quantification-reactive-oxygen-species-using-27-dichlorofluorescein

Quantification of Reactive Oxygen Species Using 2&#8242;,7&#8242;-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in M&#252;ller Glial Cells

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the ...

The development of an effective antitumor immune response to control tumor growth is influenced by the tumor cell itself and/or by the tumor microenvironment. Tumor invasion and tumor cell spreading require a finely tuned regulation of the formation and loosening of adhesive contacts of tumor cells with the extracellular matrix (ECM). In our laboratory, a rat tumor cell line derived from a spontaneous rat sarcoma revealed, by flow cytometry, a high frequency of intercellular adhesion molecule-1 (ICAM-1, 70.1 +/- 8.7%) and urokinase-type plaminogen activator receptor (uPAR, 51.2 +/- 5.2%) positive cells, while a weak expression of MHC class II (IA, 2.2 +/- 0.2% and IE, 17.4 +/- 3.7%) and B7 (12.1 +/- 2.2%) antigens was detected. In our tumor experimental model, after implantation of tumor cells, visible tumor masses were present at days 5-7 with a relatively fast tumor growth until day 15 (progressive phase) followed by a suppression of the tumor growth (regressive phase). Here we present data that correlates a significant decrease in the frequency of ICAM-1 and uPAR expressing tumor cells with the appearance of tumor cells in sites distant from that of the primary tumor. In addition we describe the development of a cellular immune response which controls the tumor progression and is associated with an increase in the expression of major histocompatibility complex (MHC) class II IA antigen during tumor development. The histological examination at tumor progressive and regressive time points revealed the relevant presence of polymorphonuclear neutrophils (PMNs) evidencing colliquative necrosis in tumor growth areas. Taken together, these results support the idea that the balance between adhesive interactions, proteolytic activity and tumorigenicity may lead to a tumor invasive phenotype.

Decreased expression of intercellular adhesion molecule-1 (ICAM-1) and urokinase-type plasminogen activator receptor (uPAR) is associated with tumor cell spreading in vivo.

Tumor cells cannot activate T lymphocytes, since they do not usually express major histocompatibility complex (MHC) class II molecules. Thus, tumor antigens can only be presented indirectly to T cells through professional antigen-presenting cells (APC). In our laboratory, we have treated a tumor cell line (Tu1-A) - derived from an induced rat mammary sarcoma - in order to increase the expression of MHC class I and class II molecules. In our tumor model, the transference of these induced cells into normal rats generated a tumor mass that exhibited a lower tumor growth rate and an earlier regression as compared to those observed in rats inoculated with wild-type Tu1-A cells. This earlier tumor regression was associated with the development of an antigen-specific immune response. 85-87% of the rats in both groups rejected the tumor and were alive at day 60 after tumor cell inoculation. However, in rats treated with wild-type cells the rejection was delayed and took place after tumor ulceration. Rats that had rejected tumors were rechallenged with wild-type cells in order to assay the presence of a long-lived antitumor immunity. All the animals were resistant to the second tumor challenge. We conclude that the development of a specific immune response could be achieved by the superexpression of MHC molecules on tumor cells or when tumor ulceration promotes APC to take up necrotic cells and tumor antigens are presented to T lymphocytes.

Immune control of tumors by antigen presentation improvement.

The low-density lipoprotein receptor-related protein-1 (LRP-1) is an endocytic receptor of activated forms of the proteinase inhibitor alpha(2)-macroglobulin (alpha(2)M*). It has been proposed that alpha(2)M* and LRP-1 modulate diverse cellular processes, including cell adhesion, proliferation, and migration, which are involved in inflammation and tumor progression. However, relatively little is known about the role of alpha(2)M*/LRP-1 interaction on these processes. In this work, we demonstrate that alpha(2)M* binding to LRP-1 induces cell proliferation and MAPK activation in the J774 macrophage-derived cell line, which were blocked by RAP, an antagonist of LRP-1-binding ligands, and by PD980059, a specific inhibitor for the Mek1-ERK1/2 pathway. In addition, we demonstrate that LPS, a bacterial product that it is known to down-regulate the LRP-1 expression on macrophage, abrogated the signaling activity triggered by alpha(2)M* on LPS-treated J774 cells. These results suggest that alpha(2)M*/LRP-1 interaction constitutes a key role in the macrophage functioning during inflammation and cancer.

