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.

Cholecystokinin Outcome on Markers of Intestinal IgA Antibody Response.

Cholecystokinin 8 (CCK8) is an entero-octapeptide that participates in crosstalk with components of intestinal immunity via the CCK receptor (CCKR), but its role in modulation of the IgA response is not fully known under physiological conditions. Male eight-week-old BALB/c mice each were intraperitoneally injected once during 7 days with CCK8, devazapide (CCKR1 antagonist), L365,260 (CCKR2 antagonist) or vehicle (sham group). In intestinal lavages, total and secretory IgA (SIgA) were determined by ELISA; in lamina propria, IgA+ B lymphocytes and IgA+ plasma cells were analyzed by flow cytometry; mRNA levels of polymeric immunoglobulin receptor (pIgR) in epithelial cells and α chain, interleukins (ILs) in lamina propria cells were assessed by qRTPCR. Regarding the sham conditions, IgA+ plasma-cell percentage and IL-2, IL-5, IL-10 and transforming growth factor-ß (TGF-ß) mRNA levels were either increased by CCK8 or decreased by both CCKR antagonists. For IgA/SIgA responses, IL-4/IL-6 mRNA levels were decreased by all drugs and pIgR mRNA was increased by CCK8 and reduced by L365,260. IgA+ B cell percentage and α chain mRNA levels were elicited by CCK8 and L365,260. Data suggested a presumable differential role of CCK/CCKR on the IgA-response; outcome of L365,260 on the elicitation of IgA+ B cells and α chain mRNA needs further examination.

(-)-Epicatechin protects from amebic liver abscess development in hamster.

In this work the effect of (-)-epicatechin on the development of amebic liver abscess in hamsters was evaluated. (-)-epicatechin is a flavonoid present in plants that possesses various biological properties, including its activity against some protozoal parasites; however its antiamebic activity in a living model had not been evaluated. Syrian golden hamsters were intrahepatically inoculated with 1x106E. histolytica trophozoites, three days after inoculation they received nine intraperitoneal doses of (-)-epicatechin (10 mg/100 g) every 48 h. Animals without treatments and treated with metronidazole were included as controls. Macroscopic characteristics of the hepatic abscess, histopathological analysis of the tissue and the levels of inflammatory cytokines were determined. (-)-epicatechin produced a decrease in liver abscess progression being observed only 9.49% of damage compared to 84% shown by untreated animals. During treatment with (-)-epicatechin hepatic tissue showed signs of liver repair and absence of amoebae. Additionally, (-)-epicatechin produced a modulating effect on inflammatory cytokines TNF-α, IL-1ß and IL-10. All these events observed in animals treated with (-)-epicatechin could contribute to the elimination of trophozoites and liver healing.

Intestinal Homeostasis under Stress Siege.

Intestinal homeostasis encompasses a complex and balanced interplay among a wide array of components that collaborate to maintain gut barrier integrity. The appropriate function of the gut barrier requires the mucus layer, a sticky cushion of mucopolysaccharides that overlays the epithelial cell surface. Mucus plays a critical anti-inflammatory role by preventing direct contact between luminal microbiota and the surface of the epithelial cell monolayer. Moreover, mucus is enriched with pivotal effectors of intestinal immunity, such as immunoglobulin A (IgA). A fragile and delicate equilibrium that supports proper barrier function can be disturbed by stress. The impact of stress upon intestinal homeostasis results from neuroendocrine mediators of the brain-gut axis (BGA), which comprises a nervous branch that includes the enteric nervous system (ENS) and the sympathetic and parasympathetic nervous systems, as well as an endocrine branch of the hypothalamic-pituitary-adrenal axis. This review is the first to discuss the experimental animal models that address the impact of stress on components of intestinal homeostasis, with special emphasis on intestinal mucus and IgA. Basic knowledge from animal models provides the foundations of pharmacologic and immunological interventions to control disturbances associated with conditions that are exacerbated by emotional stress, such as irritable bowel syndrome.

