Publications by authors named "Rossana C N Melo"

87 Publications

Vesicular trafficking of immune mediators in human eosinophils revealed by immunoelectron microscopy.

Exp Cell Res 2016 10 22;347(2):385-90. Epub 2016 Aug 22.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, CLS 943, Boston, MA 02215, USA.

Electron microscopy (EM)-based techniques are mostly responsible for our current view of cell morphology at the subcellular level and continue to play an essential role in biological research. In cells from the immune system, such as eosinophils, EM has helped to understand how cells package and release mediators involved in immune responses. Ultrastructural investigations of human eosinophils enabled visualization of secretory processes in detail and identification of a robust, vesicular trafficking essential for the secretion of immune mediators via a non-classical secretory pathway associated with secretory (specific) granules. This vesicular system is mainly organized as large tubular-vesicular carriers (Eosinophil Sombrero Vesicles - EoSVs) actively formed in response to cell activation and provides a sophisticated structural mechanism for delivery of granule-stored mediators. In this review, we highlight the application of EM techniques to recognize pools of immune mediators at vesicular compartments and to understand the complex secretory pathway within human eosinophils involved in inflammatory and allergic responses.
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http://dx.doi.org/10.1016/j.yexcr.2016.08.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5028316PMC
October 2016

Lipid Body Organelles within the Parasite Trypanosoma cruzi: A Role for Intracellular Arachidonic Acid Metabolism.

PLoS One 2016 4;11(8):e0160433. Epub 2016 Aug 4.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora (UFJF), Juiz de Fora, MG, Brazil.

Most eukaryotic cells contain varying amounts of cytosolic lipidic inclusions termed lipid bodies (LBs) or lipid droplets (LDs). In mammalian cells, such as macrophages, these lipid-rich organelles are formed in response to host-pathogen interaction during infectious diseases and are sites for biosynthesis of arachidonic acid (AA)-derived inflammatory mediators (eicosanoids). Less clear are the functions of LBs in pathogenic lower eukaryotes. In this study, we demonstrated that LBs, visualized by light microscopy with different probes and transmission electron microscopy (TEM), are produced in trypomastigote forms of the parasite Trypanosoma cruzi, the causal agent of Chagas' disease, after both host interaction and exogenous AA stimulation. Quantitative TEM revealed that LBs from amastigotes, the intracellular forms of the parasite, growing in vivo have increased size and electron-density compared to LBs from amastigotes living in vitro. AA-stimulated trypomastigotes released high amounts of prostaglandin E2 (PGE2) and showed PGE2 synthase expression. Raman spectroscopy demonstrated increased unsaturated lipid content and AA incorporation in stimulated parasites. Moreover, both Raman and MALDI mass spectroscopy revealed increased AA content in LBs purified from AA-stimulated parasites compared to LBs from unstimulated group. By using a specific technique for eicosanoid detection, we immunolocalized PGE2 within LBs from AA-stimulated trypomastigotes. Altogether, our findings demonstrate that LBs from the parasite Trypanosoma cruzi are not just lipid storage inclusions but dynamic organelles, able to respond to host interaction and inflammatory events and involved in the AA metabolism. Acting as sources of PGE2, a potent immunomodulatory lipid mediator that inhibits many aspects of innate and adaptive immunity, newly-formed parasite LBs may be implicated with the pathogen survival in its host.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160433PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4973985PMC
July 2017

Host Lipid Bodies as Platforms for Intracellular Survival of Protozoan Parasites.

Front Immunol 2016 3;7:174. Epub 2016 May 3.

Laboratory of Cellular Biology, Department of Biology, Institute of Biological Sciences (ICB), Federal University of Juiz de Fora (UFJF) , Juiz de Fora, Minas Gerais , Brazil.

Pathogens induce several changes in the host cell signaling and trafficking mechanisms in order to evade and manipulate the immune response. One prominent pathogen-mediated change is the formation of lipid-rich organelles, termed lipid bodies (LBs) or lipid droplets, in the host cell cytoplasm. Protozoan parasites, which contribute expressively to the burden of infectious diseases worldwide, are able to induce LB genesis in non-immune and immune cells, mainly macrophages, key players in the initial resistance to the infection. Under host-parasite interaction, LBs not only accumulate in the host cytoplasm but also relocate around and move into parasitophorous vacuoles. There is increasing evidence that protozoan parasites may target host-derived LBs either for gaining nutrients or for escaping the host immune response. Newly formed, parasite-induced LBs may serve as lipid sources for parasite growth and also produce inflammatory mediators that potentially act in the host immune response deactivation. In this mini review, we summarize current knowledge on the formation and role of host LBs as sites exploited by intracellular protozoan parasites as a strategy to maintain their own survival.
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http://dx.doi.org/10.3389/fimmu.2016.00174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4853369PMC
May 2016

CD63 is tightly associated with intracellular, secretory events chaperoning piecemeal degranulation and compound exocytosis in human eosinophils.

J Leukoc Biol 2016 08 10;100(2):391-401. Epub 2016 Mar 10.

Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, UFJF, Juiz de Fora, Brazil; Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA; and

Eosinophil activation leads to secretion of presynthesized, granule-stored mediators that determine the course of allergic, inflammatory, and immunoregulatory responses. CD63, a member of the transmembrane-4 glycoprotein superfamily (tetraspanins) and present on the limiting membranes of eosinophil-specific (secretory) granules, is considered a potential surface marker for eosinophil degranulation. However, the intracellular secretory trafficking of CD63 in eosinophils and other leukocytes is not understood. Here, we provide a comprehensive investigation of CD63 trafficking at high resolution within human eosinophils stimulated with inflammatory stimuli, CCL11 and tumor necrosis factor α, which induce distinctly differing secretory processes in eosinophils: piecemeal degranulation and compound exocytosis, respectively. By using different transmission electron microscopy approaches, including an immunonanogold technique, for enhanced detection of CD63 at subcellular compartments, we identified a major intracellular pool of CD63 that is directly linked to eosinophil degranulation events. Transmission electron microscopy quantitative analyses demonstrated that, in response to stimulation, CD63 is concentrated within granules undergoing secretion by piecemeal degranulation or compound exocytosis and that CD63 tracks with the movements of vesicles and granules in the cytoplasm. Although CD63 was observed at the cell surface after stimulation, immunonanogold electron microscopy revealed that a strong CD63 pool remains in the cytoplasm. It is remarkable that CCL11 and tumor necrosis factor α triggered increased formation of CD63(+) large vesiculotubular carriers (eosinophil sombrero vesicles), which fused with granules in the process of secretion, likely acting in the intracellular translocation of CD63. Altogether, we identified active, intracellular CD63 trafficking connected to eosinophil granule-derived secretory pathways. This is important for understanding the complex secretory activities of eosinophils underlying immune responses.
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http://dx.doi.org/10.1189/jlb.3A1015-480RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6608091PMC
August 2016

Potential effects of UV radiation on photosynthetic structures of the bloom-forming cyanobacterium Cylindrospermopsis raciborskii CYRF-01.

Front Microbiol 2015 30;6:1202. Epub 2015 Oct 30.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora Juiz de Fora, Brazil.

Cyanobacteria are aquatic photosynthetic microorganisms. While of enormous ecological importance, they have also been linked to human and animal illnesses around the world as a consequence of toxin production by some species. Cylindrospermopsis raciborskii, a filamentous nitrogen-fixing cyanobacterium, has attracted considerable attention due to its potential toxicity and ecophysiological adaptability. We investigated whether C. raciborskii could be affected by ultraviolet (UV) radiation. Non-axenic cultures of C. raciborskii were exposed to three UV treatments (UVA, UVB, or UVA + UVB) over a 6 h period, during which cell concentration, viability and ultrastructure were analyzed. UVA and UVA + UVB treatments showed significant negative effects on cell concentration (decreases of 56.4 and 64.3%, respectively). This decrease was directly associated with cell death as revealed by a cell viability fluorescent probe. Over 90% of UVA + UVB- and UVA-treated cells died. UVB did not alter cell concentration, but reduced cell viability in almost 50% of organisms. Transmission electron microscopy (TEM) revealed a drastic loss of thylakoids, membranes in which cyanobacteria photosystems are localized, after all treatments. Moreover, other photosynthetic- and metabolic-related structures, such as accessory pigments and polyphosphate granules, were damaged. Quantitative TEM analyses revealed a 95.8% reduction in cell area occupied by thylakoids after UVA treatment, and reduction of 77.6 and 81.3% after UVB and UVA + UVB treatments, respectively. Results demonstrated clear alterations in viability and photosynthetic structures of C. raciborskii induced by various UV radiation fractions. This study facilitates our understanding of the subcellular organization of this cyanobacterium species, identifies specific intracellular targets of UVA and UVB radiation and reinforces the importance of UV radiation as an environmental stressor.
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http://dx.doi.org/10.3389/fmicb.2015.01202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4627488PMC
November 2015

Lipid droplets in leukocytes: Organelles linked to inflammatory responses.

Exp Cell Res 2016 Jan 29;340(2):193-7. Epub 2015 Oct 29.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, CLS 943, Boston, MA 02215, USA. Electronic address:

Studies on lipid droplets (LDs) in leukocytes have attracted attention due to their association with human diseases. In these cells, LDs are rapidly formed in response to inflammatory stimuli or allergic/inflammatory diseases including infections with parasites and bacteria. Leukocyte LDs are linked to the regulation of immune responses by compartmentalization of several proteins and lipids involved in the control and biosynthesis of inflammatory mediators (eicosanoids). In this mini review, we summarize current knowledge on the composition, structure and function of leukocyte LDs, organelles now considered as structural markers of inflammation.
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http://dx.doi.org/10.1016/j.yexcr.2015.10.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4744558PMC
January 2016

Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils.

Exp Cell Res 2015 Oct 6;337(2):129-135. Epub 2015 Aug 6.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG, Brazil.

Background: SNARE members mediate membrane fusion during intracellular trafficking underlying innate and adaptive immune responses by different cells. However, little is known about the expression and function of these proteins in human eosinophils, cells involved in allergic, inflammatory and immunoregulatory responses. Here, we investigate the expression and distribution of the Qa-SNARE syntaxin17 (STX17) within human eosinophils isolated from the peripheral blood.

Methods: Flow cytometry and a pre-embedding immunonanogold electron microscopy (EM) technique that combines optimal epitope preservation and secondary Fab-fragments of antibodies linked to 1.4 nm gold particles for optimal access to microdomains, were used to investigate STX17.

Results: STX17 was detected within unstimulated eosinophils. Immunogold EM revealed STX17 on secretory granules and on granule-derived vesiculotubular transport carriers (Eosinophil Sombrero Vesicles-EoSVs). Quantitative EM analyses showed that 77.7% of the granules were positive for STX17 with a mean±SEM of 3.9±0.2 gold particles/granule. Labeling was present on both granule outer membranes and matrices while EoSVs showed clear membrane-associated labeling. STX17 was also present in secretory granules in eosinophils stimulated with the cytokine tumor necrosis factor alpha (TNF-α) or the CC-chemokine ligand 11 CCL11 (eotaxin-1), stimuli that induce eosinophil degranulation. The number of secretory granules labeled for STX17 was significantly higher in CCL11 compared with the unstimulated group. The level of cell labeling did not change when unstimulated cells were compared with TNF-α-stimulated eosinophils.