Activated alpha(2)-macroglobulin induces cell proliferation and mitogen-activated protein kinase activation by LRP-1 in the J774 macrophage-derived cell line.

Macrophages under certain stimuli induce matrix metalloproteinase 9 (MMP-9) expression and protein secretion through the activation of MAPK-ERK and NF-κB signaling pathways. Previously, we demonstrated that activated α(2)-macroglulin (α(2)M*) through the interaction with its receptor low-density lipoprotein receptor-related protein 1 (LRP1) induces macrophage proliferation mediated by the activation of MAPK-ERK1/2. In the present work, we examined whether α(2)M*/LRP1interaction could induce the MMP-9 production in J774 and Raw264.7 macrophage-derived cell lines. It was shown that α(2)M* promoted MMP-9 expression and protein secretion by LRP1 in both macrophage-derived cell lines, which was mediated by the activation of MAPK-ERK1/2 and NF-κB. Both intracellular signaling pathways activated by α(2)M* were effectively blocked by calphostin-C, suggesting involvement of PKC. In addition, we demonstrate that α(2)M* produced extracellular calcium influx via LRP1. However, when the intracellular calcium mobilization was inhibited by BAPTA-AM, the α(2)M*-induced MAPK-ER1/2 activation was fully blocked in both macrophage cell lines. Finally, using specific pharmacological inhibitors for PKC, Mek1, and NF-κB, it was shown that the α(2)M*-induced MMP-9 protein secretion was inhibited, indicating that the MMP production promoted by the α(2)M*/LRP1 interaction required the activation of both signaling pathways. These findings may prove useful in the understanding of the macrophage LRP1 role in the vascular wall during atherogenic plaque progression.

Activated α(2) macroglobulin induces matrix metalloproteinase 9 expression by low-density lipoprotein receptor-related protein 1 through MAPK-ERK1/2 and NF-κB activation in macrophage-derived cell lines.

Nitro-conjugated linoleic acids (NO2-cLA), endogenous nitrodiene lipids which act as inflammatory signaling mediators, were isolated and single isomers purified from the biomimetic acidic nitration products of conjugated linoleic acid (CLA). Structures were elucidated by means of detailed NMR and HPLC-MS/MS spectroscopic analysis and the relative double bond configurations assigned. Additional synthetic methods produced useful quantities and similar isomeric distributions of these unusual and reactive compounds for biological studies and isotopic standards, and the potential conversion of nitro-linoleic to nitro-conjugated linoleic acids was explored via a facile base-catalyzed isomerization. This represents one of the few descriptions of naturally occurring conjugated nitro dienes (in particular, 1-nitro 1,3-diene), an unusual and highly reactive motif with few biological examples extant. 

Biomimetic nitration of conjugated linoleic acid: formation and characterization of naturally occurring conjugated nitrodienes.

Significant efforts are being made to minimize aflatoxin contamination in peanut seeds and one possible strategy is to understand and exploit the mechanisms of plant defense against fungal infection. In this study we have identified and characterized, at biochemical and molecular levels, plant protease inhibitors (PPIs) produced in peanut seeds of the resistant PI 337394 and the susceptible Forman cultivar during Aspergillus parasiticus colonization. With chromatographic methods and 2D-electrophoresis-mass spectrometry we have isolated and identified four variants of Bowman-Birk trypsin inhibitor (BBTI) and a novel Kunitz-type protease inhibitor (KPI) produced in response to A. parasiticus colonization. KPI was detected only in the resistant cultivar, while BBTI was produced in the resistant cultivar in a higher concentration than susceptible cultivar and with different isoforms. The kinetic expression of KPI and BBTI genes along with trypsin inhibitory activity was analyzed in both cultivars during infection. In the susceptible cultivar an early PPI activity response was associated with BBTI occurrence. Meanwhile, in the resistant cultivar a later response with a larger increase in PPI activity was associated with BBTI and KPI occurrence. The biological significance of PPI in seed defense against fungal infection was analyzed and linked to inhibitory properties on enzymes released by the fungus during infection, and to the antifungal effect of KPI.

