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1.
Environ Pollut ; 268(Pt B): 115863, 2020 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-33126161

RESUMEN

Cigarette smoke (CS) affects immune functions, leading to severe outcomes in smokers. Robust evidence addresses the immunotoxic effects of combustible tobacco products. As heat-not-burn tobacco products (HNBT) vaporize lower levels of combustible products, we here compared the effects of cigarette smoke (CS) and HNBT vapor on Jurkat T cells. Cells were exposed to air, conventional cigarettes or heatsticks of HNBT for 30 min and were stimulated or not with phorbol myristate acetate (PMA). Cell viability, proliferation, reactive oxygen species (ROS) production, 8-OHdG, MAP-kinases and nuclear factor κB (NFκB) activation and metallothionein expression (MTs) were assessed by flow cytometry; nitric oxide (NO) and cytokine levels were measured by Griess reaction and ELISA, respectively. Levels of metals in the exposure chambers were quantified by inductively coupled plasma mass spectrometry. MT expressions were quantified by immunohistochemistry in the lungs and liver of C57Bl/6 mice exposed to CS, HNBT or air (1 h, twice a day for five days: via inhalation). While both CS and HBNT exposures increased cell death, CS led to a higher number of necrotic cells, increased the production of ROS, NO, inflammatory cytokines and MTs when compared to HNBT-exposed cells, and led to a higher expression of MTs in mice. CS released higher amounts of metals. CS and HNBT exposures decreased PMA-induced interleukin-2 (IL-2) secretion and impaired Jurkat proliferation, effects also seen in cells exposed to nicotine. Although HNBT vapor does not activate T cells as CS does, exposure to both HNBT and CS suppressed proliferation and IL-2 release, a pivotal cytokine involved with T cell proliferation and tolerance, and this effect may be related to nicotine content in both products.

2.
Biochem Pharmacol ; 182: 114230, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-32979352

RESUMEN

L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.

3.
Cells ; 9(5)2020 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-32403233

RESUMEN

Embryo implantation into the uterine wall is a highly modulated, complex process. We previously demonstrated that Annexin A1 (AnxA1), which is a protein secreted by epithelial and inflammatory cells in the uterine microenvironment, controls embryo implantation in vivo. Here, we decipher the effects of recombinant AnxA1 in this phenomenon by using human trophoblast cell (BeWo) spheroids and uterine epithelial cells (Ishikawa; IK). AnxA1-treated IK cells demonstrated greater levels of spheroid adherence and upregulation of the tight junction molecules claudin-1 and zona occludens-1, as well as the glycoprotein mucin-1 (Muc-1). The latter effect of AnxA1 was not mediated through IL-6 secreted from IK cells, a known inducer of Muc-1 expression. Rather, these effects of AnxA1 involved activation of the formyl peptide receptors FPR1 and FPR2, as pharmacological blockade of FPR1 or FPR1/FPR2 abrogated such responses. The downstream actions of AnxA1 were mediated through the ERK1/2 phosphorylation pathway and F-actin polymerization in IK cells, as blockade of ERK1/2 phosphorylation reversed AnxA1-induced Muc-1 and claudin-1 expression. Moreover, FPR2 activation by AnxA1 induced vascular endothelial growth factor (VEGF) secretion by IK cells, and the supernatant of AnxA1-treated IK cells evoked angiogenesis in vitro. In conclusion, these data highlight the role of the AnxA1/FPR1/FPR2 pathway in uterine epithelial control of blastocyst implantation.

4.
Cell Death Discov ; 5: 135, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31552142

RESUMEN

Annexin A1 (AnxA1) modulates neutrophil life span and bone marrow/blood cell trafficking thorough activation of formyl-peptide receptors (FPRs). Here, we investigated the effect of exogenous AnxA1 on haematopoiesis in the mouse. Treatment of C57BL/6 mice with recombinant AnxA1 (rAnxA1) reduced the granulocyte-macrophage progenitor (GMP) population in the bone marrow, enhanced the number of mature granulocytes Gr-1+Mac-1+ in the bone marrow as well as peripheral granulocytic neutrophils and increased expression of mitotic cyclin B1 on hematopoietic stem cells (HSCs)/progenitor cells (Lin-Sca-1+c-Kit+: LSK). These effects were abolished by simultaneous treatment with Boc-2, an FPR pan-antagonist. In in vitro studies, rAnxA1 reduced both HSC (LSKCD90lowFLK-2-) and GMP populations while enhancing mature cells (Gr1+Mac1+). Moreover, rAnxA1 induced LSK cell proliferation (Ki67+), increasing the percentage of cells in the S/G2/M cell cycle phases and reducing Notch-1 expression. Simultaneous treatment with WRW4, a selective FPR2 antagonist, reversed the in vitro effects elicited by rAnxA1. Treatment of LSK cells with rAnxA1 led to phosphorylation of PCLγ2, PKC, RAS, MEK, and ERK1/2 with increased expression of NFAT2. In long-term bone marrow cultures, rAnxA1 did not alter the percentage of LSK cells but enhanced the Gr-1+Mac-1+ population; treatment with a PLC (U73122), but not with a PKC (GF109203), inhibitor reduced rAnxA1-induced phosphorylation of ERK1/2 and Elk1. Therefore, we identify here rAnxA1 as an inducer of HSC/progenitor cell differentiation, favouring differentiation of the myeloid/granulocytic lineage, via Ca2+/MAPK signalling transduction pathways.

