Dietary glutamine supplementation affects macrophage function, hematopoiesis and nutritional status in early weaned mice

Background Et aims: To investigate the effect that early weaning associated with the ingestion of either a glutamine-free or supplemented diet has on the functioning of peritoneal. macrophages, hematopoiesis and nutritional status of mice. Methods: Swiss Webster mice were early weaned on their 14th day of life and distributed to two groups, being fed either a glutamine-free diet (-GLN) or a glutamine-supplemented diet (+GLN). Animals belonging to a control group (CON) were weaned on their 21st day of life. Results: The -GLN and +GLN groups had a lower lean body mass, carcass protein and ash content, plasma glutamine concentration and lymphocyte counts both in the peripheral blood and bone marrow when compared to the CON group (P < 0.05). Dietary supplementation with glutamine reversed both the lower concentrations of protein and DNA in the muscle and liver, as well. as the reduced capacity of spreading and synthesizing nitric oxide, hydrogen peroxide, TNF-alpha, IL-1 beta and IL-6 in cultures of peritoneal. macrophages obtained from the -GLN group (P < 0.05). Conclusion: These data indicate that the ingestion of glutamine modulates the function of peritoneal macrophages in early weaned mice. However, a glutamine-supplemented diet cannot substitute maternal milk in respect to immunological and metabolic parameters. (C) 2008 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Glutamine in vitro supplementation partly reverses impaired macrophage function resulting from early weaning in mice

Objective: Glutamine is one of the most abundant amino acids found in maternal milk, and its concentration increases throughout lactation. Because glutamine is essential for macrophage functionality, it is hereby suggested that early weaning in conjunction with the absence of glutamine consumption impairs the functioning of macrophages, which could in turn be reversed with an in vitro supplementation with glutamine. Methods: Swiss Webster mice were early weaned at 14 d of age (EW group) or at 21 d of age (control group, n = 8 per group). The EW group was fed a glutamine-free diet from days 14 to 21 of life. Results: Mice in the EW group presented a significant decrease in plasma and muscle concentrations of glutamine and an increase in the activity of glutamine synthetase in the muscle. Peritoneal macrophages obtained from animals in the EW group presented a significant increase in the production of interleukin (IL)-10 and a significant decrease in the synthesis of IL-1 beta, IL-6, tumor necrosis factor-a, nitric oxide, and hydrogen peroxide and in their ability to adhere, spread, phagocytize, and kill fungi. Glutamine in vitro supplementation reversed the decrease in IL-6, nitric oxide, and hydrogen peroxide synthesis and the decrease in the capacity to adhere, spread, and phagocytize in animals of the EW group. Conclusion: These new. data may imply that a lack of glutamine intake in early weaned mice hampers the functioning of peritoneal macrophages. (C) 2008 Elsevier Inc. All rights reserved.

Dietary glutamine supplementation increases the activity of peritoneal macrophages and hemopoiesis in early-weaned mice inoculated with Mycobacterium bovis bacillus Calmette-Guerin

Infants who are breast-fed have been shown to have a lower incidence of certain infectious diseases compared with formula-fed infants. Glutamine is one of the most abundant amino acids found in maternal milk and it is essential for the function of immune system cells such as macrophages. The purpose of this study was to investigate the effect of glutamine supplementation on the function of peritoneal macrophages and on hemopoiesis in early-weaned mice inoculated with Mycobacterium bovis bacillus Calmette-Guerin (BCG). Mice were wearied at 14 d of age and distributed to 2 groups and fed either a glutamine-free diet (n = 16) or a glutamine-supplemented diet (+Gln (n = 16). Both diets were isonitrogenous (with addition of a mixture of nonessential amino acids) and isocaloric. At d 21, 2 subgroups of mice (n = 16) were intraperitoneally injected with BCG and all mice were killed at d 28. Plasma, muscle and liver glutamine concentrations and muscle glutamine synthetase activity were not affected by diet or inoculation with BCG. The +GIn diet led to increased leukocyte and lymphocyte counts in the peripheral blood (P < 0.05) and granulocyte and lymphocyte counts in the bone marrow and spleen (P < 0.05). The +GIn diet increased spreading and adhesion capacities, hydrogen peroxide, nitric oxide, and tumor necrosis factor-alpha (TNF alpha) syntheses and the phagocytic and fungicidal activity of peritoneal macrophages (P < 0.05). The interaction between the +GIn diet and BCG inoculation increased the area under the curve of interleukin (IL)-1 beta and TNF alpha syntheses (P < 0.05). In conclusion, the intake of glutamine increases the function of peritoneal macrophages and hemopoiesis in early-weaned and BCG-inoculated mice. These data have important implications for the design of breast milk substitutes for human infants.