Peanut Seed Cultivars with Contrasting Resistance to Aspergillus parasiticus Colonization Display Differential Temporal Response of Protease Inhibitors.

Macrophages are the primary immune cells that reside within the myocardium, suggesting that these mononuclear phagocytes are essential in the orchestration of cardiac immunity and homeostasis. Independent of the nature of the injury, the heart triggers leukocyte activation and recruitment. However, inflammation is harmful to this vital terminally differentiated organ with extremely poor regenerative capacity. As such, cardiac tissue has evolved particular strategies to increase the stress tolerance and minimize the impact of inflammation. In this sense, growing evidences show that mononuclear phagocytic cells are particularly dynamic during cardiac inflammation or infection and would actively participate in tissue repair and functional recovery. They respond to soluble mediators such as metabolites or cytokines, which play central roles in the timing of the intrinsic cardiac stress response. During myocardial infarction two distinct phases of monocyte influx have been identified. Upon infarction, the heart modulates its chemokine expression profile that sequentially and actively recruits inflammatory monocytes, first, and healing monocytes, later. In the same way, a sudden switch from inflammatory macrophages (with microbicidal effectors) toward anti-inflammatory macrophages occurs within the myocardium very shortly after infection with , the causal agent of Chagas cardiomyopathy. While in sterile injury, healing response is necessary to stop tissue damage; during an intracellular infection, the anti-inflammatory milieu in infected hearts would promote microbial persistence. The balance of mononuclear phagocytic cells seems to be also dynamic in atherosclerosis influencing plaque initiation and fate. This review summarizes the participation of mononuclear phagocyte system in cardiovascular diseases, keeping in mind that the immune system evolved to promote the reestablishment of tissue homeostasis following infection/injury, and that the effects of different mediators could modulate the magnitude and quality of the immune response. The knowledge of the effects triggered by diverse mediators would serve to identify new therapeutic targets in different cardiovascular pathologies.

New Insights into the Immunobiology of Mononuclear Phagocytic Cells and Their Relevance to the Pathogenesis of Cardiovascular Diseases.

 The cardiovascular disease (CVD) frequently developed during metabolic syndrome and type-2 diabetes mellitus is associated with increased levels of aggregation-prone small LDL particles. Aggregated LDL (aggLDL) internalization is mediated by low-density lipoprotein receptor-related protein-1 (LRP1) promoting intracellular cholesteryl ester (CE) accumulation. Additionally, LRP1 plays a key function in the regulation of insulin receptor (IR) and glucose transporter type 4 (GLUT4) activities. Nevertheless, the link between LRP1, CE accumulation, and insulin response has not been previously studied in cardiomyocytes. We aimed to identify mechanisms through which aggLDL, by its interaction with LRP1, produce CE accumulation and affects the insulin-induced intracellular signaling and GLUT4 trafficking in HL-1 cells. We demonstrated that LRP1 mediates the endocytosis of aggLDL and promotes CE accumulation in these cells. Moreover, aggLDL reduced the molecular association between IR and LRP1 and impaired insulin-induced intracellular signaling activation. Finally, aggLDL affected GLUT4 translocation to the plasma membrane and the 2-NBDG uptake in insulin-stimulated cells. We conclude that LRP1 is a key regulator of the insulin response, which can be altered by CE accumulation through LRP1-mediated aggLDL endocytosis.

LRP1-Mediated AggLDL Endocytosis Promotes Cholesteryl Ester Accumulation and Impairs Insulin Response in HL-1 Cells.