5.
Blood ; 134(2): 101-103, 2019 07 11.
Artículo en Inglés | MEDLINE | ID: mdl-31296540
6.
PLoS One ; 13(11): e0205535, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30395570

RESUMEN

Paraquat (PQ) is one of the most widely employed herbicides that is used worldwide and it causes severe toxic effects in humans and animals. A PQ exposition can lead to pulmonary fibrosis (PF) and the mechanisms seem to be linked to oxidative stress, although other pathways have been suggested. Antioxidants can be useful as a therapy, although interventions with this kind of system are still controversial. Hence, this study has investigated the role of ascorbic acid (vitamin C) post-treatment on PQ-induced PF in male C57/BL6 mice. Pulmonary fibrosis was induced by a single PQ injection (10mg/kg; i.p.). The control group received a PQ vehicle. Seven days after the PQ or vehicle injections, the mice received vitamin C (150 mg/kg, ip, once a day) or the vehicle, over the following 7 days. Twenty-four hours after the last dose of vitamin C or the vehicle, the mice were euthanized and their bronchoalveolar lavage fluid (BALF) and their lungs were collected. The data obtained showed that vitamin C reduced the cellular recruitment, the secretion of IL-17 -a cytokine involved in neutrophils migration, TGF-ß-a pro-fibrotic mediator and the collagen deposition. Moreover, vitamin C elevated the superoxide dismutase (SOD) and catalase levels, both antioxidant enzymes, but it did not alter the tracheal contractile response that was evoked by methacholine. Therefore, the researchers have highlighted the mechanisms of vitamin C as being non-invasive and have suggested it as a promising tool to treat lung fibrosis when it is induced by a PQ intoxication.


Asunto(s)
Ácido Ascórbico/uso terapéutico , Paraquat/toxicidad , Fibrosis Pulmonar/tratamiento farmacológico , Animales , Antioxidantes/metabolismo , Ácido Ascórbico/farmacología , Líquido del Lavado Bronquioalveolar , Recuento de Células , Colágeno/metabolismo , Citocinas/metabolismo , Pulmón/efectos de los fármacos , Pulmón/enzimología , Pulmón/metabolismo , Pulmón/patología , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Cloruro de Metacolina/farmacología , Ratones Endogámicos C57BL , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo
7.
Biomed Pharmacother ; 105: 947-955, 2018 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-30021389

RESUMEN

Neutrophils are the first line of defence during inflammatory processes; nevertheless, exacerbated influx and actions of neutrophils in terms of uncontrolled inflammation are harmful to the host. Hence, neutrophil activity is the target of drugs seeking to address undesired inflammation. Here, we investigated the mechanisms of action of a ligand of the three isoforms of peroxisome proliferator-activated receptors (PPAR; (5Z)-5-[(5-bromo-1H-indole-3-yl)methylene]-3-(4-chlorobenzyl)-thiazolidine-2,4-dione), dubbed LYSO-7, on neutrophils activated by N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP), an agonist of G-protein coupled receptors (GPCRs) that binds to membrane-formylated peptide and activates intracellular inflammation pathways. Neutrophils were collected from the peritoneal cavity of male Wistar rats four hours after oyster glycogen injection. Afterwards, the neutrophils were incubated with saline or LYSO-7 (1 or 10 µM, 30 min), washed and stimulated with fMLP (10-7 µM, 1 h). LYSO-7 treatment inhibited gene and protein expression of adhesion molecules, CD62 L and CD18, abolished adhesion of neutrophils to endothelial cells, impaired chemotaxis, blocked the enhancement of intracellular calcium levels, induced the expression of PPARγ as well as PPARßδ and reduced nuclear translocation of nuclear factor κB (NF-κB). Moreover, topical application of LYSO-7 (10 mM) prior to local application of fMLP (10-7 µM) diminished the in vivo leukocyte-endothelial interactions in the mesentery microcirculation of rats. Together, our data highlight the effectiveness of anti-inflammatory actions of LYSO-7 on neutrophils activated by GPCRs, depending, at least in part, on impaired of NF-κB activation and induction of PPAR expression.


