involved in the pathogenesis of ALA in humans, most studies have been conducted with animal models (e.g., mice, gerbils, and hamsters). Histopathological findings reveal that the chronic phase of ALA in humans corresponds to lytic or liquefactive necrosis, whereas in rodent models there is granulomatous inflammation. However, the use of animal models has provided important information on molecules and mechanisms of the host/parasite interaction. Hence, the present review discusses the possible role of neutrophils in the effector immune response in ALA in rodents. Properly activated neutrophils are probably successful in eliminating amebas through oxidative and non-oxidative mechanisms, including neutrophil degranulation, the generation of free radicals (O2−, H2O2, HOCl) and peroxynitrite, the activation of NADPH-oxidase and myeloperoxidase (MPO) enzymes, and the formation of neutrophil extracellular traps (NETs). On the other hand, if amebas are not eliminated in the early stages of infection, they trigger a prolonged and exaggerated inflammatory response that apparently causes ALAs. Genetic differences in animals and humans are likely to be key to a successful host immune response

Different behavior of myeloperoxidase in two rodent amoebic liver abscess models

<div><p>The protozoan <i>Entamoeba histolytica</i> is the etiological agent of amoebiasis, which can spread to the liver and form amoebic liver abscesses. Histological studies conducted with resistant and susceptible models of amoebic liver abscesses (ALAs) have established that neutrophils are the first cells to contact invasive amoebae at the lesion site. Myeloperoxidase is the most abundant enzyme secreted by neutrophils. It uses hydrogen peroxide secreted by the same cells to oxidize chloride ions and produce hypochlorous acid, which is the most efficient microbicidal system of neutrophils. In a previous report, our group demonstrated that myeloperoxidase presents amoebicidal activity <i>in vitro</i>. The aim of the current contribution was to analyze <i>in vivo</i> the role of myeloperoxidase in a susceptible (hamsters) and resistant (Balb/c mice) animal models of ALAs. In liver samples of hamsters and mice inoculated intraportally with <i>Entamoeba histolytica</i> trophozoites, the number of neutrophils in ALAs was determined by enzymatic activity. The presence of myeloperoxidase was observed by staining, and its expression and activity were quantified <i>in situ</i>. A significant difference existed between the two animal models in the number of neutrophils and the expression and activity of myeloperoxidase, which may explain the distinct evolution of amoebic liver abscesses. Hamsters and mice were treated with an MPO inhibitor (4-aminobenzoic acid hydrazide). Hamsters treated with ABAH showed no significant differences in the percentage of lesions or in the percentage of amoebae damaged compared with the untreated hamsters. ABAH treated mice versus untreated mice showed larger abscesses and a decreased percentage of damaged amoebae in these lesion at all stages of evolution. Further studies are needed to elucidate the host and amoebic mechanisms involved in the adequate or inadequate activation and modulation of myeloperoxidase.</p></div

Neutrophils positive to MPO in hamster and mice ALAs.

<p>Liver tissue was processed by immunohistochemistry to detect the presence of MPO in ALA of hamsters (A, C, E, G, I) and mice (B, D, F, H, J) at 3, 6, 12 and 24 h post-inoculation. Negative controls was performed with an irrelevant, antibody no label was observed (A, B). (C) Hamster liver lesions, neutrophils were positive in small inflammatory foci at 3 h post-inoculation (arrowhead). (E) At 6 h post-inoculation, amoebae (arrows) were positive to MPO. (G) At 12 h, appears extensive inflammatory reaction composed by damaged neutrophils positive to MPO. (I) At 24 h, inflammatory cells lysed on the border of a necrotic area were also stained to MPO. (D) Mouse liver lesions; inflammatory cells showed MPO label at 3 h post-inoculation (arrowhead), amoeba is seen (arrow). (F) At 6 h post-inoculation, neutrophils appear surround the damaged amoeba (arrow). (H) At 12 h appear damaged amoeba (arrow); the inflammatory infiltrate was constituted by neutrophils positive to MPO (arrowhead). (J) At 24 h staining for MPO was evident, the amoeba present signs of damage (arrow). Barr = 50μm.</p

Percentage of neutrophils stained by AS-D esterase in ALAs from hamsters and Balb/c mice.

<p>Liver sections were stained with AS-D chloroacetate esterase. Neutrophils were identified by their well-known morphology and a positive stain. The total number of cells were counted in the inflammatory focus (8 infiltrates per slide/3 slides per animal), as were the number of cells positive to AS-D chloroacetate esterase (neutrophils) by using Image-Pro Plus 5.1 with 40x magnification. The percentage of neutrophils was calculated. During the evolution of hamster ALA, the percentage of neutrophils diminished progressively. In mice, the percentage of neutrophils was less compare with the hamsters but the changes showed similar behaviour, except at 24 hours. At the latter times post infection, the percentage of neutrophils was higher in mice than hamsters. Data represent the mean ± SD of the three independents experiments, (n = 5). <i>P</i>-values were determined by the Student’s <i>t</i>-test (*** p<0.001; ** p<0.01).</p

<i>E</i>. <i>histolytica</i> stimulate the MPO activity in ALA of mice and to decrease in hamsters.

<p>Mice tissue samples from ALA showed a significant higher MPO activity from 6–24 h (p<0.001). Hamster liver samples from ALAs showed a significantly reduction of MPO activity from 6 to 24 h (p<0.01). There is a statistically significant difference between the species and post-inoculation time. Data represent the mean ± SD of the three independent experiments, (n = 5). <i>P</i>-values were determined by the one-way ANOVA (***p<0.001; ** p<0.01; *p<0.05).</p

Differential expression of the <i>mpo</i> gene in hamster and mice ALAs.

<p>Mice tissue samples show a significant high expression of the <i>mpo</i> gene at all post-inoculation times compared with the control group. The difference between these two ALAs models were higher at 3 and 12 h (p<0.001). Hamsters ALA did not show expression of the <i>mpo</i> gene compared with the control group, the lowest expression was found at 6 and 12 h (p<0.001). There is a statistically significant difference between the species and post-inoculation time (p<0.001). Data represent the mean ± SD of three independent experiments, (n = 5). <i>P</i>-values were determined by the one-way ANOVA (*** p<0.001).</p