In Vitro Evaluation of the Antiamoebic Activity of Kaempferol against Trophozoites of Entamoeba histolytica and in the Interactions of Amoebae with Hamster Neutrophils.

Entamoeba histolytica (E. histolytica) is a parasite in humans that provokes amoebiasis. The most employed drug is metronidazole (MTZ); however, some studies have reported that this drug induces genotoxic effects. Therefore, it is necessary to explore new compounds without toxicity that can eliminate E. histolytica. Flavonoids are polyphenolic compounds that have demonstrated inhibition of growth and dysregulation of amoebic proteins. Despite the knowledge acquired to date, action mechanisms are not completely understood. The present work evaluates the effect of kaempferol against E. histolytica trophozoites and in the interactions with neutrophils from hamster, which is a susceptibility model. Our study demonstrated a significant reduction in the amoebic viability of trophozoites incubated with kaempferol at 150 µM for 90 min. The gene expression analysis showed a significant downregulation of Pr (peroxiredoxin), Rr (rubrerythrin), and TrxR (thioredoxin reductase). In interactions with amoebae and neutrophils for short times, we observed a reduction in ROS (reactive oxygen species), NO (nitric oxide), and MPO (myeloperoxidase) neutrophil activities. In conclusion, we confirmed that kaempferol is an effective drug against E. histolytica through the decrease in E. histolytica antioxidant enzyme expression and a regulator of several neutrophil mechanisms, such as MPO activity and the regulation of ROS and NO.

Protein Phosphatase PP2C Identification in Entamoeba spp.

Entamoeba histolytica is the causative agent of amoebiasis, and Entamoeba dispar is its noninvasive morphological twin. Entamoeba invadens is a reptilian parasite. In the present study, Western blot, phosphatase activity, immunofluorescence, and bioinformatic analyses were used to identify PP2C phosphatases of E. histolytica, E. dispar, and E. invadens. PP2C was identified in trophozoites of all Entamoeba species and cysts of E. invadens. Immunoblotting using a Leishmania mexicana anti-PP2C antibody recognized a 45.2 kDa PP2C in all species. In E. histolytica and E. invadens, a high molecular weight element PP2C at 75 kDa was recognized, mainly in cysts of E. invadens. Immunofluorescence demonstrated the presence of PP2C in membrane and vesicular structures in the cytosol of all species analyzed. The ~75 kDa PP2C of Entamoeba spp. shows the conserved domain characteristic of phosphatase enzymes (according to in silico analysis). Possible PP2C participation in the encystation process was discussed.

Impact of chronic immobilization stress on parameters of colonic homeostasis in BALB/c mice.

The intestinal epithelium is a monolayer of cells arranged side­by­side and connected by tight junction (TJ) proteins expressed at the apical extreme of the paracellular membrane. This layer prevents stress­induced inflammatory responses, thus helping to maintain gut barrier function and gut homeostasis. The aim of the present study was to evaluate the effects of chronic immobilization stress on the colonic expression of various parameters of homeostasis. A total of two groups of female BALB/c mice (n=6) were included: A stressed group (short­term immobilization for 2 h/day for 4 consecutive days) and an unstressed (control) group. Colon samples were obtained to detect neutrophils and goblet cells by optical microscopy, TJ protein expression (occludin, and claudin ­2, ­4, ­7, ­12 and ­15) by western blotting, mRNA levels of TJ genes and proinflammatory cytokines [tumor necrosis factor (TNF)­α, interleukin (IL)­1ß, ­6 and ­8] by reverse transcription­quantitative PCR, fecal lactoferrin by ELISA and the number of colony­forming units of aerobic bacteria. Compared with goblet cells in control mice, goblet cells were enlarged and reduced in number in stressed mice, whereas neutrophil cellularity was unaltered. Stressed mice exhibited reduced mRNA expression for all evaluated TJ mRNAs, with the exception of claudin­7, which was upregulated. Protein levels of occludin and all claudins (with the exception of claudin­12) were decreased in stressed mice. Fecal lactoferrin, proinflammatory cytokine mRNA levels and aerobic bacterial counts were all increased in the stressed group. These results indicated that immobilization stress induced proinflammatory and potential remodeling effects in the colon by decreasing TJ protein expression. The present study may be a useful reference for therapies aiming to regulate the effects of stress on intestinal inflammatory dysfunction.

Young adult binge drinkers have immunophenotypical disarrangements in peripheral natural killer cells.