Gastric anti-ulcer activity of leaf fractions obtained of polar extract from Wilbrandia ebracteata in mice

Leaf fractions of Wilbrandia ebracteata were investigated for anti-ulcerogenic effects in ethanol and indomethacin-induced gastric ulcer assays in mice. Protective anti-ulcer effects were detected only in the ethanol-induced ulcer assay effects after pre-treatment with MeOH extract, MeOH chlorophyll-free, chlorophyll residue, HEX, DCM, aqueous MeOH fraction, ethyl acetate (EtOAc) and aqueous fractions. A potent anti-ulcerogenic effect was determined after pre-treatment of animals with EtOAc fraction, which was fractionated for isolation of active constituents. Seven flavonoids, 3`,4`,5,6,7,8-hexahydroxyflavonol, orientin, isoorientin, vitexin, isovitexin, luteolin, 6-methoxi-luteolin were isolated from the leaves of W. ebracteata (Cucurbitaceae) by chromatographic methods and identified by their spectral data. The data suggest that flavonoids are active anti-ulcerogenic compounds from leaves of W. ebracteata. The ability of scavenging free radicals was evaluated by DPPH reduction assay by TLC of flavonoids isolated.

Evaluation of Environmental Mycobacteria Contamination in a Specific Pathogen Free Animal Facility from a Tropical Country

P>With the evidence showing the protection variability of bacille Calmette-Guerin, new potential vaccines for tuberculosis have been tested around the world. One of the general concerns in tuberculosis vaccine development is the possibility of priming the host immune system with prior exposure to environmental mycobacteria antigens, which can change the efficacy of subsequent vaccination. As there is a great homology between the species from Mycobacterium genera, the previous contact of experimental animals with environmental mycobacteria could sensitize the mice and, in this way, could influence subsequent vaccine research. The aim of our study was to investigate critical points in an animal facility to search for environmental mycobacteria that eventually could be in direct or indirect contact with the experimental animals. Samples were collected from surfaces of walls, floor, animal cages and shelves and analysed using the Ogawa-Kudoh decontamination method. Samples of drinking water, food and sawdust were collected for analysis by the NALC/NaOH decontamination method. Also, the samples were cultivated directly in broth medium, without any method for decontamination. After decontamination methods, we observed bacterial colony growth in 4.31% of the total of samples analysed. These samples were stained with Ziehl-Neelsen and we did not detect any acid-fast bacilli, suggesting that the animal facility analysed is free from contamination by environmental mycobacteria and is not a source of mycobacterial antigens. Furthermore, our study showed a new paradigm in tuberculosis vaccine development: concern about the animal facility environment in terms of immune system priming of experimental animals by nascent bacterial contaminants.

Evaluation of the Experimental Inoculation of Cryptococcus albidus and Cryptococcus laurentii in Normal Mice: Virulence Factors and Molecular Profile Before and After Animal Passage

The genus Cryptococcus includes free-developing species, a few of which are of medical importance. Some, such as C. neoformans and C. gattii, cause infections in man frequently and C. albidus and C. laurentii cause less so. The aims of this study were to evaluate organ colonization after inoculation of C. albidus and C. laurentii isolates in normal BALB/c mice, the virulence factors (growth at 37A degrees C, capsule, melanin, proteinase, and phospholipase production) and the molecular profile (PCR-fingerprinting) of the yeasts before and after infection. The importance of different profiles (virulence and molecular) was considered in relation to the distribution in different organs and to the time intervals of isolation from organs. C. albidus was isolated from animal organs 2 to 10 days after inoculation and C. laurentii from 2 to 120 days. Most isolates of the two species kept the virulence factors showed before inoculation. The high homogeneity of the molecular profile of C. albidus and the high heterogeneity of C. laurentii were kept through the passages in animals. It is concluded that most isolates of both species were recovered from the animal organs after 5 or more days, and phenotypes were not altered by inoculation. No molecular alteration was detected and the virulence factors were not related to the time intervals before isolation from organs.

