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1.
Med ; 5(7): 759-779.e7, 2024 Jul 12.
Article in English | MEDLINE | ID: mdl-38593812

ABSTRACT

BACKGROUND: The treatment of melanoma, the deadliest form of skin cancer, has greatly benefited from immunotherapy. However, many patients do not show a durable response, which is only partially explained by known resistance mechanisms. METHODS: We performed single-cell RNA sequencing of tumor immune infiltrates and matched peripheral blood mononuclear cells of 22 checkpoint inhibitor (CPI)-naive stage III-IV metastatic melanoma patients. After sample collection, the same patients received CPI treatment, and their response was assessed. FINDINGS: CPI responders showed high levels of classical monocytes in peripheral blood, which preferentially transitioned toward CXCL9-expressing macrophages in tumors. Trajectories of tumor-infiltrating CD8+ T cells diverged at the level of effector memory/stem-like T cells, with non-responder cells progressing into a state characterized by cellular stress and apoptosis-related gene expression. Consistently, predicted non-responder-enriched myeloid-T/natural killer cell interactions were primarily immunosuppressive, while responder-enriched interactions were supportive of T cell priming and effector function. CONCLUSIONS: Our study illustrates that the tumor immune microenvironment prior to CPI treatment can be indicative of response. In perspective, modulating the myeloid and/or effector cell compartment by altering the described cell interactions and transitions could improve immunotherapy response. FUNDING: This research was funded by Roche Pharma Research and Early Development.


Subject(s)
Immune Checkpoint Inhibitors , Melanoma , Skin Neoplasms , Tumor Microenvironment , Humans , Melanoma/drug therapy , Melanoma/immunology , Melanoma/pathology , Immune Checkpoint Inhibitors/therapeutic use , Immune Checkpoint Inhibitors/pharmacology , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunology , Skin Neoplasms/immunology , Skin Neoplasms/drug therapy , Skin Neoplasms/pathology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/metabolism , Male , Female , Myeloid Cells/immunology , Myeloid Cells/drug effects , Myeloid Cells/metabolism , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/metabolism , Middle Aged , Killer Cells, Natural/immunology , Killer Cells, Natural/drug effects
2.
Clin Cancer Res ; 28(4): 770-780, 2022 02 15.
Article in English | MEDLINE | ID: mdl-34782366

ABSTRACT

PURPOSE: Disease progression in BRAF V600E/K positive melanomas to approved BRAF/MEK inhibitor therapies is associated with the development of resistance mediated by RAF dimer inducing mechanisms. Moreover, progressing disease after BRAFi/MEKi frequently involves brain metastasis. Here we present the development of a novel BRAF inhibitor (Compound Ia) designed to address the limitations of available BRAFi/MEKi. EXPERIMENTAL DESIGN: The novel, brain penetrant, paradox breaker BRAFi is comprehensively characterized in vitro, ex vivo, and in several preclinical in vivo models of melanoma mimicking peripheral disease, brain metastatic disease, and acquired resistance to first-generation BRAFi. RESULTS: Compound Ia manifested elevated potency and selectivity, which triggered cytotoxic activity restricted to BRAF-mutated models and did not induce RAF paradoxical activation. In comparison to approved BRAFi at clinical relevant doses, this novel agent showed a substantially improved activity in a number of diverse BRAF V600E models. In addition, as a single agent, it outperformed a currently approved BRAFi/MEKi combination in a model of acquired resistance to clinically available BRAFi. Compound Ia presents high central nervous system (CNS) penetration and triggered evident superiority over approved BRAFi in a macro-metastatic and in a disseminated micro-metastatic brain model. Potent inhibition of MAPK by Compound Ia was also demonstrated in patient-derived tumor samples. CONCLUSIONS: The novel BRAFi demonstrates preclinically the potential to outperform available targeted therapies for the treatment of BRAF-mutant tumors, thus supporting its clinical investigation.