Asunto(s)
Inhibidores de la Ciclooxigenasa/farmacología , Indoles/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Tiazolidinedionas/farmacología , Animales , Relación Dosis-Respuesta a Droga , Ligandos , Masculino , Receptores Activados del Proliferador del Peroxisoma/agonistas , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G/agonistas , Tiazolidinas/farmacología
8.
J Cell Physiol ; 233(9): 6591-6603, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29115663

RESUMEN

Annexin A1 (AnxA1) is a glucocorticoid-regulated anti-inflammatory protein secreted by phagocytes and other specialised cells. In the endocrine system, AnxA1 controls secretion of steroid hormones and it is abundantly expressed in the testis, ovaries, placenta and seminal fluid, yet its potential modulation of fertility has not been described. Here, we observed that AnxA1 knockout (KO) mice delivered a higher number of pups, with a higher percentage of female offsprings. This profile was not dependent on the male features, as sperm from KO male mice did not present functional alterations, and had an equal proportion of Y and X chromosomes, comparable to wild type (WT) male mice. Furthermore, mismatched matings of male WT mice with female KO yielded a higher percentage of female pups per litter, a phenomenon which was not observed when male KO mice mated with female WT animals. Indeed, AnxA1 KO female mice displayed several differences in parameters related to gestation including (i) an arrested estrous cycle at proestrus phase; (ii) increased sites of implantation; (iii) reduced pre- and post-implantation losses; (iv) exacerbated features of the inflammatory reaction in the uterine fluid during implantation phase; and (v) enhanced plasma progesterone in the beginning of pregnancy. In summary, herein we highlight that AnxA1 pathway as a novel determinant of fundamental non-redundant regulatory functions during early pregnancy.


Asunto(s)
Anexina A1/metabolismo , Implantación del Embrión/fisiología , Animales , Ciclo Estral/metabolismo , Ciclo Estral/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Modelos Animales , Embarazo , Proestro/metabolismo , Proestro/fisiología , Razón de Masculinidad , Útero/metabolismo , Útero/fisiología , Cromosoma X/metabolismo , Cromosoma X/fisiología , Cromosoma Y/metabolismo , Cromosoma Y/fisiología
9.
Front Pharmacol ; 8: 766, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29163156

RESUMEN

TSPO (Translocator 18 KDa; tryptophan-rich sensory protein oxygen sensor) is a constitutive outer mitochondrial membrane protein overexpressed in inflammatory cells during local or systemic processes. Despite its expression is characterized, role of TSPO in inflammation remains elusive. For this study, we investigated the role of TSPO ligands on neutrophil functions elicited by two different inflammatory pathways. Peritoneal neutrophils were isolated from male Balb-C mice, treated with TSPO ligand diazepam, Ro5-4864 or PK11195 (1,100 or 1000 nM; 2 h) and further stimulated with lipopolysaccharide from Escherichia coli (LPS), a binding for Toll-Like Receptor-4 (TLR4), or leukotriene B4 (LTB4), a G-protein coupled receptor (GPCR) ligand. LPS treatment did not lead to overexpression of TSPO on neutrophils, and pre-treatment with any TSPO ligand did not alter cytokine expression, adhesion molecule expression, or the production of reactive oxygen and nitrogen species caused by LPS stimulation. Conversely, all TSPO ligands impaired LTB4's actions, as visualized by reductions in L-selectin shedding, ß2 integrin overexpression, neutrophil chemotaxis, and actin filament assembly. TSPO ligands showed distinct intracellular effects on LTB4-induced neutrophil locomotion, with diazepam enhancing cofilin but not modifying Arp2/3 expression, and Ro5-4864 and PK11195 reducing both cofilin and Arp2/3 expression. Taken together, our data exclude a direct role of TSPO ligands in TLR4-elicited pathways, and indicate that TSPO activation inhibits GPCR inflammatory pathways in neutrophils, with a relevant role in neutrophil influx into inflammatory sites.