Synthetic nanoemulsion resembling a protein-free model of 7-ketocholesterol containing low density lipoprotein: In vitro and in vivo studies

7-ketocholesterol (7-KC) differs from cholesterol by a functional ketone group at C7. It is an oxygenated cholesterol derivative (oxysterol), commonly present in oxidized low-density lipoprotein (LDL). Oxysterols are generated and participate in several physiologic and pathophysiologic processes. For instance, the cytotoxic effects of oxidized LDL have been widely attributed to bioactive compounds like oxysterols. The toxicity is in part due to 7-KC. Here we aimed to demonstrate the possibility of incorporating 7-KC into the synthetic nanoemulsion LDE, which resembles LDL in composition and behavior. This would provide a suitable artificial particle resembling LDL to study 7-KC metabolism. We were able to incorporate 7-KC in several amounts into LDE. The incorporation was evaluated and confirmed by several methods, including gel filtration chromatography, using radiolabeled lipids. The incorporation did not change the main lipid composition characteristics of the new nanoparticle. Particle sizes were also evaluated and did not differ from LDE. In vivo studies were performed by injecting the nanoemulsion into mice. The plasma kinetics and the targeted organs were the same as described for LDE. Therefore, 7-KC-LDE maintains composition, size and some functional characteristics of LDE and could be used in experiments dealing with 7-ketocholesterol metabolism in lipoproteins.

Characterization and in vitro activities of cell-free antigens from Histoplasma capsulatum-loaded biodegradable microspheres

In the last decades, the incidence of histoplasmosis, a pulmonary fungal disease caused by Histoplasma capsulatum, has increased worldwide. In this context, vaccines for the prevention of this infection or therapies are necessary. Cell-free antigens (CFAgs) from H. capsulatum when administered for murine immunization purposes are able to confer protection and control of the infection, since they activate cellular immunity. However the most of vaccination procedures need several anti, gens administrations and immunoadjuvants, which are not approved for use in humans. The aim of this study was to develop and characterize a vaccination approach using biodegradable PLGA microspheres (MS) that could allow the controlled and/or sustained release of the encapsulated antigens from H. capsulatum. CFAgs-loaded MS presented a size less than 10 mu m, were marked engulfed by bone marrow-derived macrophages (BMDM phi) and induced the nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by these cells. Our data show that CFAgs-loaded MS induce cell activation, suggesting an immunostimulant effect to be further investigated during immunization procedures. CFAgs-loaded MS present potential to be used as vaccine in order to confer protection against H. capsulatum infection. (C) 2009 Elsevier B.V. All rights reserved.

Pregnancy outcome in ethanol-treated mice with folic acid supplementation in saccharose

Introduction Maternal folic acid deficiency is the most important metabolic factor in the etiology of neural tube defects (NTD) and is reduced by ethanol, which is extensively consumed by young women. Objective The objective of the study was to determine whether folic acid supplementation in dietary saccharose is efficient in the prevention NTD induced by ethanol in fetuses of Swiss mice. Materials and methods Pregnant mice were divided into four groups of six animals each: control (C), ethanol (E), deficient-supplemented (DS), and deficient-supplemented+ethanol (DSE). Groups C and E received commercial mouse chow (containing 3 mg/kg folic acid) throughout the experiment, while groups DS and DSE received a folic acid-free diet with the addition of saccharose supplemented with folic acid (2 mg/kg folic acid) in water. Group E and DSE animals received ethanol (4 g/kg) administered intraperitoneally from the seventh to the ninth gestational day (gd) and were euthanized on the 18th gd, while groups C and DS received saline. Results Congenital anomalies were observed in groups E and DSE. The fetal weight and length of the animals in group E were lower than in groups C and DS and, in group DSE, were lower than in groups C and DS. The placental diameter of group E was smaller than that of group C, and the placental weight of group C animals was lower than that of groups E, DSE, and DS. Conclusion The study demonstrated that dietary supplementation with folate in saccharose is an accessible means of consumption that could be further diffused but in an increased dose than recommended to reduce the teratogenic effects of ethanol.

Atm knock-in mice harboring an in-frame deletion corresponding to the human ATM 7636del9 common mutation exhibit a variant phenotype

ATM, the gene mutated in the human immunodeficiency disorder ataxia-telangiectasia (A-T), plays a central role in recognizing ionizing radiation damage in DNA and in controlling several cell cycle checkpoints. We describe here a murine model in which a nine-nucleotide in-frame deletion has been introduced into the Atm gene by homologous recombination followed by removal of the selectable marker cassette by Cre-loxP site-specific, recombination-mediated excision. This mouse, Abm-Delta SRI, was designed as a model of one of the most common deletion mutations (7636de19) found in A-T patients. The murine Atm deletion results in the loss of three amino acid residues (SRI; 2556-2558) but produces near full-length detectable Atm protein that lacks protein kinase activity. Radiosensitivity was observed in Atm-Delta SRI mice, whereas the immunological profile of these mice showed greater heterogeneity of T-cell subsets than observed in Atm(-/-) mice. The life span of Atm-Delta SRI mice was significantly longer than that of Atm(-/-) mice when maintained under nonspecific pathogen-free conditions. This can be accounted for by a lower incidence of thymic lymphomas in Atm-Delta SRI mice up to 40 weeks, after which time the animals died of other causes. The thymic lymphomas in Atm-Delta SRI mice were characterized by extensive apoptosis, which appears to be attributable to an increased number of cells expressing Fas ligand. A variety of other tumors including B-cell lymphomas, sarcomas, and carcinomas not seen in Atm(-/-) mice were observed in older Atm-Delta SRI animals. Thus, expression of mutant protein in Atm-Delta SRI knock-in mice gives rise to a discernibly different phenotype to Atm(-/-) mice, which may account for the heterogeneity seen in A-T patients with different mutations.