Subject(s)
Melanoma , Proto-Oncogene Proteins B-raf , Brain/pathology , Cell Line, Tumor , Drug Resistance, Neoplasm , Humans , Melanoma/drug therapy , Melanoma/genetics , Melanoma/pathology , Molecular Targeted Therapy , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use
3.
PLoS Comput Biol ; 17(9): e1009426, 2021 09.
Article in English | MEDLINE | ID: mdl-34529654

ABSTRACT

Bee-mediated pollination greatly increases the size and weight of tomato fruits. Therefore, distinguishing between the local set of bees-those that are efficient pollinators-is essential to improve the economic returns for farmers. To achieve this, it is important to know the identity of the visiting bees. Nevertheless, the traditional taxonomic identification of bees is not an easy task, requiring the participation of experts and the use of specialized equipment. Due to these limitations, the development and implementation of new technologies for the automatic recognition of bees become relevant. Hence, we aim to verify the capacity of Machine Learning (ML) algorithms in recognizing the taxonomic identity of visiting bees to tomato flowers based on the characteristics of their buzzing sounds. We compared the performance of the ML algorithms combined with the Mel Frequency Cepstral Coefficients (MFCC) and with classifications based solely on the fundamental frequency, leading to a direct comparison between the two approaches. In fact, some classifiers powered by the MFCC-especially the SVM-achieved better performance compared to the randomized and sound frequency-based trials. Moreover, the buzzing sounds produced during sonication were more relevant for the taxonomic recognition of bee species than analysis based on flight sounds alone. On the other hand, the ML classifiers performed better in recognizing bees genera based on flight sounds. Despite that, the maximum accuracy obtained here (73.39% by SVM) is still low compared to ML standards. Further studies analyzing larger recording samples, and applying unsupervised learning systems may yield better classification performance. Therefore, ML techniques could be used to automate the taxonomic recognition of flower-visiting bees of the cultivated tomato and other buzz-pollinated crops. This would be an interesting option for farmers and other professionals who have no experience in bee taxonomy but are interested in improving crop yields by increasing pollination.


Subject(s)
Bees/classification , Bees/physiology , Machine Learning , Pollination/physiology , Solanum lycopersicum/growth & development , Acoustics , Algorithms , Animals , Computational Biology , Crops, Agricultural/growth & development , Flowers/physiology , Solanum lycopersicum/physiology
4.
Nat Commun ; 11(1): 123, 2020 01 08.
Article in English | MEDLINE | ID: mdl-31913317

ABSTRACT

Induction of DNA double-strand breaks (DSBs) in ribosomal DNA (rDNA) repeats is associated with ATM-dependent repression of ribosomal RNA synthesis and large-scale reorganization of nucleolar architecture, but the signaling events that regulate these responses are largely elusive. Here we show that the nucleolar response to rDNA breaks is dependent on both ATM and ATR activity. We further demonstrate that ATM- and NBS1-dependent recruitment of TOPBP1 in the nucleoli is required for inhibition of ribosomal RNA synthesis and nucleolar segregation in response to rDNA breaks. Mechanistically, TOPBP1 recruitment is mediated by phosphorylation-dependent interactions between three of its BRCT domains and conserved phosphorylated Ser/Thr residues at the C-terminus of the nucleolar phosphoprotein Treacle. Our data thus reveal an important cooperation between TOPBP1 and Treacle in the signaling cascade that triggers transcriptional inhibition and nucleolar segregation in response to rDNA breaks.


Subject(s)
Ataxia Telangiectasia Mutated Proteins/metabolism , Carrier Proteins/metabolism , Cell Nucleolus/genetics , DNA, Ribosomal/genetics , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Amino Acid Motifs , Ataxia Telangiectasia Mutated Proteins/genetics , Carrier Proteins/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Nucleolus/metabolism , DNA Breaks, Double-Stranded , DNA, Ribosomal/metabolism , DNA-Binding Proteins/genetics , Humans , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Phosphoproteins/chemistry , Phosphoproteins/genetics , Protein Binding , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism
5.
Crit Rev Biochem Mol Biol ; 51(1): 7-14, 2016.
Article in English | MEDLINE | ID: mdl-26585314

ABSTRACT

Macrophages are multi-faceted phagocytic effector cells that derive from circulating monocytes and undergo differentiation in target tissues to regulate key aspects of the inflammatory process. Macrophages produce and degrade a variety of lipid mediators that stimulate or suppress pain and inflammation. Among the analgesic and anti-inflammatory lipids released from these cells are the fatty acid ethanolamides (FAEs), which produce their effects by engaging nuclear peroxisome proliferator activated receptor-α (PPAR-α). Two members of this lipid family, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), have recently emerged as important intrinsic regulators of nociception and inflammation. These substances are released from the membrane precursor, N-acylphosphatidylethanolamine (NAPE), by the action of a NAPE-specific phospholipase D (NAPE-PLD), and in macrophage are primarily deactivated by the lysosomal cysteine amidase, N-acylethanolamine acid amidase (NAAA). NAPE-PLD and NAAA regulate FAE levels, exerting a tight control over the ability of these lipid mediators to recruit PPAR-α and attenuate the inflammatory response. This review summarizes recent findings on the contribution of the FAE-PPAR-α signaling complex in inflammation, and on NAAA inhibition as a novel mechanistic approach to treat chronic inflammatory disorders.