Alcohol consumption is an issue of worldwide relevance and a problem of national scale in Mexico. The consumption pattern of large amounts of alcohol on the weekends is rapidly increasing in young adults between 18 and 29 years. Despite various studies that have focused on the noxious effect of alcohol in immunity, the changes in the immunoprofiles of peripheral blood cells have not been completely described. Natural killer cells (NKCs) are lymphoid-origin cells of the immune system that are responsible for defense against tumors, among other functions. In homeostatic conditions, they are found to be in a state of "dynamic balance" between activation and inhibition stimuli, which, if broken, may lead to immunosuppression or activation of cytotoxic mechanisms. In this study, we evaluated the immunoprofile of peripheral NKCs of 54 young adults, 29 of whom were binge drinkers and 25 of whom were low risk (LR), as classified by validated tools. Drinking habits were assessed. Blood samples were collected to perform hematic biometry and liver enzyme tests. Peripheral NKCs were identified by FACS, and stained for CCR2, CCR4, CCR5, CXCR4, CD69, CD127, CD137, TLR4, and Granzyme B. The data were analyzed using the t test and Mann-Whitney's U test for contrasts, and the effect size was obtained in order to evaluate the impact of each immunoprofile. The binge group showed increased expression of CCR5 and PD-1 in NKCs, respective to the LR group, and decreased expression of TLR4, along with fewer CCR4+ cells. Moreover, the increase found in CCR5 and PD-1 expression was correlated with the number of drinks in the last drinking session. Our findings show that young binge drinkers have different immunoprofiles that could suggest an early status of immunosuppression and trafficking of NKCs to the liver, which could be related to the onset of early liver damage, early in a subject's lifespan.

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.

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.

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.

Nasal IgA secretion in a murine model of acute stress. The possible role of catecholamines.

Stress stimuli affect the immune system of the mucosa, and in particular IgA secretion. It is well documented that intense psychological and physical stress can increase susceptibility to infection by diverse pathogens in the upper respiratory tract. Our workgroup reported that chronic stress caused by immobilization elicits a decrease in nasal IgA levels in mice. Here, we explore how acute stress (caused by 4h of immobilization) affects IgA secretion in the nasal mucosa, and the possible role of the sympathetic nervous system in this effect. Nine-week-old male CD1 mice were divided into four groups: control, chemical sympathectomy (with 6-OHDA) and treatment with nadolol (5mg/kg) or phentolamine (15mg/kg). All these groups were subdivided into stressed and unstressed animals. The parameters evaluated included plasma corticosterone and epinephrine (only in control groups), SIgA levels (by ELISA) and SIgA expression (by Western Blot) in nasal fluid, percentage of IgA+ plasma cells, and mRNA expression of heavy alpha chain, pIgR, TNFα and TGFß in nasal mucosa. Acute stress reduced the percentage of IgA+ cells while increasing the levels of IgA, the two hormones, and the mRNA expression of heavy alpha chain, pIgR, TNFα and TGFß, which resulted in greater synthesis and transport of IgA. The treatments with 6-OHDA and α- and ß-adrenergic receptor blockers suggest that sympathetic innervation by both types of adrenergic receptors is important for the control of SIgA secretion in nasal mucosa during acute stress. The increase in this parameter depended on the cytokines involved in IgA synthesis and transport.

Oxidant/antioxidant state in tissue of prymary and recurrent pterygium.

BACKGROUND: Pterygium is a disorder of the ocular surface induced by chronic exposure to UV-light. Abundant data is available from patients with primary pterygium, but scarce from those with recurrent pterygium. The present study aimed to explore the oxidant/antioxidant status in tissue of primary and recurrent pterigium in men and women. METHODS: Pathological tissue samples were taken during surgery on patients with primary and recurrent pterygium. Healthy conjunctive tissue samples were taken during cataract surgery. After homogenization of 77 tissue samples, evaluation was made of thiobarbituric reactive substances (TBARS), nitric oxide (NO), total antioxidant status (TAS) and the activity of the three main antioxidant enzymes: glutathione peroxidase, superoxide dismutase and catalase. Gender differences were evaluated. RESULTS: Compared to the control group, in the primary pterygium group there was an increase in NO and TAS, and a tendency to a decrease of all antioxidant enzymes, indicating an increase in non-enzymatic antioxidant activity. Compared to the control group, in the recurrent pterygium group there was a significant decrease in the level of TAS and antioxidant enzymes. A high positive correlation was found between most of measured parameters within the control group and the recurrent pterygium group, but not within the primary pterygium group. Compared to men, a significant difference was observed in the elevated NO level and low TAS level of women in the prymary pterygium group. CONCLUSIONS: The diminished antioxidant defense in the recurrent pterygium group, possibly determined mainly by decreased non-enzymatic activity, supports the idea that oxidative stress plays an important role in the recurrence of this disorder.