Conclusions: The present study clearly shows by immunanonogold EM that STX17 is localized in eosinophil secretory granules and transport vesicles and might be involved in the transport of granule-derived cargos.
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http://dx.doi.org/10.1016/j.yexcr.2015.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561602PMC
October 2015

Eosinophil secretion of granule-derived cytokines.

Front Immunol 2014 27;5:496. Epub 2014 Oct 27.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School , Boston, MA , USA.

Eosinophils are tissue-dwelling leukocytes, present in the thymus, and gastrointestinal and genitourinary tracts of healthy individuals at baseline, and recruited, often in large numbers, to allergic inflammatory foci and sites of active tissue repair. The biological significance of eosinophils is vast and varied. In health, eosinophils support uterine and mammary gland development, and maintain bone marrow plasma cells and adipose tissue alternatively activated macrophages, while in response to tissue insult eosinophils function as inflammatory effector cells, and, in the wake of an inflammatory response, promote tissue regeneration, and wound healing. One common mechanism driving many of the diverse eosinophil functions is the regulated and differential secretion of a vast array of eosinophil-derived cytokines. Eosinophils are distinguished from most other leukocytes in that many, if not all, of the over three dozen eosinophil-derived cytokines are pre-synthesized and stored within intracellular granules, poised for very rapid, stimulus-induced secretion. Eosinophils engaged in cytokine secretion in situ utilize distinct pathways of cytokine release that include classical exocytosis, whereby granules themselves fuse with the plasma membrane and release their entire contents extracellularly; piecemeal degranulation, whereby granule-derived cytokines are selectively mobilized into vesicles that emerge from granules, traverse the cytoplasm and fuse with the plasma membrane to release discrete packets of cytokines; and eosinophil cytolysis, whereby intact granules are extruded from eosinophils, and deposited within tissues. In this latter scenario, extracellular granules can themselves function as stimulus-responsive secretory-competent organelles within the tissue. Here, we review the distinctive processes of differential secretion of eosinophil granule-derived cytokines.
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http://dx.doi.org/10.3389/fimmu.2014.00496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4209865PMC
November 2014

Pre-embedding immunogold labeling to optimize protein localization at subcellular compartments and membrane microdomains of leukocytes.

Nat Protoc 2014 Oct 11;9(10):2382-94. Epub 2014 Sep 11.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

Precise immunolocalization of proteins within a cell is central to understanding cell processes and functions such as intracellular trafficking and secretion of molecules during immune responses. Here we describe a protocol for ultrastructural detection of proteins in leukocytes. The method uses a pre-embedding approach (immunolabeling before standard processing for transmission electron microscopy (TEM)). This protocol combines several strategies for ultrastructure and antigen preservation, robust blocking of nonspecific binding sites, as well as superior antibody penetration for detecting molecules at subcellular compartments and membrane microdomains. A further advantage of this technique is that electron microscopy (EM) processing is quick. This method has been used to study leukocyte biology, and it has helped demonstrate how activated leukocytes deliver specific cargos. It may also potentially be applied to a variety of different cell types. Excluding the initial time required for sample preparation (15 h) and the final resin polymerization step (16 h), the protocol (immunolabeling and EM procedures) can be completed in 8 h.
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http://dx.doi.org/10.1038/nprot.2014.163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4204927PMC
October 2014

Unraveling the complexity of lipid body organelles in human eosinophils.

J Leukoc Biol 2014 Nov 10;96(5):703-12. Epub 2014 Sep 10.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA

Lipid-rich organelles are common in many cell types. In cells, such as adipocytes, these organelles are termed LDs, whereas in other cells, such as leukocytes, they are called LBs. The study of leukocyte LBs has attracted attention as a result of their association with human diseases. In leukocytes, such as eosinophils, LB accumulation has been documented extensively during inflammatory conditions. In these cells, LBs are linked to the regulation of immune responses by compartmentalization of several proteins and lipids involved in the control and biosynthesis of inflammatory mediators (eicosanoids). However, it has been unclear how diverse proteins, including membrane-associated enzymes involved in eicosanoid formation, incorporate into LBs, especially if the internal content of LBs is assumed to consist solely of stores of neutral lipids, as present within adipocyte LDs. Studies of the formation, function, and ultrastructure of LBs in eosinophils have been providing insights pertinent to LBs in other leukocytes. Here, we review current knowledge of the composition and function of leukocyte LBs as provided by studies of human eosinophil LBs, including recognitions of the internal architecture of eosinophil LBs based on 3D electron tomographic analyses.
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http://dx.doi.org/10.1189/jlb.3RU0214-110RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4197557PMC
November 2014

A new approach for optimal morphological identification and immunolabeling of spermatogonial cells.

Microsc Microanal 2014 Aug 16;20(4):1304-11. Epub 2014 May 16.

1Laboratório de Biologia Estrutural e Reprodução,Departamento de Morfologia,Universidade Federal de Minas Gerais,31.270-901 Belo Horizonte,Brasil.