Subject(s)
Inflammation/prevention & control , Lipids/pharmacology , Macrophages/metabolism , PPAR alpha/agonists , Animals , Humans , Lipids/physiology
6.
ACS Chem Biol ; 10(8): 1838-46, 2015 Aug 21.
Article in English | MEDLINE | ID: mdl-25874594

ABSTRACT

Fatty acid ethanolamides such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are lipid-derived mediators that potently inhibit pain and inflammation by ligating type-α peroxisome proliferator-activated receptors (PPAR-α). These bioactive substances are preferentially degraded by the cysteine hydrolase, N-acylethanolamine acid amidase (NAAA), which is highly expressed in macrophages. Here, we describe a new class of ß-lactam derivatives that are potent, selective, and systemically active inhibitors of intracellular NAAA activity. The prototype of this class deactivates NAAA by covalently binding the enzyme's catalytic cysteine and exerts profound anti-inflammatory effects in both mouse models and human macrophages. This agent may be used to probe the functions of NAAA in health and disease and as a starting point to discover better anti-inflammatory drugs.


Subject(s)
Amidohydrolases/antagonists & inhibitors , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Inflammation/drug therapy , Macrophage Activation/drug effects , beta-Lactams/chemistry , beta-Lactams/pharmacology , Amidohydrolases/immunology , Animals , Anti-Inflammatory Agents/therapeutic use , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Humans , Inflammation/enzymology , Inflammation/immunology , Macrophages/drug effects , Macrophages/enzymology , Macrophages/immunology , Male , Mice, Inbred C57BL , beta-Lactams/therapeutic use
7.
Angew Chem Int Ed Engl ; 54(2): 485-9, 2015 Jan 07.
Article in English | MEDLINE | ID: mdl-25395373

ABSTRACT

The ceramides are a family of bioactive lipid-derived messengers involved in the control of cellular senescence, inflammation, and apoptosis. Ceramide hydrolysis by acid ceramidase (AC) stops the biological activity of these substances and influences survival and function of normal and neoplastic cells. Because of its central role in the ceramide metabolism, AC may offer a novel molecular target in disorders with dysfunctional ceramide-mediated signaling. Here, a class of benzoxazolone carboxamides is identified as the first potent and systemically active inhibitors of AC. Prototype members of this class inhibit AC with low nanomolar potency by covalent binding to the catalytic cysteine. Their metabolic stability and high in vivo efficacy suggest that these compounds may be used as probes to investigate the roles of ceramide in health and disease, and that this scaffold may represent a promising starting point for the development of novel therapeutic agents.


Subject(s)
Amides/chemistry , Benzoxazoles/chemistry , Ceramidases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology
8.
ChemMedChem ; 10(2): 380-95, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25338703

ABSTRACT

Inhibition of fatty acid amide hydrolase (FAAH) activity is under investigation as a valuable strategy for the treatment of several disorders, including pain and drug addiction. A number of potent FAAH inhibitors belonging to different chemical classes have been disclosed to date; O-aryl carbamates are one of the most representative families. In the search for novel FAAH inhibitors, a series of O-(1,2,3-triazol-4-yl)methyl carbamate derivatives were designed and synthesized exploiting a copper- catalyzed [3+2] cycloaddition reaction between azides and alkynes (click chemistry). Exploration of the structure-activity relationships within this new class of compounds identified potent inhibitors of both rat and human FAAH with IC50 values in the single-digit nanomolar range. In addition, these derivatives showed improved stability in rat plasma and kinetic solubility in buffer with respect to the lead compound. Based on the results of the study, the novel analogues identified can be considered to be promising starting point for the development of new FAAH inhibitors with improved drug-like properties.


