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Biomedical subjects

Rossana C N Melo

Publications and source records attributed to Rossana C N Melo.

13 recordsLinked to original sources
PubMed2006

Roles and origins of leukocyte lipid bodies: proteomic and ultrastructural studies.

Lipid bodies (LBs), multifunctional organelles present in most eukaryotic cells, are sites of eicosanoid formation in leukocytes; but little is known about the composition of leukocyte LBs or the biogenesis and internal structures of LBs from mammalian cells. Proteomic analyses of LBs purified from human monocytic U937 cells detected, common to LBs in other cells, proteins involved in cholesterol and triglyceride metabolism, Rab GTPases, and many membrane and endoplasmic reticulum (ER)-associated proteins. Newly lipid body (LB)-associated proteins included MRP-14, potentially involved in arachidonate transport, and ribosomal subunit proteins and translation regulatory proteins. Ultrastructurally, in U937 cells as well as human neutrophils and eosinophils, ribosomes are attached to and distributed within LBs, and LBs contain extensive ER-like membranes. The presence of ribosomes, ER-like membranes and many membrane-associated and ER luminal proteins within LBs, supports a new model by which enveloped ER-membranes and domains form LBs and indicates that LBs may be sites of protein synthesis.

Chromatography, High Pressure Liquid↗
PubMed2006

Leukocyte lipid bodies regulation and function: contribution to allergy and host defense.

Lipid bodies are lipid-rich organelles found in the cytoplasm of a variety of cells, including leukocytes. Lipid body morphology, its ability to interact with other organelles and its functions are dictated by its lipid arrangement, as well as its protein composition. Both may vary according to the cell type and with the specific lipid body biogenic stimulatory pathways. Nascent lipid bodies, which are formed in vivo in the course of a variety of immunopathological conditions, are sites of enzyme localization, eicosanoid production, as well as, sites for cytokine storage in inflammatory leukocytes, suggesting that lipid bodies function as inducible intracellular platforms for spatial segregation and organization of signaling leading to inflammatory mediator secretion during inflammation. The emerging role of lipid bodies as inflammatory organelles raises lipid body status to critical regulators of different inflammatory diseases, key markers of leukocyte activation and attractive targets for novel anti-inflammatory therapies.

Animals↗
PubMed2006

Leukocyte lipid bodies: inflammation-related organelles are rapidly detected by wet scanning electron microscopy.

Leukocyte lipid bodies are dynamic, functionally active organelles with central roles in inflammation. Here, we report that leukocyte lipid bodies are facilely detected by a versatile, potent technique, termed wet scanning electron microscopy (SEM), which combines the rapid preparation of light microscopy with the resolution of SEM. Using as leukocyte models resting and agonist-stimulated human eosinophils, cells that generate prominent numbers of lipid bodies in inflammatory conditions, we demonstrated that lipid bodies can be rapidly imaged as bright, highly contrasted structures under wet SEM and scored by computerized image processing. Critical advantages of this approach are that it permits cell observation in a fully hydrated system and facilitates lipid preservation. These attributes are especially important because lipid bodies are degraded during routine dehydration processes. Moreover, this technology is advantageous over lipophilic fluorescent probes because it allows sustained detection of lipid bodies in contrast to short-lived fluorescent labeling of these organelles. The value of wet SEM in enabling rapid and large-scale lipid body imaging and scoring within leukocytes is particularly important because lipid bodies are organelles underlying the heightened functions of inflammatory cells. Wet SEM technology provides new approaches and opportunities for delineations of lipid bodies in inflammatory diseases, including allergic inflammation.

Cell Survival↗
PubMed2006

Mycobacterium bovis bacillus Calmette-Guérin induces TLR2-mediated formation of lipid bodies: intracellular domains for eicosanoid synthesis in vivo.

