Identification of Novel Immunoregulatory Molecules in Human Thymic Regulatory CD4(+)CD25(+) T Cells by Phage Display

Thymic CD4(+)CD25(+) cells play an important role in immune regulation and are continuously developed in the thymus as an independent lineage. How these cells are generated, what are their multiple pathways of suppressive activity and which are their specific markers are questions that remain unanswered. To identify molecules involved in the function and development of human CD4(+)CD25(+) T regulatory cells we targeted thymic CD4(+)CD25(+) cells by peptide phage display. A phage library containing random peptides was screened ex vivo for binding to human thymic CD4(+)CD25(+) T cells. After four rounds of selection on CD4(+)CD25(+) enriched populations of thymocytes, we sequenced several phage displayed peptides and selected one with identity to the Vitamin D Receptor (VDR). We confirmed the binding of the VDR phage to active Vitamin D in vitro, as well as the higher expression of VDR in CD4(+)CD25(+) cells. We suggest that differential expression of VDR on natural Tregs may be related to the relevance of Vitamin D in function and ontogeny of these cells.

Inorganic elements in the fat bodies of Diatraea saccharalis (Lepidoptera: Crambidae) larvae parasitized by Cotesia flavipes (Hymenoptera: Braconidae)

Koinobiont parasitoids use several strategies to regulate the host`s physiological processes during parasitism. Although many aspects of host-parasitoid interactions have been explored, studies that attempted to assess the effects of parasitism on the availability of inorganic elements in the host are virtually nonexistent. Therefore, we aimed to evaluate the effects of parasitism on the concentrations of inorganic elements in the fat bodies of larvae of Diatraea saccharalis (Lepidoptera: Crambidae) during the development of the parasitoid Cotesia flavipes (Hymenoptera: Braconidae), by using total reflection X-ray fluorescence (TXRF). TXRF analysis allowed comparisons of the changes in the availability of the elements P. S. K, Ca, Cr, Fe, Ni, Cu, and Zn in the fat body tissues of D. saccharalis larvae parasitized by C. flavipes. Overall, the concentration of inorganic elements was higher early in parasitoid development (1 and 3 days after parasitism) compared to non-parasitized larvae, but much lower towards the end of parasitoid development (7 and 9 days after parasitism). Ca, K, and S were reduced after the fifth day of parasitism, which affected the total abundance of inorganic elements observed in the fat bodies of the parasitized hosts. The regulatory mechanisms or pathological effects related to the observed variation of the host inorganic elements induced by the parasitoid remain unknown, but there might be a strategy to make these elements available to the parasitoid larvae at the end of their development, when higher metabolic activity of the host fat body is required to sustain parasitoid growth. The observed variation of the host`s inorganic elements could also be related to the known effects of parasitism on the host`s immune response. (C) 2010 Elsevier Inc. All rights reserved.

Feeding Dietary Mannan Oligosaccharides to Juvenile Nile Tilapis, Oreochromis niloticus, Has No Effect on Hematological Parameters and Showed Decreased Feed Consumption

Impaired immune system by environmental stressors can lead fishes to be more susceptible to diseases that limit the economic development of aquaculture systems. This study was set out to determine the effect of six levels of mannan oligosaccharides (MOS; ActiveMOS((R)); Biorigin, Lencois Paulista, Sao Paulo, Brazil) on the performance index and hematology of Nile tilapia, Oreochromis niloticus juveniles. Fish (13.62 g) were randomly distributed into 18 plastic aquaria (300 L; 20 fishes per aquarium) and fed during 45 d with a commercial diet supplemented with 0, 0.2, 0.4, 0.6, 0.8, and 1% dietary MOS, in a totally randomized design trial (n = 3); biometrical and hematological data were collected and analyzed. There were no significant differences in hematological parameters between fish fed control and MOS supplementation diets, and daily feed consumption (FC) decreased (P < 0.05) with increasing levels of dietary MOS. Dietary MOS did not increase leukocyte count and presented negative effects on FC of Nile tilapia. At 0.4% MOS supplementation, the individual weight gain was higher in absolute values but not different (P > 0.05) compared to control diet.

mRNA profile of Nellore calves after primary infection with Haemonchus placei

Haemonchus parasites are responsible for many losses in animal production. However, few studies are available, especially of zebu cattle. In this study, we investigated mRNA differences of immune response genes in naive Nellore calves infected with Haemonchus placei, relating these differences to patterns of cellular infiltrate. Calves were infected with 15,000 H. placei 13 larvae and after 7 days lymph node and abomasum tissues were collected. IL-2, IL-4, IL-8, IL-12, IL-13, IFN-gamma, MCP-1, lysozyme, pepsinogen and TNF-alpha genes were evaluated by qPCR. Mast cells, eosinophils and globular leukocytes were counted by abomasum histology. In the infected group, IL-4, IL-13 and TNF-alpha were up-regulated in the abomasal lymph node. In the abomasum, IL-13 increased and TNF-alpha was down-regulated (p < 0.05). No differences were detected for mast cells and eosinophil counts in abomasal tissue (p > 0.05). We conclude that for this infection time, there was Th2 polarization but that cellular infiltrate in abomasal tissue takes longer to develop. (C) 2010 Elsevier B.V. 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.

