Mast cell repopulation of the peritoneal cavity: contribution of mast cell progenitors versus bone marrow derived committed mast cell precursors

<p>Abstract</p> <p>Background</p> <p>Mast cells have recently gained new importance as immunoregulatory cells that are involved in numerous pathological processes. One result of these processes is an increase in mast cell numbers at peripheral sites. This study was undertaken to determine the mast cell response in the peritoneal cavity and bone marrow during repopulation of the peritoneal cavity in rats.</p> <p>Results</p> <p>Two mast cell specific antibodies, mAb AA4 and mAb BGD6, were used to distinguish the committed mast cell precursor from more mature mast cells. The peritoneal cavity was depleted of mast cells using distilled water. Twelve hours after distilled water injection, very immature mast cells could be isolated from the blood and by 48 hours were present in the peritoneal cavity. At this same time the percentage of mast cells in mitosis increased fourfold. Mast cell depletion of the peritoneal cavity also reduced the total number of mast cells in the bone marrow, but increased the number of mast cell committed precursors.</p> <p>Conclusions</p> <p>In response to mast cell depletion of the peritoneal cavity, a mast cell progenitor is released into the circulation and participates in repopulation of the peritoneal cavity, while the committed mast cell precursor is retained in the bone marrow.</p

Tissue distribution of DNA-Hsp65/TDM-loaded PLGA microspheres and uptake by phagocytic cells

This study aimed to demonstrate that microspheres, used as delivery vehicle of DNA-Hsp65/TDM [plasmid DNA encoding heat shock protein 65 (Hsp65) coencapsulated with trehalose dimycolate (TDM) into PLGA microspheres], are widely spread among several organs after intramuscular administration in BALB/c mice. In general, we showed that these particles were phagocytosed by antigen presenting cells, such as macrophages and dendritic cells. Besides, it was demonstrated herein that draining lymph node cells presented a significant increase in the number of cells expressing costimulatory molecules (CD80 and CD86) and MHC class II, and also that the administration of the DNA-Hsp65/TDM and vector/TDM formulations resulted in the up-regulation of CD80, CD86 and MHC class II expression when compared to control formulations (vector/TDM and empty). Regarding the intracellular trafficking we observed that following phagocytosis, the microspheres were not found in the late endosomes and/or lysosomes, until 15 days after internalization, and we suggest that these constructions were hydrolysed in early compartments. Overall, these data expand our knowledge on PLGA [poly (lactic-co- glycolic acid)] microspheres as gene carriers in vaccination strategies, as well as open perspectives for their potential use in clinical practice

A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells

Abstract\ud \ud \ud \ud Background\ud \ud A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. These findings have attracted considerable interest in the field of DNA vaccination. We have previously described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis. As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses.\ud \ud \ud \ud Methods\ud \ud To address this question, we analysed the activation of both human macrophages and dendritic cells (DCs) cultured with DNA-HSP65. Then, these cells stimulated with the DNA vaccine were evaluated regarding the expression of surface markers, cytokine production and microbicidal activity.\ud \ud \ud \ud Results\ud \ud It was observed that DCs and macrophages presented different ability to uptake DNA vaccine. Under DNA stimulation, macrophages, characterized as CD11b+/CD86+/HLA-DR+, produced high levels of TNF-alpha, IL-6 (pro-inflammatory cytokines), and IL-10 (anti-inflammatory cytokine). Besides, they also presented a microbicidal activity higher than that observed in DCs after infection with M. tuberculosis. On the other hand, DCs, characterized as CD11c+/CD86+/CD123-/BDCA-4+/IFN-alpha-, produced high levels of IL-12 and low levels of TNF-alpha, IL-6 and IL-10. Finally, the DNA-HSP65 vaccine was able to induce proliferation of peripheral blood lymphocytes.\ud \ud \ud \ud Conclusion\ud \ud Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans. These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.We thank Dr. Carlos Rodrigo ZárateBladés for helpful suggestions during the course of the studies. We also thank Mrs. Izaíra T. Brandão and Mrs. Ana P. Masson for technical assistance. This study was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Programa Nacional de DST/AIDS do Ministério da Saúde and Conselho Nacional de Pesquisa (CNPq).We thank Dr. Carlos Rodrigo Zárate-Bladés for helpful suggestions during the course of the studies. We also thank Mrs. Izaíra T. Brandão and Mrs. Ana P. Masson for technical assistance. This study was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Programa Nacional de DST/AIDS do Ministério da Saúde and Conselho Nacional de Pesquisa (CNPq)

