Apocynin, an NADPH Oxidase Enzyme Inhibitor, Prevents Amebic Liver Abscess in Hamster.

Amebiasis is an intestinal infection caused by Entamoeba histolytica. Amebic liver abscess (ALA) is the most common extraintestinal complication of amebiasis. In animal models of ALA, neutrophils have been shown to be the first cells to come into contact with Entamoeba histolytica during the initial phase of ALA. One of the multiple mechanisms by which neutrophils exhibit amebicidal activity is through reactive oxygen species (ROS) and the enzyme NADPH oxidase (NOX2), which generates and transports electrons to subsequently reduce molecular oxygen into superoxide anion. Previous reports have shown that ROS release in the susceptible animal species (hamster) is mainly stimulated by the pathogen, in turn provoking such an exacerbated inflammatory reaction that it is unable to be controlled and results in the death of the animal model. Apocynin is a natural inhibitor of NADPH oxidase. No information is available on the role of NOX in the evolution of ALA in the hamster, a susceptible model. Our study showed that administration of a selective NADPH oxidase 2 (NOX2) enzyme inhibitor significantly decreases the percentage of ALA, the size of inflammatory foci, the number of neutrophils, and NOX activity indicated by the reduction in superoxide anion (O2-) production. Moreover, in vitro, the apocynin damages amoebae. Our results showed that apocynin administration induces a decrease in the activity of NOX that could favor a decrease in ALA progression.

LDH-A Promotes Metabolic Rewiring in Leucocytes from the Intestine of Rats Treated with TNBS.

Although the aetiology of inflammatory bowel diseases (IBDs) is still unknown, one of their main characteristics is that the immune system chronically affects the permeability of the intestinal lamina propria, in turn altering the composition of the microbiota. In this study, the TNBS rat model of colitis was used because it contains a complex inflammatory milieu of polymorphonuclear cells (PMN) and lymphocytes infiltrating the lamina propria. The aim of the present study was to investigate six dehydrogenases and their respective adaptations in the tissue microenvironment by quantifying enzymatic activities measured under substrate saturation conditions in epithelial cells and leukocytes from the lamina propria of rats exposed to TNBS. Our results show that in the TNBS group, an increased DAI score was observed due to the presence of haemorrhagic and necrotic areas in the colon. In addition, the activities of G6PDH and GADH enzymes were significantly decreased in the epithelium in contrast to the increased activity of these enzymes and increased lactate mediated by the LDH-A enzyme in leukocytes in the lamina propria of the colon. Over the past years, evidence has emerged illustrating how metabolism supports aspect of cellular function and how a metabolic reprogramming can drive cell differentiation and fate. Our findings show a metabolic reprogramming in colonic lamina propria leukocytes that could be supported by increased superoxide anion.

Ascorbic Acid Ameriolates Liver Damage by Myeloperoxidase Oxidative Products in a Hamster Model of Amoebic Liver Abscess.

Entamoeba histolytica is a protozoan-pathogen-causing amoebic liver abscess (ALA). After amoeba establishment in the liver, it causes abundant infiltrate of neutrophils. Liver tissue damage by neutrophils results in part from anti-amoebic oxidative intermediates, including reactive oxygen species (ROS), reactive nitrogen species (RNS), and hypochlorous acid (HOCl), derived from the myeloperoxidase (MPO) enzyme. Ascorbic acid (ASC) is an antioxidant that acts as a scavenger for ROS and NOS-derived free radicals. No previous information regarding the effect of ASC concerning the participation of MPO in an experimental model of ALA in hamsters has been reported. Thus, the aim of the present work was to analyze the effect of ASC on acute ALA development and to measure the activity and gene expression of the MPO enzyme. Hamsters were treated with ASC (800 mg/kg) and then intrahepatically inoculated with E. histolytica trophozoites. Animals were sacrificed at 3, 6, and 12 h post-inoculation (p.i.), and liver samples were collected. The percentage of lesions, amoeba in situ count, MPO activity, and mpo gene expression were ascertained. Compared to ALA hamsters without ASC treatment as the control group (CT), the ALA group treated with ASC had a significant decrease in liver lesions (all p.i. hours) and viable amoeba count (12 h p.i.) and an increase in MPO activity (12 h p.i.) and mpo gene expression (6 h/12 h p.i.). These data suggest that ASC ameliorated liver damage caused by oxidizing products via modulation of mpo expression and activity.

CD4+/IL­4+ lymphocytes of the lamina propria and substance P promote colonic protection during acute stress.