Early Effects on T lymphocyte Response to Iron Deficiency in Mice. Short Communication

Iron deficiency is a common nutritional disorder, affecting about 30% of the world population. Deficits in iron functional compartments have suppressive effects on the immune system. Environmental problems, age, and other nutrient deficiencies are some of the situations which make human studies difficult and warrant the use of animal models. This study aimed to investigate alterations in the immune system by inducing iron deficiency and promoting recuperation in a mouse model. Hemoglobin concentration, hematocrit, liver iron store, and flow cytometry analyses of cell-surface transferrin receptor (CD71) on peripheral blood and spleen CD4+ and CD8+ T lymphocyte were performed in the control (C) and the iron-deficient (ID) groups of animals at the beginning and end of the experiment. Hematological indices of C and ID mice were not different but the iron stores of ID mice were significantly reduced. Although T cell subsets were not altered, the percentage of T cells expressing CD71 was significantly increased by ID. The results suggest that iron deficiency induced by our experimental model would mimic the early events in the onset of anemia, where thymus atrophy is not enough to influence subset composition of T cells, which can still respond to iron deficiency by upregulating the expression of transferrin receptor.

Effects of repetitive stress during the acute phase of Trypanosoma cruzi infection on chronic Chagas` disease in rats

The effect of repetitive stress during acute infection with Trypanosoma cruzi (T. cruzi) on the chronic phase of ensuing Chagas` disease was the focus of this investigation. The aim of this study was to evaluate in Wistar rats the influence of repetitive stress during the acute phase of infection (7 days) with the Y strain of T. cruzi on the chronic phase of the infection (at 180 days). Exposure to ether vapor for 1min twice a day was used as a stressor. Repetitive stress enhanced the number of circulating parasites and cardiac tissue disorganization, from a moderate to a severe diffuse mononuclear inflammatory process and the presence of amastigote burden in the cardiac fibers. Immunological parameters revealed that repetitive stress triggered a reduced concanavalin A induced splenocyte proliferation in vitro with major effects on the late chronic phase. Serum interleukin-12 concentration decreased in both stressed and infected rats in the early phase of infection although it was higher on 180 days post-infection. These results suggest that repetitive stress can markedly impair the host`s immune system and enhance the pathological process during the chronic phase of Chagas` disease.

Influence sexual dimorphism on the persistence of blood parasites in infected Calomys callosus

Gender has long been known to be a contributory factor in the incidence and progression of disorders associated with immune system disregulation. The aims of this experiment were to verify the influences of sexual dimorphism on the persistence of blood parasites out of the acute phase of infection. Male and female Calomys callosus were separated and infected with two strains of Trypanosoma cruzi, and let age until 120 days. Xenogiagnostic, culture of organs and blood, histopathology and lytic antibody percentages were evaluated on late chronic phase. Xenodiagnosis, hemoculture and lytic antibody percentages were positive front 45 until 120 days. For both strains in adrenal and heart, amastigote burdens were present until 45 days, scarcely found on 60 days and absent on 120 days. Steroid hormones, although having a protective role, does not enable animals to get completely rid of the infection. Even without showing apparent signs of pathological unbalance, parasite persists, hidden throughout the host`s body. (C) 2008 Elsevier Ltd. All rights reserved.

Prostaglandin E(2)-loaded microspheres as strategy to inhibit phagocytosis and modulate inflammatory mediators release

PGE(2), an arachidonic acid metabolite produced by various type of cells regulates a broad range of physiological activities in the endocrine, cardiovascular, gastrointestinal, and immune systems, and is involved in maintaining the local homeostasis. In the immune system, PGE(2) is mainly produced by APCs and it can suppress the Th1-mediated immune responses. The aim of this study was to develop PGE(2)-loaded biodegradable MS that prolong and sustain the in vivo release of this mediator. An o/w emulsion solvent extraction-evaporation method was chosen to prepare the MS. We determined their diameters, evaluated the in vitro release of PGE(2), using enzyme immunoassay and MS uptake by peritoneal macrophages. To assess the preservation of biological activities of this mediator, we determined the effect of PGE(2) released from MS on LPS-induced TNF-alpha release by murine peritoneal macrophages. We also analyzed the effect of encapsulated PGE(2) on inflammatory mediators release from HUVECs. Finally, we studied the effect of PGE(2) released from biodegradable MS in sepsis animal model. The use of this formulation can provide an alternative strategy for treating infections, by modulating or inhibiting inflammatory responses, especially when they constitute an exacerbated profile. (C) 2008 Elsevier B.V. All rights reserved.