10.
Bioorg Chem ; 72: 199-207, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28463767

RESUMEN

Novel N-triazolyl maleimide derivatives were synthesized by azide-alkyne Huisgen cycloaddition (1,3-dipolar cycloaddition) and tested for cytotoxicity against a cell line derived from human melanomas SK-Mel-28 and SK-Mel-103, and human umbilical vein endothelial cell lines (HUVEC). The 4l was chose to be biologically tested due to incorporation of benzyl triazolic to the nitrogen of maleimide has not been tested before, and due the satisfactory yield. The analysis of cell metabolism, using the MTT method, showed that the compound 4l impaired cell metabolism in HUVEC only in high concentration (100µM). A lower concentration of compound 4l, whether in association or not with paclitaxel, was required to cause toxicity in both SK-Mel-28 and SK-Mel-103 cells in comparison with HUVEC cells. Moreover, the ability of 4l to cause cell death was evaluated by flow cytometry, and the data obtained highlighted the apoptotic action of 4l and paclitaxel co-treatment on Sk-Mel-28 cells only, which corroborated the greater efficacy of maleimide compounds against cancer cells. Together, our data provide promising data on the selectivity of maleimide compounds to cancer cells, and suggest that novel maleimide-substituted compounds may be synthesized and tested on different cancer cell lines, as primary or co-adjuvant agents of cancer cell toxicity.


Asunto(s)
Maleimidas/farmacología , Muerte Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Maleimidas/síntesis química , Maleimidas/química , Estructura Molecular , Relación Estructura-Actividad
11.
Proc Natl Acad Sci U S A ; 113(52): E8415-E8424, 2016 12 27.
Artículo en Inglés | MEDLINE | ID: mdl-27956610

RESUMEN

Although neutrophils are known to be fundamental in controlling innate immune responses, their role in regulating adaptive immunity is just starting to be appreciated. We report that human neutrophils exposed to pregnancy hormones progesterone and estriol promote the establishment of maternal tolerance through the induction of a population of CD4+ T cells displaying a GARP+CD127loFOXP3+ phenotype following antigen activation. Neutrophil-induced T (niT) cells produce IL-10, IL-17, and VEGF and promote vessel growth in vitro. Neutrophil depletion during murine pregnancy leads to abnormal development of the fetal-maternal unit and reduced empbryo development, with placental architecture displaying poor trophoblast invasion and spiral artery development in the maternal decidua, accompanied by significantly attenuated niT cell numbers in draining lymph nodes. Using CD45 congenic cells, we show that induction of niT cells and their regulatory function occurs via transfer of apoptotic neutrophil-derived proteins, including forkhead box protein 1 (FOXO1), to T cells. Unlike in women with healthy pregnancies, neutrophils from blood and placental samples of preeclamptic women fail to induce niT cells as a direct consequence of their inability to transfer FOXO1 to T cells. Finally, neutrophil-selective FOXO1 knockdown leads to defective placentation and compromised embryo development, similar to that resulting from neutrophil depletion. These data define a nonredundant function of neutrophil-T cell interactions in the regulation of vascularization at the maternal-fetal interface.


Asunto(s)
Neovascularización Fisiológica , Neutrófilos/citología , Placenta/fisiología , Linfocitos T Reguladores/citología , Adulto , Animales , Decidua/fisiología , Femenino , Proteína Forkhead Box O1/fisiología , Técnicas de Silenciamiento del Gen , Voluntarios Sanos , Humanos , Sistema Inmunológico , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fenotipo , Embarazo , Adulto Joven
12.
Sci Rep ; 6: 36239, 2016 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-27824095

RESUMEN

L-asparaginase (L-ASNase) (EC 3.5.1.1) is an important enzyme for the treatment of acute lymphoblastic leukaemia. Currently, the enzyme is obtained from bacteria, Escherichia coli and Erwinia chrysanthemi. The bacterial enzymes family is subdivided in type I and type II; nevertheless, only type II have been employed in therapeutic proceedings. However, bacterial enzymes are susceptible to induce immune responses, leading to a high incidence of adverse effects compromising the effectiveness of the treatment. Therefore, alternative sources of L-ASNase may be useful to reduce toxicity and enhance efficacy. The yeast Saccharomyces cerevisiae has the ASP1 gene responsible for encoding L-asparaginase 1 (ScASNase1), an enzyme predicted as type II, like bacterial therapeutic isoforms, but it has been poorly studied. Here we characterised ScASNase1 using a recombinant enzyme purified by affinity chromatography. ScASNase1 has specific activity of 196.2 U/mg and allosteric behaviour, like type I enzymes, but with a low K0.5 = 75 µM like therapeutic type II. We showed through site-directed mutagenesis that the T64-Y78-T141-K215 residues are involved in catalysis. Furthermore, ScASNase1 showed cytotoxicity for the MOLT-4 leukemic cell lineage. Our data show that ScASNase1 has characteristics described for the two subfamilies of l-asparaginase, types I and II, and may have promising antineoplastic properties.