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.

Mouse neutrophils release extracellular traps in response to Naegleria fowleri.

Naegleria fowleri is a free-living amoeba, which is able to infect humans through the nasal mucosa causing a disease in the central nervous system known as primary amoebic meningoencephalitis (PAM). Polymorphonuclear cells (PMNs) play a critical role in the early phase of N fowleri infection. Recently, a new biological defence mechanism called neutrophil extracellular traps (NETs) has been attracting attention. These structures represent an important strategy to immobilize and kill invading microorganisms. In this work, we evaluate the capacity of N fowleri to induce the NETs release by PMNs cells in mice in vitro and in vivo. In vitro: Neutrophils from bone marrow were cocultured with N fowleri trophozoites. In vivo: we employed a mouse model of PAM. We evaluated DNA, histone and myeloperoxidase (MPO) and the formation of NETs by confocal microscopy. Our results showed N fowleri induce both NETs and MPO release by PMNs cells in mice after trophozoite exposure, which increased through time, in vitro and in vivo. These results demonstrate that NETs are somehow associated with the amoebas. We suggest PMNs release their traps trying to avoid N fowleri attachment at the apical side of the nasal epithelium.

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.

Indices of anti-dengue immunoglobulin G subclasses in adult Mexican patients with febrile and hemorrhagic dengue in the acute phase.

Heterologous secondary infections are at increased risk of developing dengue hemorrhagic fever (DHF) because of antibody-dependent enhancement (ADE). IgG subclasses can fix and activate complement and bind to Fcɣ receptors. These factors may also play an important role in the development of ADE and thus in the pathogenesis of DHF. The aim of this study was to analyze the indices of anti-dengue IgG subclasses in adult patients with febrile and hemorrhagic dengue in the acute phase. In 2013, 129 patients with dengue fever (DF) and 57 with DHF in Veracruz, Mexico were recruited for this study and anti-dengue IgM and IgG determined by capture ELISA. Anti-dengue IgG subclasses were detected by indirect ELISA. Anti-dengue IgG2 and IgG3 subclasses were detected in patients with dengue. IgG1 increased significantly in the sera of patients with both primary and secondary infections and DHF, but was higher in patients with secondary infections. The IgG4 subclass index was significantly higher in the sera of patients with DHF than in that of those with DF, who were in the early and late acute phase of both primary and secondary infection. In conclusion, indices of subclasses IgG1 and IgG4 were higher in patients with DHF.

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.

Determining Structural Changes for Ligand Recognition between Human and Rat Phosphorylated BACE1 in Silico and Its Phosphorylation by GSK3ß at Thr252 by in Vitro Studies.

Alzheimer's disease (AD) is a neurodegenerative disease affecting older adults. AD pathogenesis involves the production of the highly neurotoxic amyloid-ß peptide 1-42 (Aß1-42) from ß-site amyloid precursor protein cleaving enzyme 1 (BACE1). The phosphorylation of BACE1 at Thr252 increases its enzymatic activity. This study examined the phosphorylation of BACE1 from human and rat BACE1 in silico through phosphorylation predictors. Besides, we explored how phosphorylation at various sites affected the BACE1 structure and its affinity with amyloid precursor protein (APP) and six BACE1 inhibitors. Additionally, we evaluated the phosphorylation of Thr252-BACE1 by glycogen synthase kinase 3 ß (GSK3ß) in vitro. The phosphorylation predictors showed that Thr252, Ser59, Tyr76, Ser71, and Ser83 could be phosphorylated. Also, Ser127 in rat BACE1 can be phosphorylated, but human BACE1 has a Gly at this position. Molecular dynamics simulations showed that Ser127 plays an important role in the open and closed BACE1 conformational structures. Docking studies and the molecular mechanics generalized Born surface area (MMGBSA) approach showed that human BACE1 phosphorylated at Thr252 and rat BACE1 phosphorylated at Ser71 have the best binding and free energy with APP, forming hydrogen bonds with Asp672. Importantly, inhibitors have a higher affinity for the phosphorylated rat BACE1 than for its human counterpart, which could explain their failure during clinical trials. Finally, in vitro experiments showed that GSK3ß could phosphorylate BACE1. In conclusion, BACE1 phosphorylation influences the BACE1 conformation and its recognition of ligands and substrates. Thus, these features should be carefully considered in the design of BACE1 inhibitors.