High quality fixation often inactivates epitopes and gentler fixation can fail to preserve biological structure at the required resolution. For studies of male reproduction, immunofluorescence techniques using paraformaldehyde fixation associated with paraffin as an embedding medium gives good epitope preservation, although the cell becomes morphologically compromised. On the other hand, glutaraldehyde associated with a plastic resin has been used with success to recognize and distinguish each spermatogonial cell subtype, but the antigenic sites become inaccessible to antibodies. Here we describe a new method that provides excellent morphological details of testicular cells while preserving the binding capacity of epitopes. Using a combination of glutaraldehyde and paraformaldehyde as a fixative and LR White resin for embedding, we show that it is possible to clearly recognize spermatogonial subtypes (Aund, A-A4, In and B spermatogonia) on 1-μm thick-sections and to label epitopes such as bromodeoxyuridine, a marker used for cellular cycle studies in the testis. The information gained from this procedure can be critical for understanding spermatogonial process of self-renewal and differentiation.
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http://dx.doi.org/10.1017/S1431927614000889DOI Listing
August 2014

The intriguing ultrastructure of lipid body organelles within activated macrophages.

Microsc Microanal 2014 Jun 1;20(3):869-78. Epub 2014 May 1.

1Laboratory of Cellular Biology,Department of Biology,Federal University of Juiz de Fora (UFJF),Juiz de Fora,MG 36036-900,Brazil.

Macrophages are widely distributed immune system cells with essential functions in tissue homeostasis, apoptotic cell clearance, and first defense in infections. A distinguishing feature of activated macrophages participating in different situations such as inflammatory and metabolic diseases is the presence of increased numbers of lipid-rich organelles, termed lipid bodies (LBs) or lipid droplets, in their cytoplasm. LBs are considered structural markers of activated macrophages and are involved in different functions such as lipid metabolism, intracellular trafficking, and synthesis of inflammatory mediators. In this review, we revisit the distinct morphology of LB organelles actively formed within macrophages in response to infections and cell clearance, taking into account new insights provided by ultrastructural studies. We also discuss the LB interactions within macrophages, revealed by transmission electron microscopy, with a focus on the remarkable LB-phagosome association and discuss potential links between structural aspects and function.
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http://dx.doi.org/10.1017/S143192761400066XDOI Listing
June 2014

Human Eosinophil Leukocytes Express Protein Disulfide Isomerase in Secretory Granules and Vesicles: Ultrastructural Studies.

J Histochem Cytochem 2014 Jun 26;62(6):450-459. Epub 2014 Mar 26.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG, Brazil (FFD,KBA,LASC,RCNM)Department of Pathology (AMD)Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts (RS,PFW,RCNM)

Protein disulfide isomerase (PDI) has fundamental roles in the oxidative folding of proteins in the endoplasmic reticulum (ER) of eukaryotic cells. The study of this molecule has been attracting considerable attention due to its association with other cell functions and human diseases. In leukocytes, such as neutrophils, PDI is involved with cell adhesion, signaling and inflammation. However, the expression of PDI in other leukocytes, such as eosinophils, important cells in inflammatory, allergic and immunomodulatory responses, remains to be defined. Here we used different approaches to investigate PDI expression within human eosinophils. Western blotting and flow cytometry demonstrated high PDI expression in both unstimulated and CCL11/eotaxin-1-stimulated eosinophils, with similar levels in both conditions. By using an immunogold electron microscopy technique that combines better epitope preservation and secondary Fab-fragments of antibodies linked to 1.4-nm gold particles for optimal access to microdomains, we identified different intracellular sites for PDI. In addition to predictable strong PDI labeling at the nuclear envelope, other unanticipated sites, such as secretory granules, lipid bodies and vesicles, including large transport vesicles (eosinophil sombrero vesicles), were also labeled. Thus, we provide the first identification of PDI in human eosinophils, suggesting that this molecule may have additional/specific functions in these leukocytes.
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http://dx.doi.org/10.1369/0022155414531437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4174628PMC
June 2014

Mycobacterium leprae intracellular survival relies on cholesterol accumulation in infected macrophages: a potential target for new drugs for leprosy treatment.

Cell Microbiol 2014 Jun 21;16(6):797-815. Epub 2014 Mar 21.

Laboratório de Microbiologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, 21040-900, RJ, Brazil.

We recently showed that Mycobacterium leprae (ML) is able to induce lipid droplet formation in infected macrophages. We herein confirm that cholesterol (Cho) is one of the host lipid molecules that accumulate in ML-infected macrophages and investigate the effects of ML on cellular Cho metabolism responsible for its accumulation. The expression levels of LDL receptors (LDL-R, CD36, SRA-1, SR-B1, and LRP-1) and enzymes involved in Cho biosynthesis were investigated by qRT-PCR and/or Western blot and shown to be higher in lepromatous leprosy (LL) tissues when compared to borderline tuberculoid (BT) lesions. Moreover, higher levels of the active form of the sterol regulatory element-binding protein (SREBP) transcriptional factors, key regulators of the biosynthesis and uptake of cellular Cho, were found in LL skin biopsies. Functional in vitro assays confirmed the higher capacity of ML-infected macrophages to synthesize Cho and sequester exogenous LDL-Cho. Notably, Cho colocalized to ML-containing phagosomes, and Cho metabolism impairment, through either de novo synthesis inhibition by statins or depletion of exogenous Cho, decreased intracellular bacterial survival. These findings highlight the importance of metabolic integration between the host and bacteria to leprosy pathophysiology, opening new avenues for novel therapeutic strategies to leprosy.
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http://dx.doi.org/10.1111/cmi.12279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262048PMC
June 2014

Visualizing aquatic bacteria by light and transmission electron microscopy.

Antonie Van Leeuwenhoek 2014 Jan 17;105(1):1-14. Epub 2013 Oct 17.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora (UFJF), Juiz de Fora, MG, 36036-900, Brazil.