Asunto(s)
Antineoplásicos/farmacología , Asparagina/genética , Asparagina/metabolismo , Saccharomyces cerevisiae/enzimología , Regulación Alostérica , Antineoplásicos/química , Asparagina/química , Asparagina/farmacología , Dominio Catalítico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cromatografía de Afinidad , Humanos , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/farmacología
13.
Eur J Med Chem ; 114: 1-7, 2016 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-26974369

RESUMEN

We report a method to obtain biaryl dipeptide tyrosine via Suzuki-Miyaura and alkynyl dipeptide tyrosine by Sonogashira cross-coupling reactions. Analysis of the biological action of biaryl dipeptide tyrosine 4d compound showed its ability to impair the metabolism and proliferation of SK-Mel-28 human melanoma lineage cells, independently of mitochondrial membrane depolarization, apoptosis and necrosis. Moreover, 4d compound did not cause toxicity to human umbilical vein endothelial cells (HUVEC), suggesting its toxic specificity to cancer cells.


Asunto(s)
Dipéptidos/toxicidad , Células Endoteliales de la Vena Umbilical Humana/citología , Neoplasias/patología , Tirosina/toxicidad , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Dipéptidos/síntesis química , Dipéptidos/química , Relación Dosis-Respuesta a Droga , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Estructura Molecular , Neoplasias/metabolismo , Relación Estructura-Actividad , Tirosina/síntesis química , Tirosina/química
14.
Sci Rep ; 5: 14917, 2015 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-26449762

RESUMEN

PCB126 is a dioxin-like polychlorinated biphenyl (PCB) environmental pollutant with a significant impact on human health, as it bioaccumulates and causes severe toxicity. PCB126-induced immune toxicity has been described, although the mechanisms have not been fully elucidated. In this study, an in vivo protocol of PCB126 intoxication into male Wistar rats by intranasal route was used, which has not yet been described. The intoxication was characterised by PCB126 accumulation in the lungs and liver, and enhanced aryl hydrocarbon receptor expression in the liver, lungs, kidneys, and adipose tissues. Moreover, an innate immune deficiency was characterised by impairment of adhesion receptors on blood leukocytes and by reduced blood neutrophil locomotion and oxidative burst activation elicited by ex vivo G protein-coupled receptor (GPCR) activation. Specificity of PCB126 actions on the GPCR pathway was shown by normal burst oxidative activation evoked by Toll-like receptor 4 and protein kinase C direct activation. Moreover, in vivo PCB180 intoxication did not alter adhesion receptors on blood leukocytes either blood neutrophil locomotion, and only partially reduced the GPCR-induced burst oxidative activation on neutrophils. Therefore, a novel mechanism of in vivo PCB126 toxicity is described which impairs a pivotal inflammatory pathway to the host defence against infections.


Asunto(s)
Inmunidad Innata/efectos de los fármacos , Bifenilos Policlorados/toxicidad , Receptores Acoplados a Proteínas G/metabolismo , Sistema Respiratorio/efectos de los fármacos , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/inmunología , Tejido Adiposo/metabolismo , Administración Intranasal , Animales , Western Blotting , Moléculas de Adhesión Celular/sangre , Ensayo de Inmunoadsorción Enzimática , Riñón/efectos de los fármacos , Riñón/inmunología , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/inmunología , Hígado/metabolismo , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/metabolismo , Masculino , Absorción Nasal , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Bifenilos Policlorados/administración & dosificación , Bifenilos Policlorados/farmacocinética , Ratas Wistar , Receptores de Hidrocarburo de Aril/metabolismo , Estallido Respiratorio/efectos de los fármacos , Sistema Respiratorio/inmunología , Sistema Respiratorio/metabolismo
15.
Respir Res ; 16: 18, 2015 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-25848680