Effects of vitamin C supplementation on acute phase Chagas disease in experimentally infected mice with Trypanosoma cruzi QM1 strain

The tissue changes that occur in Chagas disease are related to the degree of oxidative stress and antioxidant capacity of affected tissue. Studies with vitamin C supplementation did not develop oxidative damage caused by Chagas disease in the host, but other studies cite the use of peroxiredoxins ascorbate - dependent on T. cruzi to offer protection against immune reaction. Based on these propositions, thirty "Swiss" mice were infected with T. cruzi QM1 strain and treated with two different vitamin C doses in order to study the parasitemia evolution, histopathological changes and lipid peroxidation biomarkers during the acute phase of Chagas disease. The results showed that the parasite clearance was greater in animals fed with vitamin C overdose. There were no significant differences regarding the biomarkers of lipid peroxidation and inflammatory process or the increase of myocardium in animals treated with the recommended dosage. The largest amount of parasite growth towards the end of the acute phase suggests the benefit of high doses of vitamin C for trypomastigotes. The supplementation doesn't influence the production of free radicals or the number of amastigote nests in the acute phase of Chagas disease.

Tbx1 and Bmp2 in the development of the ear, neural crest and pharyngeal system in mice

Dissertation presented to obtain the Ph.D degree in Biology

Vitamin C effects in mice experimentally infected with Trypanosoma cruzi QM2 strain

INTRODUCTION: To evaluate the efficacy of vitamin C in reducing the consequences generated by the production of free radicals in the acute and chronic phases of Chagas disease, two different doses of ascorbic acid were administered orally to 60 mice infected by Trypanosoma cruzi QM2 strain. METHODS: The animals were divided into six groups: G1, G2, and G3 for the acute phase study, and G'1, G'2, and G'3 for the chronic stage. The groups G1 and G'1 received 8.6x10-4mg/g of vitamin C daily, whereas G2 and G'2 received 7.14x10-3mg/g daily. The other groups, G3 and G'3, were considered placebos and received 10µL of mineral water. RESULTS: The study of the acute phase showed statistically significant differences between G1 and the other groups at various count days of the parasitemia evolution. The multiplying parasite was slower in G1 until the 11th day, but on the 22nd day it had greater parasitemia than in G2 and G3, and from the 36th day on, parasitemia stabilized at higher levels. However, when the histopathology of acute and chronic phases is considered, one does not note significant differences. CONCLUSIONS: The administration of two different doses of vitamin C was not able to protect mice and to contain the oxidative stress caused by free radicals formed by the metabolism of oxygen (reactive oxygen species) and nitrogen (reactive nitrogen species).

Pre-clinical trials for Machado-Joseph Disease: Hypothesis-based and hypothesis-free therapeutic approaches

Tese de Doutoramento em Ciências da Saúde

Histopathological alterations induced by non-viable cells and biochemical fractions from Paracoccidioides brasiliensis in mice

Non-viable cells and biochemical fractions from Paracoccidioides brasiliens were obtained for experimental inoculation in mice and posterior histopatological analysis. Dead total fungus, total fungus disrupted by sonorous waves, lipids of the fungus, supernatant of the lipid purification, integral and disrupted fungus free of lipids were obtained. The six preparations arised from masses of lyophilized yeasts of a recent isolate of P. brasiliensis (strain JT-1) and from a "Pool" equitably constituted by four strains maintained in laboratory for a long time (SN, 2, 18 and 192). Different doses of the 12 preparations were intraperitonially inoculated and histopathological analysis were done 30 days later. This analysis showed that all the inoculated preparations gave origin to inflamatory foci, except the one designated "supernatant of lipid purification". The alterations were detected exclusively in the liver of the animals and occurred from the smallest dose tested (1 mg), with exception of the lipids of the fungus, where the foci appeared only from a 3 mg dose onwards. No difference in the capacity of inducing histopathological alterations was found between the preparations obtained from the recent isolate (JT-1) and from the older ones ("Pool"). On the other hand, an increase of the number of inflammatory foci in function of the inoculated dose was observed.