The understanding of the functional role of aquatic bacteria in microbial food webs is largely dependent on methods applied to the direct visualization and enumeration of these organisms. While the ultrastructure of aquatic bacteria is still poorly known, routine observation of aquatic bacteria by light microscopy requires staining with fluorochromes, followed by filtration and direct counting on filter surfaces. Here, we used a new strategy to visualize and enumerate aquatic bacteria by light microscopy. By spinning water samples from varied tropical ecosystems in a cytocentrifuge, we found that bacteria firmly adhere to regular slides, can be stained by fluorochoromes with no background formation and fast enumerated. Significant correlations were found between the cytocentrifugation and filter-based methods. Moreover, preparations through cytocentrifugation were more adequate for bacterial viability evaluation than filter-based preparations. Transmission electron microscopic analyses revealed a morphological diversity of bacteria with different internal and external structures, such as large variation in the cell envelope and capsule thickness, and presence or not of thylakoid membranes. Our results demonstrate that aquatic bacteria represent an ultrastructurally diverse population and open avenues for easy handling/quantification and better visualization of bacteria by light microscopy without the need of filter membranes.
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http://dx.doi.org/10.1007/s10482-013-0047-6DOI Listing
January 2014

The internal architecture of leukocyte lipid body organelles captured by three-dimensional electron microscopy tomography.

PLoS One 2013 26;8(3):e59578. Epub 2013 Mar 26.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG, Brazil.

Lipid bodies (LBs), also known as lipid droplets, are complex organelles of all eukaryotic cells linked to a variety of biological functions as well as to the development of human diseases. In cells from the immune system, such as eosinophils, neutrophils and macrophages, LBs are rapidly formed in the cytoplasm in response to inflammatory and infectious diseases and are sites of synthesis of eicosanoid lipid mediators. However, little is known about the structural organization of these organelles. It is unclear whether leukocyte LBs contain a hydrophobic core of neutral lipids as found in lipid droplets from adipocytes and how diverse proteins, including enzymes involved in eicosanoid formation, incorporate into LBs. Here, leukocyte LB ultrastructure was studied in detail by conventional transmission electron microscopy (TEM), immunogold EM and electron tomography. By careful analysis of the two-dimensional ultrastructure of LBs from human blood eosinophils under different conditions, we identified membranous structures within LBs in both resting and activated cells. Cyclooxygenase, a membrane inserted protein that catalyzes the first step in prostaglandin synthesis, was localized throughout the internum of LBs. We used fully automated dual-axis electron tomography to study the three-dimensional architecture of LBs in high resolution. By tracking 4 nm-thick serial digital sections we found that leukocyte LBs enclose an intricate system of membranes within their "cores". After computational reconstruction, we showed that these membranes are organized as a network of tubules which resemble the endoplasmic reticulum (ER). Our findings explain how membrane-bound proteins interact and are spatially arranged within LB "cores" and support a model for LB formation by incorporating cytoplasmic membranes of the ER, instead of the conventional view that LBs emerge from the ER leaflets. This is important to understand the functional capabilities of leukocyte LBs in health and during diverse diseases in which these organelles are functionally involved.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0059578PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3608657PMC
September 2013

Eosinophil extracellular DNA trap cell death mediates lytic release of free secretion-competent eosinophil granules in humans.

Blood 2013 Mar 9;121(11):2074-83. Epub 2013 Jan 9.

Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

Eosinophils release their granule proteins extracellularly through exocytosis, piecemeal degranulation, or cytolytic degranulation. Findings in diverse human eosinophilic diseases of intact extracellular eosinophil granules, either free or clustered, indicate that eosinophil cytolysis occurs in vivo, but the mechanisms and consequences of lytic eosinophil degranulation are poorly understood. We demonstrate that activated human eosinophils can undergo extracellular DNA trap cell death (ETosis) that cytolytically releases free eosinophil granules. Eosinophil ETosis (EETosis), in response to immobilized immunoglobulins (IgG, IgA), cytokines with platelet activating factor, calcium ionophore, or phorbol myristate acetate, develops within 120 minutes in a reduced NADP (NADPH) oxidase-dependent manner. Initially, nuclear lobular formation is lost and some granules are released by budding off from the cell as plasma membrane-enveloped clusters. Following nuclear chromatolysis, plasma membrane lysis liberates DNA that forms weblike extracellular DNA nets and releases free intact granules. EETosis-released eosinophil granules, still retaining eosinophil cationic granule proteins, can be activated to secrete when stimulated with CC chemokine ligand 11 (eotaxin-1). Our results indicate that an active NADPH oxidase-dependent mechanism of cytolytic, nonapoptotic eosinophil death initiates nuclear chromatolysis that eventuates in the release of intact secretion-competent granules and the formation of extracellular DNA nets.
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http://dx.doi.org/10.1182/blood-2012-05-432088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3596967PMC
March 2013

LQB-118, an orally active pterocarpanquinone, induces selective oxidative stress and apoptosis in Leishmania amazonensis.

J Antimicrob Chemother 2013 Apr 3;68(4):789-99. Epub 2013 Jan 3.

Laboratório de Bioquímica de Tripanosomatídeos, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil.

Objectives: The pterocarpanquinone LQB-118, previously demonstrated to be effective in vivo via oral delivery, was investigated for its mechanism in selective parasite killing.

Methods: Oxidative stress in Leishmania amazonensis was analysed by evaluating reactive oxygen species (ROS) production (2',7'-dichlorodihydrofluorescein diacetate) and the loss of mitochondrial membrane potential (ΔΨm) using rhodamine, JC-1 and MitoCapture. Ultrastructural analysis was performed using transmission electron microscopy (TEM). DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL).