RESUMEN

BACKGROUND: Diesel exhaust particles (DEPs) are deposited into the respiratory tract and are thought to be a risk factor for the development of diseases of the respiratory system. In healthy individuals, the timing and mechanisms of respiratory tract injuries caused by chronic exposure to air pollution remain to be clarified. METHODS: We evaluated the effects of chronic exposure to DEP at doses below those found in a typical bus corridor in Sao Paulo (150 µg/m3). Male BALB/c mice were divided into mice receiving a nasal instillation: saline (saline; n = 30) and 30 µg/10 µL of DEP (DEP; n = 30). Nasal instillations were performed five days a week, over a period of 90 days. Bronchoalveolar lavage (BAL) was performed, and the concentrations of interleukin (IL)-4, IL-10, IL-13 and interferon-gamma (INF-γ) were determined by ELISA-immunoassay. Assessment of respiratory mechanics was performed. The gene expression of Muc5ac in lung was evaluated by RT-PCR. The presence of IL-13, MAC2+ macrophages, CD3+, CD4+, CD8+ T cells and CD20+ B cells in tissues was analysed by immunohistochemistry. Bronchial thickness and the collagen/elastic fibers density were evaluated by morphometry. We measured the mean linear intercept (Lm), a measure of alveolar distension, and the mean airspace diameter (D0) and statistical distribution (D2). RESULTS: DEP decreased IFN-γ levels in BAL (p = 0.03), but did not significantly alter IL-4, IL-10 and IL-13 levels. MAC2+ macrophage, CD4+ T cell and CD20+ B cell numbers were not altered; however, numbers of CD3+ T cells (p ≤ 0.001) and CD8+ T cells (p ≤ 0.001) increased in the parenchyma. Although IL-13 (p = 0.008) expression decreased in the bronchiolar epithelium, Muc5ac gene expression was not altered in the lung of DEP-exposed animals. Although respiratory mechanics, elastic and collagen density were not modified, the mean linear intercept (Lm) was increased in the DEP-exposed animals (p ≤ 0.001), and the index D2 was statistically different (p = 0.038) from the control animals. CONCLUSION: Our data suggest that nasal instillation of low doses of DEP over a period of 90 days results in alveolar enlargement in the pulmonary parenchyma of healthy mice.


Asunto(s)
Contaminantes Atmosféricos/toxicidad , Neumonía/inducido químicamente , Alveolos Pulmonares/efectos de los fármacos , Emisiones de Vehículos/toxicidad , Animales , Brasil , Líquido del Lavado Bronquioalveolar/inmunología , Colágeno/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Tejido Elástico/metabolismo , Mediadores de Inflamación/metabolismo , Subgrupos Linfocitarios/efectos de los fármacos , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones Endogámicos BALB C , Mucina 5AC/genética , Mucina 5AC/metabolismo , Neumonía/inmunología , Neumonía/metabolismo , Neumonía/patología , Neumonía/fisiopatología , Alveolos Pulmonares/inmunología , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Alveolos Pulmonares/fisiopatología , ARN Mensajero/metabolismo , Mecánica Respiratoria/efectos de los fármacos , Factores de Tiempo
16.
PLoS One ; 8(10): e76894, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24124600

RESUMEN

The present study aimed to show the in vivo mechanisms of action of an indole-thiazolidine molecule peroxisome-proliferator activated receptor pan-agonist (PPAR pan) and cyclooxygenase (COX) inhibitor, LYSO-7, in an ethanol/HCl-induced (Et/HCl) gastric lesion model. Swiss male mice were treated with vehicle, LYSO-7 or Bezafibrate (p.o.) 1 hour before oral administration of Et/HCl (60%/0.03M). In another set of assays, animals were injected i.p. with an anti-granulocyte antibody, GW9962 or L-NG-nitroarginine methyl ester (L-NAME) before treatment. One hour after Et/HCl administration, neutrophils were quantified in the blood and bone marrow and the gastric microcirculatory network was studied in situ. The gastric tissue was used to quantify the percentage of damaged area, as well as myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) protein and PPARγ protein and gene expression. Acid secretion was evaluated by the pylorus ligation model. LYSO-7 or Bezafibrate treatment reduced the necrotic area. LYSO-7 treatment enhanced PPARγ gene and protein expression in the stomach, and impaired local neutrophil influx and stasis of the microcirculatory network caused by Et/HCl administration. The effect seemed to be due to PPARγ agonist activity, as the LYSO-7 effect was abolished in GW9962 pre-treated mice. The reversal of microcirculatory stasis, but not neutrophil influx, was mediated by nitric oxide (NO), as L-NAME pre-treatment abolished the LYSO-7-mediated reestablishment of microcirculatory blood flow. This effect may depend on enhanced eNOS protein expression in injured gastric tissue. The pH and concentration of H(+) in the stomach were not modified by LYSO-7 treatment. In addition, LYSO-7 may induce less toxicity, as 28 days of oral treatment did not induce weight loss, as detected in pioglitazone treated mice. Thus, we show that LYSO-7 may be an effective treatment for gastric lesions by controlling neutrophil influx and microcirculatory blood flow mediated by NO.