Otimização da análise da atividade da redutase do nitrato e sua caracterização em folhas de cana-de-açúcar

O objetivo deste trabalho foi padronizar e caracterizar as condições para determinação da atividade da redutase do nitrato em tecido foliar de cana-de-açúcar, com uso do método in vivo. Amostras foliares foram coletadas de uma lavoura de primeira soqueira da cultivar IACSP 933046, com idade de seis meses. Foram estudadas diferentes condições de preparo das amostras foliares e do meio de incubação. O material que possibilitou a maior atividade da redutase do nitrato foi obtido pela amostragem de 25 discos de 1 cm de diâmetro, coletados às 13h, do centro da folha do tipo +1 sem nervura. O meio de incubação otimizado para a determinação da atividade dessa enzima em folhas de cana-de-açúcar deve ser composto por: 2,5 mL de KNO3 300 mmol L-1; 2,5 mL de tampão fosfato 285 mmol L-1 pH 7,3; 1,0 mL de Tween 20 a 0,6% (v/v); e 4,0 mL de água deionizada. A maior atividade da redutase do nitrato é obtida pela incubação das amostras por 90 min, a 32ºC, no escuro; é observada em plantas jovens formadas pela brotação da soqueira; e alcança o valor mínimo na fase de maturação das plantas

The lectin ArtinM activates RBL-2H3 mast cells without inducing degranulation.

Mast cells are connective tissue resident cells with morphological and functional characteristics that contribute to their role in allergic and inflammatory processes, host defense and maintenance of tissue homeostasis. Mast cell activation results in the release of pro-inflammatory mediators which are largely responsible for the physiological functions of mast cells. The lectin ArtinM, extracted from Artocarpus heterophyllus (jackfruit), binds to D-manose, thus inducing degranulation of mast cells. ArtinM has several immunomodulatory properties including acceleration of wound healing, and induction of cytokine release. The aim of the present study was to investigate the role of ArtinM in the activation and proliferation of mast cells. The rat mast cell line RBL-2H3 was used throughout this study. At a low concentration (0.25μg/mL), ArtinM induced mast cell activation and the release of IL-6 without stimulating the release of pre-formed or newly formed mediators. Additionally, when the cells were activated by ArtinM protein tyrosine phosphorylation was stimulated. The low concentration of ArtinM also activated the transcription factor NFkB, but not NFAT. ArtinM also affected the cell cycle and stimulated cell proliferation. Therefore, ArtinM may have therapeutic applications by modulating immune responses due to its ability to activate mast cells and promote the release of newly synthesized mediators. Additionally, ArtinM could have beneficial effects at low concentrations without degranulating mast cells and inducing allergic reactions

Lack of galectin-3 alters the balance of innate immune cytokines and confers resistance to Rhodococcus equi infection

Galectin-3 is a p-galactoside-binding lectin implicated in the fine-tuning of innate immunity. Rhodococcus equi, a facultative intracellular bacterium of macrophages, causes severe granulomatous bronchopneumonia in young horses and immunocompromised humans. The aim of this study is to investigate the role of galectin-3 in the innate resistance mechanism against R. equi infection. The bacterial challenge of galectin-3-deficient mice (gal3(-/-)) and their wild-type counterpart (gal3(+/+)) revealed that the LD50 for the gal3(-/-) mice was about seven times higher than that for the gal3(+/+) mice. When challenged with a sublethal dose, gal3(-/-) mice showed lower bacteria counts and higher production of IL-12 and IFN-gamma production, besides exhibiting a delayed although increased inflammatory reaction. Gal3(-/-) macrophages exhibited a decreased frequency of bacterial replication and survival, and higher transcript levels of IL-1 beta, IL-6, IL-10, TLR2 and MyD88. R. equi-infected gal3(+/+) macrophages showed decreased expression of TLR2, whereas R. equi-infected gal3(-/-) macrophages showed enhanced expression of this receptor. Furthermore, galectin-3 deficiency in macrophages may be responsible for the higher IL-1 beta serum levels detected in infected gal3(-/-) mice. Therefore galectin-3 may exert a regulatory role in innate immunity by diminishing IL-1 beta production and thus affecting resistance to R. equi infection.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Pesquisa Cientifica e Tecnologica (CNPq