Subject(s)
Amidohydrolases/antagonists & inhibitors , Carbamates/chemistry , Enzyme Inhibitors/chemistry , Amidohydrolases/genetics , Amidohydrolases/metabolism , Animals , Carbamates/chemical synthesis , Carbamates/pharmacokinetics , Catalysis , Click Chemistry , Copper/chemistry , Cycloaddition Reaction , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacokinetics , HEK293 Cells , Half-Life , Humans , Kinetics , Male , Mice , Microsomes, Liver/metabolism , Protein Binding , Rats , Rats, Sprague-Dawley , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Structure-Activity Relationship
9.
PLoS One ; 8(10): e77706, 2013.
Article in English | MEDLINE | ID: mdl-24204926

ABSTRACT

Endocannabinoid signaling is terminated by enzymatic hydrolysis, a process that, for 2-Arachidonoylglycerol (2-AG), is mediated by monoacylglycerol lipase (MAGL). The piperidine carbamate, 4-nitrophenyl- 4-(dibenzo[d] [1,3]dioxol-5-yl (hydroxy) methyl) piperidine- 1-carboxylate (JZL184), is a drug that inhibits MAGL and presents high potency and selectivity. Thus, JZL184 increases the levels of 2-AG, an endocannabinoid that acts on the CB1 and CB2 cannabinoid receptors. Here, we investigated the effects of MAGL inhibition, with a single dose (16 mg/kg, intraperitoneally (i.p.)) of JZL184, in a murine model of lipopolysaccharide (LPS) -induced acute lung injury (ALI) 6, 24 and 48 hours after the inflammatory insult. Treatment with JZL184 decreased the leukocyte migration into the lungs as well as the vascular permeability measured through the bronchoalveolar lavage fluid (BAL) and histological analysis. JZL184 also reduced the cytokine and chemokine levels in the BAL and adhesion molecule expression in the blood and BAL. The CB1 and CB2 receptors were considered involved in the anti-inflammatory effects of JZL184 because the AM281 selective CB1 receptor antagonist (1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide) and the AM630 selective CB2 receptor antagonist ([6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)-methanone) blocked the anti-inflammatory effects previously described for JZL184. It was concluded that MAGL inhibition, and consequently the increase in 2-AG levels, produced anti-inflammatory effects in a murine model of LPS-induced ALI, a finding that was considered a consequence of the activation of the CB1 and CB2 receptors.


Subject(s)
Acute Lung Injury/drug therapy , Acute Lung Injury/metabolism , Monoacylglycerol Lipases/antagonists & inhibitors , Monoacylglycerol Lipases/metabolism , Animals , Arachidonic Acids/metabolism , Benzodioxoles/pharmacology , Bronchoalveolar Lavage Fluid , Capillary Permeability/drug effects , Cell Adhesion Molecules/drug effects , Cell Adhesion Molecules/metabolism , Endocannabinoids/metabolism , Glycerides/metabolism , Indoles , Inflammation/drug therapy , Inflammation/metabolism , Leukocytes/drug effects , Leukocytes/metabolism , Male , Mice , Mice, Inbred C57BL , Piperidines/pharmacology , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/antagonists & inhibitors , Receptor, Cannabinoid, CB2/metabolism
10.
Int Immunopharmacol ; 17(3): 633-7, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23994465

ABSTRACT

Early life experiences, particularly during the gestational period, are homeostatic determinants for an individual's brain development. However, recent data suggest that the immune response of the offspring is also affected by events during the gestational period. Here, we evaluated the impact of prenatal immune activation on the innate and adaptive immune responses of adult offspring. Pregnant Swiss mice received saline or lipopolysaccharide (LPS) on gestational day 17. In adulthood, male offspring were analyzed using 2 experimental techniques: in vitro analysis of cytokine production and immune cell activity and development of the delayed-type hypersensitivity (DTH) responses of ovalbumin-sensitized mice. We analyzed Th1/Th2/Th17 cytokine production in vitro, neutrophil and dendritic cell function, and the DTH response. Offspring from LPS-treated dams displayed increased cell-mediated immunity as indicated by increased IL-12 production by cultured antigen-presenting cells and an enhanced DTH response as well as impaired production of the regulatory cytokine IL-10. This study provides new insights regarding the influence of immune activation during late gestation on the immunological homeostasis of offspring, particularly on Th1 immunity.