Asunto(s)
Inhibidores de la Ciclooxigenasa/farmacología , Gastritis/metabolismo , Gastritis/patología , Indoles/farmacología , Microcirculación/efectos de los fármacos , Receptores Activados del Proliferador del Peroxisoma/agonistas , Tiazolidinedionas/farmacología , Animales , Peso Corporal/efectos de los fármacos , Inhibidores de la Ciclooxigenasa/administración & dosificación , Etanol/efectos adversos , Ácido Gástrico/metabolismo , Gastritis/inducido químicamente , Gastritis/tratamiento farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Ácido Clorhídrico/efectos adversos , Indoles/administración & dosificación , Masculino , Ratones , Infiltración Neutrófila/efectos de los fármacos , Óxido Nítrico/metabolismo , PPAR gamma/genética , PPAR gamma/metabolismo , Receptores Activados del Proliferador del Peroxisoma/genética , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Flujo Sanguíneo Regional/efectos de los fármacos , Tiazolidinedionas/administración & dosificación
17.
Bioorg Med Chem ; 21(14): 4225-32, 2013 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-23721916

RESUMEN

A small library of compounds was prepared by a combination of toluene dioxygenase (TDO)-catalyzed enzymatic dihydroxylation and copper(I)-catalyzed Hüisgen cycloaddition. Some compounds were obtained by coupling an alkyne and a conduritol derivative, while more complex structures were obtained by a double Hüisgen reaction of a dialkyne and two molecules of the cyclitol. The compounds were fully characterized and subjected to preliminary biological screening.


Asunto(s)
Ciclitoles/síntesis química , Ciclitoles/farmacología , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antifúngicos/síntesis química , Antifúngicos/química , Antifúngicos/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ciclitoles/química , Reacción de Cicloadición , Inmunosupresores/síntesis química , Inmunosupresores/química , Inmunosupresores/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/química , Triazoles/síntesis química , Triazoles/química , Triazoles/farmacología
18.
J Immunol ; 190(11): 5689-701, 2013 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-23645879

RESUMEN

Annexin A1 (AnxA1) is a protein that displays potent anti-inflammatory properties, but its expression in eye tissue and its role in ocular inflammatory diseases have not been well studied. We investigated the mechanism of action and potential uses of AnxA1 and its mimetic peptide (Ac2-26) in the endotoxin-induced uveitis (EIU) rodent model and in human ARPE-19 cells activated by LPS. In rats, analysis of untreated EIU after 24 and 48 h or EIU treated with topical applications or with a single s.c. injection of Ac2-26 revealed the anti-inflammatory actions of Ac2-26 on leukocyte infiltration and on the release of inflammatory mediators; the systemic administration of Boc2, a formylated peptide receptor (fpr) antagonist, abrogated the peptide's protective effects. Moreover, AnxA1(-/-) mice exhibited exacerbated EIU compared with wild-type animals. Immunohistochemical studies of ocular tissue showed a specific AnxA1 posttranslational modification in EIU and indicated that the fpr2 receptor mediated the anti-inflammatory actions of AnxA1. In vitro studies confirmed the roles of AnxA1 and fpr2 and the protective effects of Ac2-26 on the release of chemical mediators in ARPE-19 cells. Molecular analysis of NF-κB translocation and IL-6, IL-8, and cyclooxygenase-2 gene expression indicated that the protective effects of AnxA1 occur independently of the NF-κB signaling pathway and possibly in a posttranscriptional manner. Together, our data highlight the role of AnxA1 in ocular inflammation, especially uveitis, and suggest the use of AnxA1 or its mimetic peptide Ac2-26 as a therapeutic approach.


Asunto(s)
Anexina A1/genética , Antiinflamatorios/farmacología , Péptidos/farmacología , Uveítis/genética , Animales , Anexina A1/administración & dosificación , Anexina A1/química , Anexina A1/metabolismo , Anexina A1/farmacología , Antiinflamatorios/administración & dosificación , Humor Acuoso/citología , Humor Acuoso/inmunología , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Citocinas/biosíntesis , Citocinas/genética , Citocinas/inmunología , Modelos Animales de Enfermedad , Endotoxinas/efectos adversos , Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/inmunología , Masculino , Ratones , Ratones Noqueados , Modelos Biológicos , FN-kappa B/metabolismo , Infiltración Neutrófila/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Oligopéptidos/farmacología , Péptidos/administración & dosificación , Fosforilación , Transporte de Proteínas/efectos de los fármacos , Ratas , Receptores de Formil Péptido/genética , Receptores de Formil Péptido/metabolismo , Uveítis/inducido químicamente , Uveítis/inmunología
19.
Eur J Pharm Sci ; 48(4-5): 689-97, 2013 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-23305993