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.

Outcomes of nicotinic modulation on markers of intestinal IgA antibody response.

Acetylcholine (ACh), as a ligand of nicotinic acetylcholine receptors (nAChRs), plays a key role in the cholinergic anti-inflammatory pathway; however, its role in the immunoglobulin A (IgA) response remains unknown. Therefore, the present study aimed to investigate the role of ACh in the intestinal biomarkers involved in IgA synthesis and the polymeric immunoglobulin receptor (pIgR) involved in IgA transcytosis. Groups of mice were administered GTS-21 (an α7nAChR agonist) or mecamylamine (a non-selective nAChR antagonist) intraperitoneally for 7 days. Intestinal fluids were used for antibody concentration assessment by ELISA, cell suspensions from Peyer's patches and the lamina propria were obtained for flow cytometric analysis of plasma cells, and CD4+ T-cells expressing intracellular transforming growth factor (TGF)-ß and IgA-producing interleukin (IL)-4, -5, -6 and -10, and isolated epithelial cells to determine the levels of pIgR mRNA using reverse transcription-quantitative PCR. Regarding to the untreated control group, the concentration of IgA was reduced in the mecamylamine group and unaltered in the GTS-21 group while IgM levels exhibited no differences; the percentage of IgA+ plasma cells from Peyer's patches and the lamina propria, and the percentage of TGF-ß+/CD4+ T-cells from Peyer's patches were greater in the GTS-21-group. In both treatment groups, the percentages of IgM+ plasma cells and IL-6+/IL-10+ CD4+ T cells were greater in both compartments; pIgR mRNA expression levels decreased in epithelial cells. The percentage of IL-4 CD4+ T-cells were greater in Peyer's patches and lower in the lamina propria in the mecamylamine group, and the percentage of IL-5 CD4+ T-cells in the lamina propria were decreased in both treatment groups. These findings require further examination to address the impact of cholinergic modulation on IgA-transcytosis via pIgR. The present study may be an experimental reference for clinical trials that address the role of nicotinic system in intestinal dysfunctions as postoperative ileus.

Riluzole, a Derivative of Benzothiazole as a Potential Anti-Amoebic Agent against Entamoeba histolytica.

Amoebiasis is produced by the parasite Entamoeba histolytica; this disease affects millions of people throughout the world who may suffer from amoebic colitis or amoebic liver abscess. Metronidazole is used to treat this protozoan, but it causes important adverse effects that limit its use. Studies have shown that riluzole has demonstrated activity against some parasites. Thus, the present study aimed, for the first time, to demonstrate the in vitro and in silico anti-amoebic activity of riluzole. In vitro, the results of Entamoeba histolytica trophozoites treated with IC50 (319.5 µM) of riluzole for 5 h showed (i) a decrease of 48.1% in amoeba viability, (ii) ultrastructural changes such as a loss of plasma membrane continuity and alterations in the nuclei followed by lysis, (iii) apoptosis-like cell death, (iv) the triggering of the production of reactive oxygen species and nitric oxide, and (v) the downregulation of amoebic antioxidant enzyme gene expression. Interestingly, docking studies have indicated that riluzole presented a higher affinity than metronidazole for the antioxidant enzymes thioredoxin, thioredoxin reductase, rubrerythrin, and peroxiredoxin of Entamoeba histolytica, which are considered as possible candidates of molecular targets. Our results suggest that riluzole could be an alternative treatment against Entamoeba histolytica. Future studies should be conducted to analyze the in vivo riluzole anti-amoebic effect on the resolution of amebic liver abscess in a susceptible model, as this will contribute to developing new therapeutic agents with anti-amoebic activity.