Results: Treatment with LQB-118 induced ROS production in the promastigotes of L. amazonensis in a concentration-dependent manner for the first 4 h and was sustained for 24 h. TEM analysis revealed several alterations typical of apoptosis. Promastigotes presented a reduction of ΔΨm after 24 h of incubation with 2.5 μM (18.7%), 5 μM (63.7%) or 10 μM (70.7%) LQB-118. A sub-G0/G1 cell cycle phenotype was observed in 21%-83% of the promastigotes incubated with 1.25-10 μM LQB-118. Concentration-dependent DNA fragmentation was observed in promastigotes treated with 2.5-10 μM LQB-118, and selective DNA fragmentation was observed in intracellular amastigotes after 72 h with 2.5 μM treatment.

Conclusions: Our results suggest that LQB-118 selectively induces ROS-triggered and mitochondria-dependent apoptosis in this parasite.
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http://dx.doi.org/10.1093/jac/dks498DOI Listing
April 2013

Lipid body-phagosome interaction in macrophages during infectious diseases: host defense or pathogen survival strategy?

PLoS Pathog 2012 5;8(7):e1002729. Epub 2012 Jul 5.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora (UFJF), Juiz de Fora, Minas Gerais, Brazil.

Phagocytosis of invading microorganisms by specialized cells such as macrophages and neutrophils is a key component of the innate immune response. These cells capture and engulf pathogens and subsequently destroy them in intracellular vacuoles-the phagosomes. Pathogen phagocytosis and progression and maturation of pathogen-containing phagosomes, a crucial event to acquire microbicidal features, occurs in parallel with accentuated formation of lipid-rich organelles, termed lipid bodies (LBs), or lipid droplets. Experimental and clinical infections with different pathogens such as bacteria, parasites, and viruses induce LB accumulation in cells from the immune system. Within these cells, LBs synthesize and store inflammatory mediators and are considered structural markers of inflammation. In addition to LB accumulation, interaction of these organelles with pathogen-containing phagosomes has increasingly been recognized in response to infections and may have implications in the outcome or survival of the microorganism within host cells. In this review, we summarize our current knowledge on the LB-phagosome interaction within cells from the immune system, with emphasis on macrophages, and discuss the functional meaning of this event during infectious diseases.
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http://dx.doi.org/10.1371/journal.ppat.1002729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3390411PMC
January 2013

Lipid bodies: inflammatory organelles implicated in host-Trypanosoma cruzi interplay during innate immune responses.

Mediators Inflamm 2012 30;2012:478601. Epub 2012 Apr 30.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora (UF JF), 36036-900 Juiz de Fora, MG, Brazil.

The flagellated protozoa Trypanosoma cruzi is the causal agent of Chagas' disease, a significant public health issue and still a major cause of morbidity and mortality in Latin America. Acute Chagas' disease elicits a strong inflammatory response. In order to control the parasite multiplication, cells of the monocytic lineage are highly mobilized. Monocyte differentiation leads to the formation of phagocytosing macrophages, which are strongly activated and direct host defense. A distinguishing feature of Chagas' disease-triggered macrophages is the presence of increased numbers of distinct cytoplasmic organelles termed lipid bodies or lipid droplets. These organelles are actively formed in response to the parasite and are sites for synthesis and storage of inflammatory mediators. This review covers current knowledge on lipid bodies elicited by the acute Chagas' disease within inflammatory macrophages and discusses the role of these organelles in inflammation. The increased knowledge of lipid bodies in pathogenic mechanisms of infections may not only contribute to the understanding of pathogen-host interactions but may also identify new targets for intervention.
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http://dx.doi.org/10.1155/2012/478601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3350868PMC
September 2012

CCL11 elicits secretion of RNases from mouse eosinophils and their cell-free granules.

FASEB J 2012 May 31;26(5):2084-93. Epub 2012 Jan 31.

Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.

Rapid secretion of eosinophil-associated RNases (EARs), such as the human eosinophilic cationic protein (ECP), from intracellular granules is central to the role of eosinophils in allergic diseases and host immunity. Our knowledge regarding allergic inflammation has advanced based on mouse experimental models. However, unlike human eosinophils, capacities of mouse eosinophils to secrete granule proteins have been controversial. To study mechanisms of mouse eosinophil secretion and EAR release, we combined an RNase assay of mouse EARs with ultrastructural studies. In vitro, mouse eosinophils stimulated with the chemokine eotaxin-1 (CCL11) secreted enzymatically active EARs (EC(50) 5 nM) by piecemeal degranulation. In vivo, in a mouse model of allergic airway inflammation, increased airway eosinophil infiltration (24-fold) correlated with secretion of active RNases (3-fold). Moreover, we found that eosinophilic inflammation in mice can involve eosinophil cytolysis and release of cell-free granules. Cell-free mouse eosinophil granules expressed functional CCR3 receptors and secreted their granule proteins, including EAR and eosinophil peroxidase in response to CCL11. Collectively, these data demonstrate chemokine-dependent secretion of EARs from both intact mouse eosinophils and their cell-free granules, findings pertinent to understanding the pathogenesis of eosinophil-associated diseases, in which EARs are key factors.
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http://dx.doi.org/10.1096/fj.11-200246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3336791PMC
May 2012

Mice spermatogonial stem cells transplantation induces macrophage migration into the seminiferous epithelium and lipid body formation: high-resolution light microscopy and ultrastructural studies.

Microsc Microanal 2011 Dec 3;17(6):1002-14. Epub 2011 Nov 3.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG, Brazil.