Subject(s)
Hypersensitivity, Delayed/immunology , Immunity, Cellular , Pregnancy/immunology , Prenatal Exposure Delayed Effects , Allergens/immunology , Animals , Cells, Cultured , Cytokines/immunology , Dendritic Cells , Female , Lipopolysaccharides/immunology , Male , Mice , Neutrophils/immunology , Ovalbumin/immunology , Spleen/cytology
11.
Int Immunopharmacol ; 16(4): 436-43, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23644142

ABSTRACT

Glutamate acts as a neurotransmitter within the Central Nervous System (CNS) and modifies immune cell activity. In lymphocytes, NMDA glutamate receptors regulate intracellular calcium, the production of reactive oxygen species and cytokine synthesis. MK-801, a NMDA receptor open-channel blocker, inhibits calcium entry into mast cells, thereby preventing mast cell degranulation. Several lines of evidence have shown the involvement of NMDA glutamate receptors in amphetamine (AMPH)-induced effects. AMPH treatment has been reported to modify allergic lung inflammation. This study evaluated the effects of MK-801 (0.25mg/kg) and AMPH (2.0mg/kg), given alone or in combination, on allergic lung inflammation in mice and the possible involvement of NMDA receptors in this process. In OVA-sensitized and challenged mice, AMPH and MK-801 given alone decreased cellular migration into the lung, reduced IL-13 and IL10 levels in BAL supernatant, reduced ICAM-1 and L-selectin expression in granulocytes in the BAL and decreased mast cell degranulation. AMPH treatment also decreased IL-5 levels. When both drugs were administered, treatment with MK-801 reversed the decrease in the number of eosinophils and neutrophils induced by AMPH in the BAL of OVA-sensitized and challenged mice as well as the effects on the expression of L-selectin and ICAM-1 in granulocytes, the IL-10, IL-5 and IL-13 levels in BAL supernatants and increased mast cell degranulation. At the same time, treatment with MK-801, AMPH or with MK-801+AMPH increased corticosterone serum levels in allergic mice. These results are discussed in light of possible indirect effects of AMPH and MK-801 via endocrine outflow from the CNS (i.e., HPA-axis activity) to the periphery and/or as a consequence of the direct action of these drugs on immune cell activity, with emphasis given to mast cell participation in the allergic lung response of mice.


Subject(s)
Amphetamine/therapeutic use , Dizocilpine Maleate/pharmacology , Pneumonia/prevention & control , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Respiratory Hypersensitivity/prevention & control , Amphetamine/administration & dosage , Amphetamine/pharmacology , Animals , Bone Marrow Cells/drug effects , Bone Marrow Cells/immunology , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , Cell Degranulation/drug effects , Cell Degranulation/immunology , Chemotaxis, Leukocyte/immunology , Corticosterone/blood , Corticosterone/immunology , Disease Models, Animal , Dizocilpine Maleate/administration & dosage , Interleukins/immunology , Leukocyte Count , Male , Mast Cells/drug effects , Mast Cells/immunology , Mast Cells/physiology , Mice , Mice, Inbred BALB C , Ovalbumin/immunology , Pneumonia/immunology , Pneumonia/metabolism , Respiratory Hypersensitivity/immunology , Respiratory Hypersensitivity/metabolism
12.
Neuroimmunomodulation ; 19(4): 209-19, 2012.
Article in English | MEDLINE | ID: mdl-22441537

ABSTRACT

OBJECTIVE: 3,4-Methylenedioxymethamphetamine (MDMA), or ecstasy, is a synthetic drug used recreationally, mainly by young people. It has been suggested that MDMA has a Th cell skewing effect, in which Th1 cell activity is suppressed and Th2 cell activity is increased. Experimental allergic airway inflammation in ovalbumin (OVA)-sensitized rodents is a useful model to study Th2 response; therefore, based on the Th2 skewing effect of MDMA, we studied MDMA in a model of allergic lung inflammation in OVA-sensitized mice. METHODS: We evaluated cell trafficking in the bronchoalveolar lavage fluid, blood and bone marrow; cytokine production; L-selectin expression and lung histology. We also investigated the effects of MDMA on tracheal reactivity in vitro and mast cell degranulation. RESULTS: We found that MDMA given prior to OVA challenge in OVA-sensitized mice decreased leukocyte migration into the lung, as revealed by a lower cell count in the bronchoalveolar lavage fluid and lung histologic analysis. We also showed that MDMA decreased expression of both Th2-like cytokines (IL-4, IL-5 and IL-10) and adhesion molecules (L-selectin). Moreover, we showed that the hypothalamus-pituitary-adrenal axis is partially involved in the MDMA-induced reduction in leukocyte migration into the lung. Finally, we showed that MDMA decreased tracheal reactivity to methacholine as well as mast cell degranulation in situ. CONCLUSIONS: Thus, we report here that MDMA given prior to OVA challenge in OVA-sensitized allergic mice is able to decrease lung inflammation and airway reactivity and that hypothalamus-pituitary-adrenal axis activation is partially involved. Together, the data strongly suggest an involvement of a neuroimmune mechanism in the effects of MDMA on lung inflammatory response and cell recruitment to the lungs of allergic animals.