RESUMEN

The compound (5Z)-5-[(5-bromo-1H-indol-3-yl)methylene]-3-(4-chlorobenzyl)-thiazolidine-2,4-dione (LYSO-7) was synthesised in order to obtain a new type of anti-inflammatory drug, designed with hybrid features to inhibit cyclooxygenase (COX) and also to activate peroxisome proliferator-activated receptor (PPAR). Results obtained from docking (in silico) studies corroborated with experimental data, showing the potential affinity between the studied ligand and targets. The specificity of LYSO-7 for COX-enzymes was detected by the inhibition of COX-1 and COX-2 activities by 30% and 20%, respectively. In transactivation reporter gene assays LYSO-07 showed a pan partial agonist effect on the three PPAR subtypes (PPARγ, PPARα and PPARß/δ). The agonist action on PPARγ was also observed by a pharmacological approach, as the reduction in the Escherichia coli lipopolysaccharide (LPS)-induced interleukin 1 beta (IL-1ß) secretion and nitric oxide (NO) production by mouse neutrophils was blocked by GW9962, a specific PPARγ antagonist. Additionally, the in vivo effect was measured by reduced carrageenan-induced neutrophil influx into the subcutaneous tissue of mice. Taken together, these data show that LYSO-7 displays a potent in vivo anti-inflammatory effect during the innate acute response, which is dependent on its associated COX inhibitory activities and PPAR activation.


Asunto(s)
Antiinflamatorios/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Indoles/farmacología , Receptores Activados del Proliferador del Peroxisoma/agonistas , Tiazolidinedionas/farmacología , Animales , Carragenina , Movimiento Celular/efectos de los fármacos , Células HeLa , Humanos , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Leucocitos/efectos de los fármacos , Leucocitos/fisiología , Masculino , Ratones , Modelos Moleculares
20.
Naunyn Schmiedebergs Arch Pharmacol ; 386(1): 5-14, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23128853

RESUMEN

Chlorogenic acid (CGA) is found in many foods, including coffee, berries, potatoes, carrots, wine, apples, and various herbs, and has anti-inflammatory, antidiabetic, and antitumoral actions. The CGA is well absorbed orally, and its effects on gastric ulcer have not been previously reported. The present manuscript evaluated the effect of oral administration of CGA on ethanol/HCl (Et/HCl) or nonsteroidal anti-inflammatory drug (NSAID)-induced gastric ulcer model in male Swiss mice. Animals were pretreated with 0.2 % carboxymethylcellulose (vehicle, p.o.), omeprazole (positive control, 30 mg/kg, p.o.), carbenoxolone (antioxidant positive control, 100 mg/kg, p.o.), or CGA (5, 25, or 50 mg/kg, p.o.). One hour later, the gastric ulcer was induced by injecting Et/HCl solution (100 µL/10 g body weight; Et 60 % + HCl 0.03 M) or piroxicam (100 mg/kg, p.o). After another hour or 4 h later, gastric tissues were collected from Et/HCl or piroxicam-treated animals, respectively, to evaluate the size of the lesion, histological alterations, secretion of gastric acid, neutrophil migration, oxidative/antioxidative enzymes, markers of lipid peroxidation, or concentrations of inflammatory mediators. CGA treatment had a gastroprotective effect in both models, reducing the percentage of lesioned area. CGA treatment did not alter the secretion of gastric action but inhibited neutrophil migration and restored the levels of catalase, superoxide dismutase, glutathione peroxidase, glutathione, and thiobarbituric acid reactive substances in mice treated with Et/HCl. Additionally, CGA treatment blocked the increase of tumor necrosis factor alpha and leukotriene B4 but did not restore the reduced prostaglandin levels in the NSAID-induced ulcer. Together, the data presented herein show that CGA may be a suitable natural compound for the prevention and treatment of gastric lesions caused by a different etiology.


Asunto(s)
Antiulcerosos/farmacología , Ácido Clorogénico/farmacología , Úlcera Gástrica/tratamiento farmacológico , Animales , Antiinflamatorios no Esteroideos/toxicidad , Antiulcerosos/administración & dosificación , Antioxidantes/farmacología , Carbenoxolona/farmacología , Ácido Clorogénico/administración & dosificación , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Etanol/toxicidad , Masculino , Ratones , Omeprazol/farmacología , Úlcera Gástrica/inducido químicamente , Úlcera Gástrica/patología , Factores de Tiempo
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