Transplantation of spermatogonial stem cells (SSCs), the male germline stem cells, in experimental animal models has been successfully used to study mechanisms involved in SSC self-renewal and to restore fertility. However, there are still many challenges associated with understanding the recipient immune response for SSCs use in clinical therapies. Here, we have undertaken a detailed structural study of macrophages elicited by SSCs transplantation in mice using both high-resolution light microscopy (HRLM) and transmission electron microscopy (TEM). We demonstrate that SSCs transplantation elicits a rapid and potent recruitment of macrophages into the seminiferous epithelium (SE). Infiltrating macrophages were derived from differentiation of peritubular monocyte-like cells into typical activated macrophages, which actively migrate through the SE, accumulate in the tubule lumen, and direct phagocytosis of differentiating germ cells and spermatozoa. Quantitative TEM analyses revealed increased formation of lipid bodies (LBs), organelles recognized as intracellular platforms for synthesis of inflammatory mediators and key markers of macrophage activation, within both infiltrating macrophages and Sertoli cells. LBs significantly increased in number and size in parallel to the augmented macrophage migration during different times post-transplantation. Our findings suggest that LBs may be involved with immunomodulatory mechanisms regulating the seminiferous tubule niche after SSC transplantation.
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http://dx.doi.org/10.1017/S1431927611012098DOI Listing
December 2011

MHC Class II and CD9 in human eosinophils localize to detergent-resistant membrane microdomains.

Am J Respir Cell Mol Biol 2012 Feb 1;46(2):188-95. Epub 2011 Sep 1.

Department of Medicine, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA.

Eosinophils function in murine allergic airways inflammation as professional antigen-presenting cells (APCs). In murine professional APC cell types, optimal functioning of MHC Class II depends on its lateral association in plasma membranes and colocalization with the tetraspanin CD9 into detergent-resistant membrane microdomains (DRMs). With human eosinophils, we evaluated the localization of MHC Class II (HLA-DR) to DRMs and the functional significance of such localization. In granulocyte-macrophage colony-stimulating factor-stimulated human eosinophils, antibody cross-linked HLA-DR colocalized by immunofluorescence microscopy focally on plasma membranes with CD9 and the DRM marker ganglioside GM1. In addition, HLA-DR coimmunoprecipitates with CD9 after chemical cross-linking of CD9. HLA-DR and CD9 were localized by Western blotting in eosinophil DRM subcellular fractions. DRM disruption with the cholesterol-depleting agent methyl-β-cyclodextrin decreased eosinophil surface expression of HLA-DR and CD9. We show that CD9 is abundant on the surface of eosinophils, presenting the first electron microscopy data of the ultrastructural immunolocalization of CD9 in human eosinophils. Disruption of HLA-DR-containing DRMs decreased the ability of superantigen-loaded human eosinophils to stimulate CD4(+) T-cell activation (CD69 expression), proliferation, and cytokine production. Our results, which demonstrate that eosinophil MHC Class II localizes to DRMs in association with CD9 in a functionally significant manner, represent a novel insight into the organization of the antigen presentation complex of human eosinophils.
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http://dx.doi.org/10.1165/rcmb.2010-0335OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3297164PMC
February 2012

Host cell lipid bodies triggered by Trypanosoma cruzi infection and enhanced by the uptake of apoptotic cells are associated with prostaglandin E₂ generation and increased parasite growth.

J Infect Dis 2011 Sep;204(6):951-61

Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.

Lipid bodies (lipid droplets) are lipid-rich organelles with functions in cell metabolism and signaling. Here, we investigate the mechanisms of Trypanosoma cruzi-induced lipid body formation and their contributions to host-parasite interplay. We demonstrate that T. cruzi-induced lipid body formation in macrophages occurs in a Toll-like receptor 2-dependent mechanism and is potentiated by apoptotic cell uptake. Lipid body biogenesis and prostaglandin E₂ (PGE₂) production triggered by apoptotic cell uptake was largely dependent of α(v)β₃ and transforming growth factor-β signaling. T. cruzi-induced lipid bodies act as sites of increased PGE synthesis. Inhibition of lipid body biogenesis by the fatty acid synthase inhibitor C75 reversed the effects of apoptotic cells on lipid body formation, eicosanoid synthesis, and parasite replication. Our findings indicate that lipid bodies are highly regulated organelles during T. cruzi infection with roles in lipid mediator generation by macrophages and are potentially involved in T. cruzi-triggered escape mechanisms.
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http://dx.doi.org/10.1093/infdis/jir432DOI Listing
September 2011

Lipid bodies in inflammatory cells: structure, function, and current imaging techniques.

J Histochem Cytochem 2011 May 23;59(5):540-56. Epub 2011 Mar 23.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora (UFJF), Juiz de Fora, MG, Brazil.

Lipid bodies (LBs), also known as lipid droplets, have increasingly been recognized as functionally active organelles linked to diverse biological functions and human diseases. These organelles are actively formed in vivo within cells from the immune system, such as macrophages, neutrophils, and eosinophils, in response to different inflammatory conditions and are sites for synthesis and storage of inflammatory mediators. In this review, the authors discuss structural and functional aspects of LBs and current imaging techniques to visualize these organelles in cells engaged in inflammatory processes, including infectious diseases. The dynamic morphological aspects of LBs in leukocytes as inducible, newly formable organelles, elicitable in response to stimuli that lead to cellular activation, contribute to the evolving understanding of LBs as organelles that are critical regulators of different inflammatory diseases, key markers of leukocyte activation, and attractive targets for novel anti-inflammatory therapies.
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http://dx.doi.org/10.1369/0022155411404073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3201176PMC
May 2011

Imaging lipid bodies within leukocytes with different light microscopy techniques.