Subject(s)
Asthma/immunology , Inflammation/immunology , Leukocytes/drug effects , Lung/drug effects , N-Methyl-3,4-methylenedioxyamphetamine/pharmacology , Th2 Cells/drug effects , Animals , Bone Marrow Cells , Bronchoalveolar Lavage Fluid/cytology , Cell Movement/drug effects , Cytokines/drug effects , Cytokines/metabolism , Disease Models, Animal , Leukocyte Count , Lung/cytology , Male , Mast Cells/drug effects , Mice , N-Methyl-3,4-methylenedioxyamphetamine/immunology , Th2 Cells/physiology , Trachea/drug effects
13.
Eur J Pharmacol ; 678(1-3): 78-85, 2012 Mar 05.
Article in English | MEDLINE | ID: mdl-22265864

ABSTRACT

Acute lung injury is an inflammatory condition for which treatment is mainly supportive because effective therapies have not been developed. Cannabidiol, a non-psychotropic cannabinoid component of marijuana (Cannabis sativa), has potent immunosuppressive and anti-inflammatory properties. Therefore, we investigated the possible anti-inflammatory effect of cannabidiol in a murine model of acute lung injury. Analysis of total inflammatory cells and differential in bronchoalveolar lavage fluid was used to characterize leukocyte migration into the lungs; myeloperoxidase activity of lung tissue and albumin concentration in the bronchoalveolar lavage fluid were analyzed by colorimetric assays; cytokine/chemokine production in the bronchoalveolar lavage fluid was also analyzed by Cytometric Bead Arrays and Enzyme-Linked Immunosorbent Assay (ELISA). A single dose of cannabidiol (20mg/kg) administered prior to the induction of LPS (lipopolysaccharide)-induced acute lung injury decreases leukocyte (specifically neutrophil) migration into the lungs, albumin concentration in the bronchoalveolar lavage fluid, myeloperoxidase activity in the lung tissue, and production of pro-inflammatory cytokines (TNF and IL-6) and chemokines (MCP-1 and MIP-2) 1, 2, and 4days after the induction of LPS-induced acute lung injury. Additionally, adenosine A(2A) receptor is involved in the anti-inflammatory effects of cannabidiol on LPS-induced acute lung injury because ZM241385 (4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol) (a highly selective antagonist of adenosine A(2A) receptor) abrogated all of the anti-inflammatory effects of cannabidiol previously described. Thus, we show that cannabidiol has anti-inflammatory effects in a murine model of acute lung injury and that this effect is most likely associated with an increase in the extracellular adenosine offer and signaling through adenosine A(2A) receptor.


Subject(s)
Acute Lung Injury/drug therapy , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Cannabidiol/pharmacology , Cannabinoids/pharmacology , Receptor, Adenosine A2A/physiology , Acute Lung Injury/metabolism , Adenosine A2 Receptor Antagonists/pharmacology , Animals , Bone Marrow Cells/drug effects , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , Cannabidiol/antagonists & inhibitors , Cannabidiol/therapeutic use , Cannabinoids/therapeutic use , Capillary Permeability/drug effects , Chemokines/metabolism , Cytokines/metabolism , Disease Models, Animal , Drug Evaluation, Preclinical/methods , Leukocytes/drug effects , Male , Mice , Mice, Inbred C57BL , Peroxidase/metabolism , Triazines/pharmacology , Triazoles/pharmacology
14.
Brain Behav Immun ; 26(1): 32-9, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21787859

ABSTRACT

Cohabitation for 14 days with Ehrlich tumor-bearing mice was shown to increase locomotor activity, to decrease hypothalamic noradrenaline (NA) levels, to increase NA turnover and to decrease innate immune responses and decrease the animals' resistance to tumor growth. Cage mates of a B16F10 melanoma-bearer mice were also reported to show neuroimmune changes. Chemosignals released by Ehrlich tumor-bearing mice have been reported to be relevant for the neutrophil activity changes induced by cohabitation. The present experiment was designed to further analyze the effects of odor cues on neuroimmune changes induced by cohabitation with a sick cage mate. Specifically, the relevance of chemosignals released by an Ehrlich tumor-bearing mouse was assessed on the following: behavior (open-field and plus maze); hypothalamic NA levels and turnover; adrenaline (A) and NA plasmatic levels; and host resistance induced by tumor growth. To comply with such objectives, devices specifically constructed to analyze the influence of chemosignals released from tumor-bearing mice were employed. The results show that deprivation of odor cues released by Ehrlich tumor-bearing mice reversed the behavioral, neurochemical and immune changes induced by cohabitation. Mice use scents for intraspecies communication in many social contexts. Tumors produce volatile organic compounds released into the atmosphere through breath, sweat, and urine. Our results strongly suggest that volatile compounds released by Ehrlich tumor-injected mice are perceived by their conspecifics, inducing the neuroimmune changes reported for cohabitation with a sick companion.