Differentiation of macrophages into foamy (lipid-laden) macrophages is a common pathological observation in tuberculous granulomas both in experimental settings as well as in clinical conditions; however, the mechanisms that regulate intracellular lipid accumulation in the course of mycobacterial infection and their significance to pathophysiology of tuberculosis are not well understood. In this study, we investigated the mechanisms of formation and function of lipid-laden macrophages in a murine model of tuberculosis. Mycobacterium bovis bacillus Calmette-Guérin (BCG), but not Mycobacterium smegmatis, induced a dose- and time-dependent increase in lipid body-inducible nonmembrane-bound cytoplasmic lipid domain size and numbers. Lipid body formation was drastically inhibited in TLR2-, but not in TLR4-deficient mice, indicating a role for TLR2 in BCG recognition and signaling to form lipid bodies. Increase in lipid bodies during infection correlated with increased generation of PGE2 and localization of cyclooxygenase-2 within lipid bodies. Moreover, we demonstrated by intracellular immunofluorescent localization of newly formed eicosanoid that lipid bodies were the predominant sites of PGE2 synthesis in activated macrophages. Our findings demonstrated that BCG-induced lipid body formation is TLR2 mediated and these structures function as signaling platforms in inflammatory mediator production, because compartmentalization of substrate and key enzymes within lipid bodies has impact on the capacity of activated leukocytes to generate increased amounts of eicosanoids during experimental infection by BCG.

Animals↗
PubMed2006

Cytokine receptor-mediated trafficking of preformed IL-4 in eosinophils identifies an innate immune mechanism of cytokine secretion.

Although leukocytes of the innate immune system, including eosinophils, contain within their granules preformed stores of cytokines available for selective and rapid release, little is known about the mechanisms governing the mobilization and secretion of these cytokines. Here we show that a cytokine receptor, the IL-4 receptor alpha chain, mediates eotaxin-stimulated mobilization of preformed IL-4 from eosinophil granules into secretory vesicles. Eosinophils contain substantial intracellular quantities of several granule- and vesicle-associated cytokine receptors, including IL-4, IL-6, and IL-13 receptors as well as CCR3. Both IL-4 and IL-4 receptor alpha chain colocalized in eosinophil granules; and after eotaxin-stimulation, IL-4 receptor alpha chain, bearing bound IL-4, was mobilized into secretory vesicles. These findings indicate that intracellular cytokine receptors within secretory vesicles transport their cognate cytokines requisite for the secretion of cytokines preformed in innate immune leukocytes.

Cells, Cultured↗
PubMed2005

Intragranular vesiculotubular compartments are involved in piecemeal degranulation by activated human eosinophils.

Eosinophils, leukocytes involved in allergic, inflammatory and immunoregulatory responses, have a distinct capacity to rapidly secrete preformed granule-stored proteins through piecemeal degranulation (PMD), a secretion process based on vesicular transport of proteins from within granules for extracellular release. Eosinophil-specific granules contain cytokines and cationic proteins, such as major basic protein (MBP). We evaluated structural mechanisms responsible for mobilizing proteins from within eosinophil granules. Human eosinophils stimulated for 30-60 min with eotaxin, regulated on activation, normal, T-cell expressed and secreted (RANTES) or platelet activating factor exhibited ultrastructural features of PMD (e.g. losses of granule contents) and extensive vesiculotubular networks within emptying granules. Brefeldin A inhibited granule emptying and collapsed intragranular vesiculotubular networks. By immunonanogold ultrastructural labelings, CD63, a tetraspanin membrane protein, was localized within granules and on vesicles outside of granules, and mobilization of MBP into vesicles within and extending from granules was demonstrated. Electron tomography with three dimension reconstructions revealed granule internal membranes to constitute an elaborate tubular network able to sequester and relocate granule products upon stimulation. We provide new insights into PMD and identify eosinophil specific granules as organelles whose internal tubulovesicular networks are important for the capacity of eosinophils to secrete, by vesicular transport, their content of preformed and granule-stored cytokines and cationic proteins.