Life stress may influence symptom onset and severity in certain gastrointestinal disorders in association with a dysregulated intestinal barrier. It has been widely accepted that stress triggers the hypothalamus­pituitary­adrenal (HPA) axis, releasing corticosterone, which promotes intestinal permeability. In response, colonic inflammation alters mucosal immune homeostasis and destroys the colonic architecture, leading to severe intestinal diseases. Endogenous substance P (SP) does not inhibit the initial extent of the HPA axis response to restraint stress, but it reduces the duration of the stress, suggesting that SP plays an important role in the transition between acute and chronic stress. The present study aimed to investigate the effect of two groups of mice exposed to stress, including acute and chronic stress. The corticosterone was evaluated by ELISA, colon samples were obtained to detected polymorphonuclear cells by hematoxylin and eosin staining, goblet and mast cells were identified by immunocytochemistry and cytokine­producing CD4+ T cells were analyzed by flow cytometry assays, adhesion proteins in the colon epithelium by western blotting and serum SP levels by ELISA. The results demonstrated an increase in the number of polymorphonuclear, goblet and mast cells, a decrease in claudin­1 expression and an elevation in E­cadherin expression during acute stress. Increased E­cadherin expression was also detected during chronic stress. Moreover, it was found that acute stress caused a shift towards a predominantly anti­inflammatory immune response (T helper 2 cells), as shown by the increase in the percentage of CD4+/IL­6+ and CD4+/IL4+ lymphocytes in the lamina propria and the increase in serum SP. In conclusion, this response promoted colonic protection during acute stress.

Muscarinic receptors control markers of inflammation in the small intestine of BALB/c mice.

Muscarinic-acetylcholine-receptors (mAChRs) modulate intestinal homeostasis, but their role in inflammation is unclear; thus, this issue was the focus of this study. BALB/c mice were treated for 7 days with muscarine (mAChR/agonist), atropine (mAChR/antagonist) or saline. Small-intestine samples were collected for histology and cytofluorometric assays in Peyer's patches (PP) and lamina propria (LP) cell-suspensions. In LP, goblet-cells/leukocytes/neutrophils/MPO+ cells and MPO/activity were increased in the muscarine group. In PP, IFN-γ+/CD4+ T or IL-6+/CD4+ T cell numbers were higher in the muscarine or atropine groups, respectively. In LP, TNF-α+/CD4+ T cell number was higher in the muscarine group and lower in the atropine.

Restraint stress increased the permeability of the nasal epithelium in BALB/c mice.

Stress seems to affect the onset and evolution of diverse illnesses with an inflammatory substrate. Whether physiological or psychological, stress increases epithelial permeability. In the mucosa of the nasal cavity and upper respiratory tract, the epithelial barrier is regulated in large part by bicellular and tricellular tight junctions (bTJs and tTJs, respectively). The junctional complexes are composed of multiple membrane proteins: claudins, tight-junction-associated MARVEL proteins (TAMs: occludin, tricellulin and marvelD3), and scaffolding proteins such as ZO-1, -2 and -3. The aim of the present study was to examine the possible modification of nasal permeability and TJ protein expression in a mouse model of acute psychological stress (a 4-h immobility session). Serum corticosterone was quantified from plasma samples to verify the onset of stress. Evaluation was made of the relative concentration of key proteins in nasal mucosa by using Western blot, and of changes in permeability by analyzing FITC-Dextran leakage from the nose to the blood. Compared to the control, the stressed group showed a greater epithelial permeability to FITC-Dextran, a reduced expression of occludin and tricellulin, and an elevated expression of ZO-2 and claudin-4. This evidence points to increased paracellular flow of large molecules through an altered structure of tTJs. Apparently, the structure of bTJs remained unchanged. The current findings could provide insights into the relation of stress to the onset/exacerbation of respiratory infections and/or allergies.

Neutrophil extracellular traps and MPO in models of susceptibility and resistance against Entamoeba histolytica.

The main effector mechanisms of neutrophils are the release of neutrophil extracellular traps (NETs) and myeloperoxidase (MPO). In this work, we evaluated the role of NETs and the activity of MPO in the interactions of rodent neutrophils with amoebae and in amoebic liver abscess (ALA)-resistant and ALA-susceptible models. We showed with in vitro assays that mice produced greater amounts of NETs and MPO than did hamsters, and the elastase activity was high in both models. However, the inhibition of NETs and MPO promoted an increase in amoeba viability in the mice. The mouse ALAs showed a more profound presence of NETs and MPO than did the hamster ALAs. We concluded that both effector mechanisms were essential for the amoebic damage and could prevent the formation of ALAs in the resistant model.

Different behavior of myeloperoxidase in two rodent amoebic liver abscess models.

The protozoan Entamoeba histolytica 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 in vitro. The aim of the current contribution was to analyze in vivo 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 Entamoeba histolytica 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 in situ. 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.

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.

Regulation of intestinal morphology and GALT by pituitary hormones in the rat.

Here, the effects of neurointermediate (NIL), anterior (AL), and total hypophysectomy (HYPOX) on ileal mucosa cells and gut-associated lymphoid tissue (GALT) are reported. Compared with the sham-operated (SHAM) rats, the villi height and goblet cells numbers were significantly decreased in all groups. Lamina propria area decreased in AL and HYPOX, but not in NIL animals. CD8(+) but not CD4(+) lymphocytes decreased in the HYPOX and NIL groups. Paneth cells did not change, while IgA cells, IgM cells, and secretory IgA were significantly decreased in all groups. NIL but not AL animals lost significant numbers of IgA cells and secretory IgA. In summary, pituitary hormones exert lobe-specific regulatory effects on the gut and on GALT.