Subject(s)
Carcinoma, Ehrlich Tumor/immunology , Carcinoma, Ehrlich Tumor/psychology , Cues , Neuroimmunomodulation/physiology , Smell/physiology , Animals , Anxiety/psychology , Behavior, Animal/physiology , Carcinoma, Ehrlich Tumor/pathology , Epinephrine/blood , Epinephrine/metabolism , Female , Hypothalamus/metabolism , Illness Behavior/physiology , Mice , Motor Activity/physiology , Neutrophil Activation/physiology , Norepinephrine/blood , Norepinephrine/metabolism , Odorants , Social Behavior
15.
Neuroimmunomodulation ; 18(3): 184-90, 2011.
Article in English | MEDLINE | ID: mdl-21311204

ABSTRACT

OBJECTIVES: In this work, we searched for maternal separation effects on serum corticosterone levels and blood neutrophil activity in adult male A/J and C57BL/6 mouse offspring. METHODS: 40 male A/J mice and 40 male C57BL/6 mice were divided within each strain into two groups. Mice in the maternal separation group were separated from their mothers (1 h/day) on postnatal days 0-13. Mice in the control group were left undisturbed. On postnatal day 45, blood was drawn from all mice and used to assess neutrophil activity by flow cytometry and serum corticosterone levels by radioimmunoassay. RESULTS: The results showed that each mouse strain responded differently to maternal separation, but in both cases, serum corticosterone levels were affected. In both strains, adult mice that experienced maternal separation showed lower serum corticosterone levels than control mice. In relation to control mice kept together with their mothers, the levels of serum corticosterone were 72.7 and 36.36% lower in A/J and C57BL/6 mice submitted to maternal separation, respectively. The current findings showed that maternal separation increased neutrophil activity in mice after reaching adulthood. The observed effects, although in the same direction, differed between A/J and C57BL/6 mice. Maternal separation increased both the percentage and intensity of phagocytosis in C57BL/6 mice, but had no effects on A/J mice. Furthermore, maternal separation increased basal and propidium iodide-labeled Staphylococcus aureus-induced oxidative burst in A/J mice but did not affect oxidative burst in C57BL/6 mice. Finally, phorbol myristate acetate-induced oxidative burst increased in both strains. CONCLUSION: These results indicate that early maternal separation increases innate immunity, most likely by modifying hypothalamus-pituitary-adrenal axis activity. This suggests that maternal separation is a good model for stress which produces long-term neuroimmune changes whatever the animal species and strain used.


Subject(s)
Corticosterone/blood , Immune Tolerance/immunology , Maternal Deprivation , Neuroimmunomodulation/immunology , Neutrophils/immunology , Stress, Psychological/blood , Stress, Psychological/pathology , Animals , Chronic Disease , Disease Models, Animal , Female , Male , Mice , Mice, Inbred A , Mice, Inbred C57BL , Neutrophils/metabolism , Neutrophils/pathology , Species Specificity , Stress, Psychological/immunology , Time
16.
Int Immunopharmacol ; 10(4): 431-9, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20093200

ABSTRACT

The endocannabinoid system has become a topic of great interest in pharmacology due to its remarkable distribution in mammal organisms and capacity to play a modulatory role on several physiological systems, including modulation of immunity. Many studies have shown that administration of cannabinoids causes inhibitory effects on immune cells, including decreased proliferation and antigen-presenting cell (APC) co-stimulatory activity. In contrast, other groups have shown that some cannabinoids might present stimulatory actions on macrophage activity and T cell activation. Therefore, we aimed to investigate whether a treatment in vivo with a low dose of anandamide (0.1mg/kg) immediately prior to sensitization would have an immunosuppressive or immunostimulatory effect on cell-mediated immunity (Th1 response) in mice. We report here that anandamide, prior to sensitization, was able to increase the Th1 response to ovalbumin in vivo and ex vivo. Anandamide increased delayed type hypersensitivity (DTH), splenocyte proliferation, and IFN-gamma production in a co-culture of adherent and non-adherent splenocytes. Moreover, anandamide prior to sensitization increased both the expression of DC co-stimulatory molecules (CD80/CD86) and IL-12/IL23 (p40) production ex vivo. We have also assessed direct effects of anandamide in the IFN-gamma/IL-4 balance of ConA-stimulated splenocytes in vitro. Anandamide at nanomolar concentrations increased the production of IFN-gamma, while such production decreased at micromolar range. Thus, anandamide induced both the increment of DC activation and IFN-gamma production, which are likely the mechanisms involved in the increase of Th1 response reported here.