Antigens, CD↗
PubMed2005

Human eosinophils secrete preformed, granule-stored interleukin-4 through distinct vesicular compartments.

Secretion of interleukin-4 (IL-4) by leukocytes is important for varied immune responses including allergic inflammation. Within eosinophils, unlike lymphocytes, IL-4 is stored in granules (termed specific granules) and can be rapidly released by brefeldin A (BFA)-inhibitable mechanisms upon stimulation with eotaxin, a chemokine that activates eosinophils. In studying eotaxin-elicited IL-4 secretion, we identified at the ultrastructural level distinct vesicular IL-4 transport mechanisms. Interleukin-4 traffics from granules via two vesicular compartments, large vesiculotubular carriers, which we term eosinophil sombrero vesicles (EoSV), and small classical spherical vesicles. These two vesicles may represent alternative pathways for transport to the plasma membrane. Loci of both secreted IL-4 and IL-4-loaded vesicles were imaged at the plasma membranes by a novel EliCell assay using a fluoronanogold probe. Three dimensional electron tomographic reconstructions revealed EoSVs to be folded, flattened and elongated tubules with substantial membrane surfaces. As documented with quantitative electron microscopy, eotaxin-induced significant formation of EoSVs while BFA pretreatment suppressed eotaxin-elicited EoSVs. Electron tomography showed that both EoSVs and small vesicles interact with and arise from granules in response to stimulation. Thus, this intracellular vesicular system mediates the rapid mobilization and secretion of preformed IL-4 by activated eosinophils. These findings, highlighting the participation of large tubular carriers, provide new insights into vesicular trafficking of cytokines.

Cells, Cultured↗
PubMed2005

A gel-based dual antibody capture and detection method for assaying of extracellular cytokine secretion: EliCell.

A distinguishing feature of eosinophils is their ability to rapidly release preformed cytokines from intracellular pools. Cytokines are delivered to the cell surface from granule stores by transport vesicles and are released in small packets at discrete locations along the cell surface through a process termed "piecemeal" degranulation. The study of this process has been hindered by lack of an assay sensitive enough to register minute protein concentrations and the inability to visualize morphology of cytokine secreting cells. These hindrances have necessitated our development of the EliCell assay, an agarose-based dual cytokine capture and detection system through which cytokine secretion and cellular morphology may be analyzed in concert. Cells are embedded within capture antibody-containing agarose and stimulated under conditions of interest. Extracellularly released cytokine is captured within the matrix at the point of release from the cell and can be labeled with a fluorochrome-conjugated antibody. Cytokine release and cellular morphology are visualized in parallel by phase contrast and fluorescence microscopy, respectively.

Antibodies↗
PubMed2004

Peripheral blood monocytes show morphological pattern of activation and decreased nitric oxide production during acute Chagas' disease in rats.

Peripheral blood monocytes (PBM) recruitment is a rapid and remarkable phenomenon during acute infection with the intracellular protozoan parasite Trypanosoma cruzi, the causative agent of Chagas' disease. The functional capabilities of these cells during the infection, however, are poorly understood. The purpose of the present study was to determine whether PBM are morphologically activated and produce nitric oxide (NO), a mediator of host cell defense when challenged with the parasite at different time points of acute disease. In parallel, the parasite load was monitored in the blood and heart, a target organ of the disease, as well as the PBM numbers. The infection did not induce NO release by PBM, although these cells exhibited a clear morphological pattern of activation characterized by irregular surface, increase of organelle amount, especially Golgi complex, and cell size. On the contrary, there was significant inhibition of NO production by PBM at the beginning (day 6) and end of acute disease (day 20). At this time, the levels of NO were inversely related to the arginase activity, an enzyme that affects the NO synthesis. The mobilization process of PBM occurred in parallel to parasite load and was associated with the resolution mechanism of parasitemia and heart parasitism. Our results showed that activated PBM are notably involved in the host response to the acute T. cruzi infection in rats. However, the in vivo NO production by these cells seems to be inhibited during the acute Chagas' disease through a mechanism involving the arginase pathway.