Methods Mol Biol 2011 ;689:149-61

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil.

Lipid bodies, also known as lipid droplets, are present in most eukaryotic cells. In leukocytes, lipid bodies are functionally active organelles with central roles in inflammation and are considered structural markers of inflammatory cells in a range of diseases. The identification of lipid bodies has methodological limitations because lipid bodies dissipate upon drying or dissolve upon fixation and staining with alcohol-based reagents. Here we discuss several techniques to detect and visualize lipid bodies within leukocytes by light microscopy. These techniques include staining with osmium or use of different fluorescent probes such as Nile red, BODIPY, Oil red, P96 and immunofluorescence labeling for adipose differentiation-related protein (ADRP).
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http://dx.doi.org/10.1007/978-1-60761-950-5_9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3659330PMC
March 2011

Histological approaches to study tissue parasitism during the experimental Trypanosoma cruzi infection.

Methods Mol Biol 2011 ;689:69-80

Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil.

During acute infection with the parasite Trypanosoma cruzi, the causal agent of Chagas' disease, tissue damage is related to intense tissue parasitism. Here we discuss histological approaches for an optimal visualization and quantification of T. cruzi nests in the heart, the main target organ of the parasite. These analyses are important to evaluate the course of the infection in different experimental models and also can be used to investigate parasite colonization and inflammatory processes in other infected tissues and biopsies.
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http://dx.doi.org/10.1007/978-1-60761-950-5_5DOI Listing
March 2011

Rat models to investigate host macrophage defense against Trypanosoma cruzi.

J Innate Immun 2011 3;3(1):71-82. Epub 2010 Nov 3.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, Brazil.

Trypanosoma cruzi is the causal agent of Chagas' disease, an infection with a great impact on public health in Latin America. One of the challenges to understand Chagas' disease lies on the complex host-parasite interaction. The understanding of this interaction requires the use of appropriate experimental models that mimic the human disease. Here, we have used two lineages of rats (Wistar and Holtzman) to comparatively evaluate the course of the acute infection (Y strain). Infection was monitored by parasitemia, cardiac and skeletal muscle parasitism and inflammation, heart ultrastructure, recruitment of monocytes/macrophages and nitric oxide, and arginase production by these cells. Although both rats were able to infect, only Holtzman rats developed a marked infection in the cardiac and skeletal muscles, in parallel to a high recruitment of first-line defense cells. A high number of inflammatory macrophages directed parasite clearance. By the end of the acute phase, Holtzman rats showed consistent disease control. Interestingly, parasite killing was not related to nitric oxide production likely inhibited by an arginase-dependent mechanism. Our work demonstrates differential responses of Holtzman and Wistar rats to T. cruzi, and highlights the use of Holtzman rats as useful models for further studies of cardiac/skeletal muscle tropism and innate immune responses that protect the host against parasite replication. This is important for the development of proper therapeutic interventions.
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http://dx.doi.org/10.1159/000320641DOI Listing
April 2011

Modulation of lipid droplets by Mycobacterium leprae in Schwann cells: a putative mechanism for host lipid acquisition and bacterial survival in phagosomes.

Cell Microbiol 2011 Feb 2;13(2):259-73. Epub 2010 Nov 2.

Laboratório de Microbiologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.

The predilection of Mycobacterium leprae (ML) for Schwann cells (SCs) leads to peripheral neuropathy, a major concern in leprosy. Highly infected SCs in lepromatous leprosy nerves show a foamy, lipid-laden appearance; but the origin and nature of these lipids, as well as their role in leprosy, have remained unclear. The data presented show that ML has a pronounced effect on host-cell lipid homeostasis through regulation of lipid droplet (lipid bodies, LD) biogenesis and intracellular distribution. Electron microscopy and immunohistochemical analysis of lepromatous leprosy nerves for adipose differentiation-related protein expression, a classical LD marker, revealed accumulating LDs in close association to ML in infected SCs. The capacity of ML to induce LD formation was confirmed in in vitro studies with human SCs. Moreover, via confocal and live-cell analysis, it was found that LDs are promptly recruited to bacterial phagosomes and that this process depends on cytoskeletal reorganization and PI3K signalling. ML-induced LD biogenesis and recruitment were found to be independent of TLR2 bacterial sensing. Notably, LD recruitment impairment by cytoskeleton drugs decreased intracellular bacterial survival. Altogether, our data revealed SC lipid accumulation in ML-containing phagosomes, which may represent a fundamental aspect of bacterial pathogenesis in the nerve.
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http://dx.doi.org/10.1111/j.1462-5822.2010.01533.xDOI Listing
February 2011

Contributions of electron microscopy to understand secretion of immune mediators by human eosinophils.

Microsc Microanal 2010 Dec 27;16(6):653-60. Epub 2010 Sep 27.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG, Brazil.

Mechanisms governing secretion of proteins underlie the biologic activities and functions of human eosinophils, leukocytes of the innate immune system, involved in allergic, inflammatory, and immunoregulatory responses. In response to varied stimuli, eosinophils are recruited from the circulation into inflammatory foci, where they modulate immune responses through the release of granule-derived products. Transmission electron microscopy (TEM) is the only technique that can clearly identify and distinguish between different modes of cell secretion. In this review, we highlight the advances in understanding mechanisms of eosinophil secretion, based on TEM findings, that have been made over the past years and that have provided unprecedented insights into the functional capabilities of these cells.
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http://dx.doi.org/10.1017/S1431927610093864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3420811PMC
December 2010
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