Subject(s)
Adjuvants, Immunologic/pharmacology , Arachidonic Acids/pharmacology , Immunity, Cellular/drug effects , Polyunsaturated Alkamides/pharmacology , Animals , Anxiety/psychology , Cell Proliferation/drug effects , Corticosterone/blood , Cytokines/biosynthesis , Dendritic Cells/drug effects , Dendritic Cells/immunology , Endocannabinoids , Enzyme-Linked Immunosorbent Assay , Exploratory Behavior/drug effects , Hypersensitivity, Delayed/immunology , Hypothalamo-Hypophyseal System/drug effects , Interferon-gamma/biosynthesis , Interleukin-4/biosynthesis , Macrophages/drug effects , Macrophages/immunology , Male , Mice , Mice, Inbred C57BL , Motor Activity/drug effects , Th1 Cells/drug effects , Th1 Cells/immunology
17.
Neuroimmunomodulation ; 16(3): 191-200, 2009.
Article in English | MEDLINE | ID: mdl-19246942

ABSTRACT

OBJECTIVE: Looking for possible neuroimmune relationships, we analyzed the effects of methylenedioxymethamphetamine (MDMA) administration on neuroendocrine, neutrophil activity and leukocyte distribution in mice. METHODS: Five experiments were performed. In the first, mice were treated with MDMA (10 mg/kg) 30, 60 min and 24 h prior to blood sample collection for neutrophil activity analysis. In the second experiment, the blood of naïve mice was collected and incubated with MDMA for neutrophil activity in vitro analysis. In the third and fourth experiments, mice were injected with MDMA (10 mg/kg) and 60 min later, blood and brain were collected to analyze corticosterone serum levels and hypothalamic noradrenaline (NA) levels and turnover. In the last experiment, mice were injected with MDMA 10 mg/kg and 60 min later, blood, bone marrow and spleen were collected for leukocyte distribution analysis. RESULTS: Results showed an increase in hypothalamic NA turnover and corticosterone serum levels 60 min after MDMA (10 mg/kg) administration, a decrease in peripheral blood neutrophil oxidative burst and a decrease in the percentage and intensity of neutrophil phagocytosis. It was further found that MDMA (10 mg/kg) treatment also altered leukocyte distribution in blood, bone marrow and spleen. In addition, no effects were observed for MDMA after in vitro exposure both in neutrophil oxidative burst and phagocytosis. CONCLUSION: The effects of MDMA administration (10 mg/kg) on neutrophil activity and leukocyte distribution might have been induced indirectly through noradrenergic neurons and/or hypothalamic-pituitary-adrenal axis activations.


Subject(s)
Hallucinogens/pharmacology , Immune Tolerance/drug effects , Immunity/drug effects , Mononuclear Phagocyte System/drug effects , N-Methyl-3,4-methylenedioxyamphetamine/pharmacology , Neutrophils/drug effects , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/immunology , Corticosterone/blood , Disease Models, Animal , Hypothalamo-Hypophyseal System/drug effects , Hypothalamo-Hypophyseal System/immunology , Hypothalamus/drug effects , Hypothalamus/metabolism , Immune Tolerance/physiology , Leukocytes/cytology , Leukocytes/drug effects , Leukocytes/immunology , Male , Mice , Mice, Inbred BALB C , Mononuclear Phagocyte System/cytology , Mononuclear Phagocyte System/immunology , Neuroimmunomodulation/drug effects , Neuroimmunomodulation/immunology , Neutrophils/cytology , Neutrophils/immunology , Norepinephrine/metabolism , Phagocytosis/drug effects , Phagocytosis/physiology , Respiratory Burst/drug effects , Respiratory Burst/physiology , Spleen/cytology , Spleen/drug effects , Spleen/immunology
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