Exercise improves intestinal IgA production by T-dependent cell pathway in adults but not in aged mice.

Introduction: Hypermutated high-affinity immunoglobulin A (IgA), neutralizes toxins and drives the diversification of bacteria communities to maintain intestinal homeostasis although the mechanism underlies the impact of moderate aerobic exercise (MAE) on the IgA-generation via T-dependent (TD) is not fully know. Therefore, the aim of this study was to determine the effect of long-time MAE on the production of IgA through the TD pathway in Peyer´s patches of the small intestine from aged mice. Methods: MAE protocol consisted of twenty 3-month-old (young) BALB/c mice running in an endless band at 0° inclination and a speed of 10 m/h for 5 days a week and resting 2 days on the weekend until reaching 6-month-old (adulthood, n=10) or 24-month-old (aging, n=10). Groups of young, adult, or elderly mice were included as sedentary controls (n=10/per group). At 6 or 24 months old, all were sacrificed, and small intestine samples were dissected to prepare intestinal lavages for IgA quantitation by ELISA and to obtain suspensions from Peyer´s patches (PP) and lamina propria (LP) cells for analysis of T, B, and plasma cell subpopulations by flow cytometry and mRNA analysis expression by RT-qPCR of molecular factors related to differentiation of B cells to IgA+ plasma cells, class switch recombination, and IgA-synthesis. Statistical analysis was computed with two-way ANOVA (factor A=age, factor B=group) and p<0.05 was considered for statistically significant differences. Results: Compared to age-matched sedentary control, in exercised elderly mice, parameters were either increased (IgA concentration, IL-21, IL-10 and RDH mRNA expression), decreased (α-chain mRNA, B cells, mIgA+ B cells, mIgM+ B cells and IL-4 mRNA) or unchanged (PP mIgA+ plasmablasts and LP cyt-IgA+ plasma cells). Regarding the exercised adult mice, they showed an up-modulation of IgA-concentration, mRNA expression IL-21, IL-10, and RDH and cells (PP B and T cells, mIgM+ plasmablasts and LP cyt-IgA+plasma cells). Conclusion: Our findings suggest that MAE restored the IgA production in adult mice via the TD cell pathway but does not in aged mice. Other studies are necessary to know in more detail the impact of long-time MAE on the TD pathway to produce IgA in aging.

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.

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.

Repeated restraint stress increases IgA concentration in rat small intestine.

The most abundant intestinal immunoglobulin and first line of specific immunological defense against environmental antigens is secretory immunoglobulin A. To better understand the effect of repeated stress on the secretion of intestinal IgA, the effects of restraint stress on IgA concentration and mRNA expression of the gene for the alpha-chain of IgA was assessed in both the duodenum and ileum of the rats. Restraint stress induced an increase in intestinal IgA, which was blocked by an adrenalectomy, suggesting a role of catecholamines and glucocorticoids. Whereas the blocking of glucocorticoid receptors by RU-486 did not affect the increased IgA concentration, it did reduce IgA alpha-chain mRNA expression in both segments, indicating a possible mediation on the part of glucocorticoids in IgA secretion by individual cells. Treatment with corticosterone significantly increased both the IgA concentration and IgA alpha-chain mRNA expression in ileum but not in duodenum, suggesting that glucocorticoids may act directly on IgA-antibody forming cells to increase IgA secretion in the former segment. A probable role by catecholamines was evidenced by the reduction in IgA concentration and IgA alpha-chain mRNA expression in both segments after a chemical sympathectomy with 6-hydroxydopamine (6-OHDA). Additionally, norepinephrine significantly reduced IgA alpha-chain mRNA levels but increased pIgR mRNA expression and IgA concentration in both intestinal segments. We propose that the increased intestinal IgA levels caused by repeated restraint stress is likely due to the effects of catecholamines on the transport of plgA across the epithelium.