Maria Célia Jamur

Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146+ perivascular cells and fibroblasts.

OBJECTIVE The relationship of multipotent mesenchymal stromal cells (MSC) with pericytes and fibroblasts has not been established thus far, although they share many markers of primitive marrow stromal cells and the osteogenic, adipogenic, and chondrogenic differentiation potentials. MATERIALS AND METHODS We compared MSCs from adult or fetal tissues, MSC differentiated in vitro, fibroblasts and cultures of retinal pericytes obtained either by separation with anti-CD146 or adhesion. The characterizations included morphological, immunophenotypic, gene-expression profile, and differentiation potential. RESULTS Osteogenic, adipocytic, and chondrocytic differentiation was demonstrated for MSC, retinal perivascular cells, and fibroblasts. Cell morphology and the phenotypes defined by 22 markers were very similar. Analysis of the global gene expression obtained by serial analysis of gene expression for 17 libraries and by reverse transcription polymerase chain reaction of 39 selected genes from 31 different cell cultures, revealed similarities among MSC, retinal perivascular cells, and hepatic stellate cells. Despite this overall similarity, there was a heterogeneous expression of genes related to angiogenesis, in MSC derived from veins, artery, perivascular cells, and fibroblasts. Evaluation of typical pericyte and MSC transcripts, such as NG2, CD146, CD271, and CD140B on CD146 selected perivascular cells and MSC by real-time polymerase chain reaction confirm the relationship between these two cell types. Furthermore, the inverse correlation between fibroblast-specific protein-1 and CD146 transcripts observed on pericytes, MSC, and fibroblasts highlight their potential use as markers of this differentiation pathway. CONCLUSION Our results indicate that human MSC and pericytes are similar cells located in the wall of the vasculature, where they function as cell sources for repair and tissue maintenance, whereas fibroblasts are more differentiated cells with more restricted differentiation potential.

Mast Cell Function: A New Vision of an Old Cell

Since first described by Paul Ehrlich in 1878, mast cells have been mostly viewed as effectors of allergy. It has been only in the past two decades that mast cells have gained recognition for their involvement in other physiological and pathological processes. Mast cells have a widespread distribution and are found predominantly at the interface between the host and the external environment. Mast cell maturation, phenotype and function are a direct consequence of the local microenvironment and have a marked influence on their ability to specifically recognize and respond to various stimuli through the release of an array of biologically active mediators. These features enable mast cells to act as both first responders in harmful situations as well as to respond to changes in their environment by communicating with a variety of other cells implicated in physiological and immunological responses. Therefore, the critical role of mast cells in both innate and adaptive immunity, including immune tolerance, has gained increased prominence. Conversely, mast cell dysfunction has pointed to these cells as the main offenders in several chronic allergic/inflammatory disorders, cancer and autoimmune diseases. This review summarizes the current knowledge of mast cell function in both normal and pathological conditions with regards to their regulation, phenotype and role.

Permeabilization of cell membranes.

In order to detect intracellular antigens, cells must first be permeabilized especially after fixation with cross-linking agents such as formaldehyde and glutaraldehyde. Permeabilization provides access to intracellular or intraorganellar antigens. Two general types of reagents are commonly used: organic solvents, such as methanol and acetone, and detergents such as saponin, Triton X-100 and Tween-20. The organic solvents dissolve lipids from cell membranes making them permeable to antibodies. Because the organic solvents also coagulate proteins, they can be used to fix and permeabilize cells at the same time. Saponin interacts with membrane cholesterol, selectively removing it and leaving holes in the membrane. The disadvantage of detergents such as Triton X-100 and Tween-20 is that they are non-selective in nature and may extract proteins along with the lipids. This chapter provides methods for the use of organic solvents and detergents to permeabilize cell membranes.

Mast Cell Degranulation Induced by Lectins: Effect on Neutrophil Recruitment

The mammalian lectin macrophage-derived neutrophil chemotactic factor (MNCF) and the plant lectin KM+ were characterized for their ability to activate and degranulate mast cells. The association between mast cell activation and the induction of neutrophil migration was also investigated. Incubation of rat peritoneal mast cells with these lectins resulted in degranulation and mediator release. By confocal microscopy, both lectins were evenly distributed on the cell surface. MNCF activated RBL-2H3 mast cells only if the cells had been sensitized with IgE. KM+ was able to activate either unsensitized or IgE sensitized RBL-2H3 cells. In microplate assays MNCF, but not KM+, bound to rat IgE. In rats that were depleted of mast cells, neutrophil recruitment by MNCF and KM+ were significantly reduced indicating that mast cell activation provides an amplification loop for the neutrophil recruitment induced by these lectins. The present study supports the concept that mammalian lectins play a fundamental role in innate immunity.

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

Galectin‐3 is a β‐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‐γ 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‐1β, 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‐1β serum levels detected in infected gal3−/− mice. Therefore galectin‐3 may exert a regulatory role in innate immunity by diminishing IL‐1β production and thus affecting resistance to R. equi infection.

Oropouche virus entry into HeLa cells involves clathrin and requires endosomal acidification

Abstract Oropouche virus (ORO), family Bunyaviridae, is the second most frequent cause of arboviral febrile illness in Brazil. Studies were conducted to understand ORO entry in HeLa cells. Chlorpromazine inhibited early steps of ORO replication cycle, consistent with entry/uncoating. The data indicate that ORO enters HeLa cells by clathrin-coated vesicles, by a mechanism susceptible to endosomal acidification inhibitors. Transmission electron microscopy and immunofluorescence indicated that ORO associates with clathrin-coated pits and can be found in association with late endosomes in a time shorter than 1h.