Trypanosoma cruzi: Effects of adrenalectomy during the acute phase of experimental infection

Glucocorticoid hormones have been implicated as an important modulator of Trypanosoma cruzi pathogenesis. Since adrenal steroid hormones play a fundamental role in modulating the immune response, we hypothesized that adrenalectomy affect the course of the experimental T. cruzi infection. This study was undertaken to determine the effects of adrenalectomy during the acute phase of T cruzi infection. Blood and tissue parasitism, macrophages, nitric oxide (NO) production and IFN-gamma were evaluated in male Wistar rats infected with the Y strain of T. cruzi. Our results show that adrenalectomized rats displayed increased number of blood and heart parasites accompanied by decreases in the total number of peritoneal macrophages and IFN-gamma when compared to controls. Adrenalectomy also reduced the levels of NO released from peritoneal macrophages of infected animals. These results suggest that adrenal corticosteroid insufficiency due to adrenalectomy could be considered an important factor during development of acute phases of experimental Chagas` disease, enhancing pathogenesis through disturbance of the host`s immune system. (C) 2008 Published by Elsevier Inc.

Melatonin enhances pro-inflammatory cytokine levels and protects against Chagas disease

Pro-inflammatory and modulatory cytokines have an essential role in host defense against human and murine Trypanosoma cruzi infection. Control of T. cruzi parasitism during the acute phase of infection is considered to be critically dependent on direct macrophage activation by cytokines. Melatonin has been proposed to regulate the immune system by affecting cytokine production in immunocompetent cells, enhancing the production of several T helper (Th)1 cytokines. The aims of this work were to evaluate in rats, the influences of exogenous melatonin treatment on T. cruzi-infected host`s immune responses. With this in mind, several immunological parameters were analyzed, including tumor necrosis factor-alpha, gamma-interferon, interleukin-12, nitric oxide (NO) and macrophage count. The melatonin therapy was provided in one of two different treatment regimens, that is, either beginning 7 days prior to infection or concomitant with the infection. Both treatments triggered an up-regulation of the immune response, with the concomitant treatment being more effective; in this case all cytokines studied, with exception of NO, displayed enhanced concentrations and there was a higher number of peritoneal macrophages, which displayed reduced concentrations under melatonin therapy. We conclude that melatonin plays a pivotal role in up-regulating the Th1 immune response thus controlling parasite replication.

Trypanosoma cruzi: Effects of social stress in Calomys callosus a natural reservoir of infection

Social environment can represent a major source of stress affecting cortisol and/or corticosterone levels, thereby altering the immune response. We have investigated the effects of social isolation on the development of Trypanosoma cruzi infection in female Calomys callosus, a natural reservoir of this protozoan parasite. Animals were divided in groups of five animals each. The animals of one group were kept together in a single cage. In a second group, four females were kept together in a cage with one male. In the final group, five individuals were kept isolated in private cages. The isolated animals showed body weight reduction, decreased numbers of peritoneal macrophages, lower global leucocytes counts, smaller lytic antibody percentage and a significantly higher level of blood parasites compared to the other animals. Their behavior was also altered. They were more aggressive than grouped females, or females exposed to the presence of a male. These results suggest that isolation creates a distinct social behavior in which immunity is impaired and pathogenesis is enhanced. (C) 2008 Elsevier Inc. All rights reserved.

Trypanosoma cruzi: The effects of zinc supplementation during experimental infection

it is well recognized that zinc is an essential trace element, influencing growth and affecting the development and integrity of the immune system. The use of oligoelements as zinc can be considered a tool in modulating the effectiveness of the immune response. In this work zinc was daily and orally supplied in male Wistar rats infected with the Y strain of Trypanosoma cruzi. Parasiternia was evaluated and a significant reduction on blood parasites was observed. In order to check some immunological parameters peritoneal macrophages were counted revealing higher percentages for zinc supplied group. Consequently enhanced concentrations of IFN-gamma was found and for the first time NO was evaluated in T cruzi infected animals under the influence of zinc therapy, revealing enhanced concentrations when compared to. unsupplied counterparts. We conclude that zinc is able to up-regulate the host`s immune response against parasite replication. (c) 2007 Elsevier Inc. All rights reserved.

Prior and concomitant dehydroepiandrosterone treatment affects immunologic response of cultured macrophages infected with Trypanosoma cruzi in vitro?

DHEA, a steroid hormone synthesized from cholesterol by cells of the adrenal cortex, plays an essential role in enhancing the host`s resistance to different experimental infections. Receptors for this hormone can be found in distinct immune cells (especially macrophages) that are known to be the first line defense against Trypanosoma cruzi infection. These cells operate through an indirect pathway releasing nitric oxide (NO) and cytokines such TNF-alpha and IL-12 which in turn trigger an enhancement of natural killer cells and lymphocytes which finally secrete pro and anti-inflammatory cytokines. The effects of pre- and post-infection DHEA treatment on production of IL-12, TNF alpha and NO were evaluated. T. cruzi infected macrophages post treated with DHEA displayed enhanced concentrations of TNF-alpha, IL-12 and NO. Probably, the mechanisms that induced the production of cytokines by infected cells are more efficient when the immune system has been stimulated first by parasite invasion, suggesting that the protective role of DHEA is greater when administered post infection. (C) 2011 Elsevier B.V. All rights reserved.

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.