Macrophages play a pivotal role in the early stages of atherosclerosis development; they excessively accumulate cholesterol in the cytosol in response to modified Low Density Lipoprotein (mLDL). The mLDL are incorporated through scavenger receptors. CD36 is a high-affinity cell surface scavenger receptor that facilitates the binding and uptake of long-chain fatty acids and mLDL into the cell. Numerous structurally diverse ligands can initiate signaling responses through CD36 to regulate cell metabolism, migration, and angiogenesis. Nitro-fatty acids are endogenous electrophilic lipid mediators that react with and modulate the function of multiple enzymes and transcriptional regulatory proteins. These actions induce the expression of several anti-inflammatory and cytoprotective genes and limit pathologic responses in experimental models of atherosclerosis, cardiac ischemia/reperfusion, and inflammatory diseases. Pharmacological and genetic approaches were used to explore the actions of nitro-oleic acid (NO-OA) on macrophage lipid metabolism. Pure synthetic NO-OA dose-dependently increased CD36 expression in RAW264.7 macrophages and this up-regulation was abrogated in BMDM from Nrf2-KO mice. Ligand binding analysis revealed that NO-OA specifically interacts with CD36, thus limiting the binding and uptake of mLDL. Docking analysis shows that NO-OA establishes a low binding energy interaction with the alpha helix containing Lys164 in CD36. NO-OA also restored autophagy flux in mLDL-loaded macrophages, thus reversing cholesterol deposition within the cell. In aggregate, these results indicate that NO-OA reduces cholesterol uptake by binding to CD36 and increases cholesterol efflux by restoring autophagy.

Nitro-oleic acid, a ligand of CD36, reduces cholesterol accumulation by modulating oxidized-LDL uptake and cholesterol efflux in RAW264.7 macrophages.

Phytochemical electrophiles are drawing significant attention due to their properties to modulate signaling pathways related to cellular homeostasis. The aim of this study was to develop new tools to examine the electrophilic activity in food and predict their beneficial effects on health. We developed a spectrophotometric assay based on the nitrobenzenethiol (NBT) reactivity, as a thiol-reactive nucleophile, to screen electrophiles in tomato fruits. The method is robust, simple, inexpensive, and could be applied to other types of food. We quantified the electrophile activity in a tomato collection and associated this activity with the pigment composition. Thus, we identified lycopene, β- and γ-carotenes, 16 by-products of carotenoid oxidation and 18 unknown compounds as NBT-reactive by HPLC-MS/MS. The potential benefits of NBT-reactive compounds on health were evaluated in the in vivo model of C. elegans where they activated the SKN-1/Nrf2 pathway, evidencing the ability of electrophilic compounds to induce a biological response.

Assessment and characterization of tomato lipophilic electrophiles and their potential contribution to nutraceutical properties via SKN-1/Nrf2 signaling activation.

Article	Year
Decreased expression of intercellular adhesion molecule-1 (ICAM-1) and urokinase-type plasminogen activator receptor (uPAR) is associated with tumor cell spreading in vivo. Clinical & experimental metastasis\| PubMed ID: 12198772	2002
Immune control of tumors by antigen presentation improvement. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine\| PubMed ID: 15001835	2003
Activated alpha(2)-macroglobulin induces cell proliferation and mitogen-activated protein kinase activation by LRP-1 in the J774 macrophage-derived cell line. Archives of biochemistry and biophysics\| PubMed ID: 17288987	2007
Activated α(2) macroglobulin induces matrix metalloproteinase 9 expression by low-density lipoprotein receptor-related protein 1 through MAPK-ERK1/2 and NF-κB activation in macrophage-derive... Journal of cellular biochemistry\| PubMed ID: 20568116	2010
Biomimetic nitration of conjugated linoleic acid: formation and characterization of naturally occurring conjugated nitrodienes. The Journal of organic chemistry\| PubMed ID: 24350701	2014
Peanut Seed Cultivars with Contrasting Resistance to Aspergillus parasiticus Colonization Display Differential Temporal Response of Protease Inhibitors. Phytopathology\| PubMed ID: 27841959	2017
New Insights into the Immunobiology of Mononuclear Phagocytic Cells and Their Relevance to the Pathogenesis of Cardiovascular Diseases. Frontiers in immunology\| PubMed ID: 29375564	2017
LRP1-Mediated AggLDL Endocytosis Promotes Cholesteryl Ester Accumulation and Impairs Insulin Response in HL-1 Cells. Cells\| PubMed ID: 31936892	2020
Nitro-oleic acid, a ligand of CD36, reduces cholesterol accumulation by modulating oxidized-LDL uptake and cholesterol efflux in RAW264.7 macrophages. Redox biology\| PubMed ID: 32531545	2020
Assessment and characterization of tomato lipophilic electrophiles and their potential contribution to nutraceutical properties via SKN-1/Nrf2 signaling activation. Food chemistry\| PubMed ID: 34284182	2022

Gustavo Bonacci

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