Expression of Mast Cell Proteases Correlates with Mast Cell Maturation and Angiogenesis during Tumor Progression

Tumor cells are surrounded by infiltrating inflammatory cells, such as lymphocytes, neutrophils, macrophages, and mast cells. A body of evidence indicates that mast cells are associated with various types of tumors. Although role of mast cells can be directly related to their granule content, their function in angiogenesis and tumor progression remains obscure. This study aims to understand the role of mast cells in these processes. Tumors were chemically induced in BALB/c mice and tumor progression was divided into Phases I, II and III. Phase I tumors exhibited a large number of mast cells, which increased in phase II and remained unchanged in phase III. The expression of mouse mast cell protease (mMCP)-4, mMCP-5, mMCP-6, mMCP-7, and carboxypeptidase A were analyzed at the 3 stages. Our results show that with the exception of mMCP-4 expression of these mast cell chymase (mMCP-5), tryptases (mMCP-6 and 7), and carboxypeptidase A (mMC-CPA) increased during tumor progression. Chymase and tryptase activity increased at all stages of tumor progression whereas the number of mast cells remained constant from phase II to III. The number of new blood vessels increased significantly in phase I, while in phases II and III an enlargement of existing blood vessels occurred. In vitro, mMCP-6 and 7 are able to induce vessel formation. The present study suggests that mast cells are involved in induction of angiogenesis in the early stages of tumor development and in modulating blood vessel growth in the later stages of tumor progression.

B-lymphocytes in bone marrow or lymph nodes can take up plasmid DNA after intramuscular delivery.

Nucleic acid vaccines are an attractive alternative to conventional protein vaccines because of their ability to induce de novo production of antigens in a given tissue after DNA delivery. Although DNA vaccines are highly effective in inducing both cell-mediated and humoral immunity, little is known about the many cell types involved in plasmid DNA uptake in vivo. Here we demonstrate, for the first time, that plasmid DNA can be taken up by both bone marrow and lymph node B cells after intramuscular immunization. Plasmid DNA was also detected in CD11b+ and CD11c+ cells. This phenomenon was not restricted to plasmid DNA encoding mycobacterial 65-kd heat shock protein (pcDNA3-hsp65) because we observed similar results with plasmid-encoding green fluorescent protein (GFP-pEGFP-2C). In addition to plasmid DNA uptake, B cells also express the encoded protein, suggesting that B cells play a role in the immune response after DNA immunization. The biodistribution of plasmid DNA in B cells opens a new perspective in B-cell gene therapy for the in vivo use of plasmid DNA.

MICROWAVE FIXATION IMPROVES ANTIGENICITY OF GLUTARALDEHYDE-SENSITIVE ANTIGENS WHILE PRESERVING ULTRASTRUCTURAL DETAIL

Microwave fixation for electron microscopy has been used primarily for post-embedding immunocytochemistry. The present study examined the ability of microwave fixation to preserve the antigenicity of glutaraldehyde-sensitive antigens for pre-embedding immunocytochemistry. Five monoclonal antibodies (MAbs) directed against cell surface components of rat mast cells were tested. The MAbs failed to show any labeling of conventionally fixed rat bone marrow-derived mast cells even at glutaraldehyde concentrations as low as 0.1%. Strong staining of mast cell plasma membranes was seen when bone marrow was initially fixed with 2% formaldehyde and then refixed in 2% glutaraldehyde/2% formaldehyde after immunostaining. However, the ultrastructural preservation of the cells was poor. Antigenicity and morphological detail were both preserved when bone marrow was fixed in 0.05% glutaraldehyde/2% formaldehyde for 4 sec in a 550-W microwave oven. With this method, mast cells in various stages of maturation as well as cells that did not contain granules were immunoreactive. This method should prove useful with antigens from many different cell types that are sensitive to glutaraldehyde fixation.

TLR2-dependent mast cell activation contributes to the control of Mycobacterium tuberculosis infection

Mast Cells (MCs) express toll-like receptor 2 (TLR2), a receptor known to be triggered by several major mycobacterial ligands and involved in resistance against Mycobacterium tuberculosis (MTB) infection. This study investigated whether adoptive transfer of TLR2 positive MCs (TLR2(+/+)) corrects the increased susceptibility of TLR2(-/-) mice to MTB infection. TLR2(-/-) mice displayed increased mycobacterial burden, diminished myeloid cell recruitment and proinflammatory cytokine production accompanied by defective granuloma formation. The reconstitution of these mice with TLR2(+/+) MCs, but not TLR2(-/-), confers better control of the infection, promotes the normalization of myeloid cell recruitment associated with reestablishment of the granuloma formation. In addition, adoptive transfer of TLR2(+/+) MC to TLR2(-/-) mice resulted in regulation of the pulmonary levels of IL-beta, IL-6, TNF-alpha, enhanced Th1 response and activated CD8(+) T cell homing to the lungs. Our results suggest that activation of MCs via TLR2 is required to compensate the defect in protective immunity and inability of TLR2(-/-) mice to control MTB infection.