Acute Disease↗
PubMed2003

EliCell assay for the detection of released cytokines from eosinophils.

Eosinophils contain several preformed cytokines within their specific granules. Therefore, without requiring them in de novo synthesis of cytokines, eosinophils can release quantities of granule-derived cytokines by highly regulated mechanisms. However, eosinophil "degranulation" is poorly understood, in part, because available methodologies did not appear appropriate for analyzing vesicular mobilization and transport of eosinophil granular contents. The EliCell assay is a microscopic methodology substantially modified from other techniques employed to detect cytokine release (i.e., ELISPOT). The method is a dual antibody capture/detection system in which viable eosinophils are incubated in a solid streptavidin-conjugated agarose matrix, which contains a biotinylated capture antibody against the cytokine of interest. Released cytokine is detected around non-permeabilized eosinophils with a separate fluorochrome-labeled detection antibody. Thus, the EliCell system captures and detects extracellular cytokines at the site of their release from eosinophils. As examples, we have used EliCell essays to detect the selective release of either IL-4 or IL-12 cytokines found preformed in eosinophils-from eotaxin- or anti-CD9-stimulated eosinophils, respectively. With appropriate pairs of antibodies, any preformed cytokine found into eosinophil granules could be studied and the mechanisms of their secretion evaluated by using the EliCell assay.

Bacterial Proteins↗
PubMed2003

Production of hydrogen peroxide by peripheral blood monocytes and specific macrophages during experimental infection with Trypanosoma cruzi in vivo.

Acute Chagas' disease triggers potent inflammatory reaction characterized by great increase of peripheral blood monocyte (PBM) and macrophage numbers. We studied the respiratory burst responses of PBM and peritoneal and splenic macrophages to in vivo infection (rats). The ultrastructure of heart inflammatory macrophages was also investigated. The infection increased the hydrogen peroxide (H2O2) production by PBM and splenic macrophages but not by peritoneal macrophages. Accordingly, the PBM and spleen cell numbers increased but the total number of peritoneal cells was similar to controls. Heart macrophages of infected rats exhibited increase (number and size) and activated morphology in parallel to high cardiomyocyte parasitism. Our data highlight the importance of innate immunity and H2O2production to host resistance during acute phase of T. cruzi infection. A novel finding is that H2O2production seems related to specific types of monocytes/macrophages that are able to release this agent when in presence of high parasite load.

Animals↗
PubMed2002

Relationship of sertoli-sertoli tight junctions to ectoplasmic specialization in conventional and en face views.

Ectoplasmic specializations are actin filament-endoplasmic reticulum complexes that occur in Sertoli cells at sites of intercellular attachment. At sites between inter-Sertoli cell attachments, near the base of the cells, the sites are also related to tight junctions. We studied the characteristics of ectoplasmic specializations from six species using conventional views in which thin sections were perpendicular to the plane of the membranes, we used rare views in which the sections were in the plane of the membrane (en face views), and we also used the freeze-fracture technique. Tissues postfixed by osmium ferrocyanide showed junctional strands (fusion points between membranes) and actin bundles, actin sheets, or both, which could be visualized simultaneously. En face views demonstrated that the majority of tight junctional strands ran parallel to actin filament bundles. Usually, two tight junctional strands were associated with each actin filament bundle. Parallel tight junctions were occasionally extremely close together ( approximately 12 nm apart). Tight junctional strands were sometimes present without an apparent association with organized actin bundles or they were tangential to actin bundles. En face views showed that gap junctions were commonly observed intercalated with tight junction strands. The results taken together suggest a relationship of organized actin with tight junction complexes. However, the occasional examples of tight junction complexes being not perfectly aligned with actin filament bundles suggest that a precise and rigidly organized actin-tight junction relationship described above is not absolutely mandatory for the presence or maintenance of tight junctions. Species variations in tight junction organization are also presented.

Actin Cytoskeleton↗