The Role of Annexin A1 in DNA Damage Response in Placental Cells: Impact on Gestational Diabetes Mellitus.

The functions of annexin A1 (ANXA1), which is expressed on membranes and in cytoplasmic granules, have been fully described. Nonetheless, the role of this protein in protecting against DNA damage in the nucleus is still emerging and requires further investigation. Here, we investigated the involvement of ANXA1 in the DNA damage response in placental cells. Placenta was collected from ANXA1 knockout mice (AnxA1-/-) and pregnant women with gestational diabetes mellitus (GDM). The placental morphology and ANXA1 expression, which are related to the modulation of cellular response markers in the presence of DNA damage, were analyzed. The total area of AnxA1-/- placenta was smaller due to a reduced labyrinth zone, enhanced DNA damage, and impaired base excision repair (BER) enzymes, which resulted in the induction of apoptosis in the labyrinthine and junctional layers. The placentas of pregnant women with GDM showed reduced expression of AnxA1 in the villous compartment, increased DNA damage, apoptosis, and a reduction of enzymes involved in the BER pathway. Our translational data provide valuable insights into the possible involvement of ANXA1 in the response of placental cells to oxidative DNA damage and represent an advancement in investigations into the mechanisms involved in placental biology.

Role of Annexin A1 Secreted by Neutrophils in Melanoma Metastasis.

Annexin A1 (AnxA1) is highly secreted by neutrophils and binds to formyl peptide receptors (FPRs) to trigger anti-inflammatory effects and efferocytosis. AnxA1 is also expressed in the tumor microenvironment, being mainly attributed to cancer cells. As recruited neutrophils are player cells at the tumor sites, the role of neutrophil-derived AnxA1 in lung melanoma metastasis was investigated here. Melanoma cells and neutrophils expressing AnxA1 were detected in biopsies from primary melanoma patients, which also presented higher levels of serum AnxA1 and augmented neutrophil-lymphocyte ratio (NLR) in the blood. Lung melanoma metastatic mice (C57BL/6; i.v. injected B16F10 cells) showed neutrophilia, elevated AnxA1 serum levels, and higher labeling for AnxA1 in neutrophils than in tumor cells at the lungs with metastasis. Peritoneal neutrophils collected from naïve mice were co-cultured with B16F10 cells or employed to obtain neutrophil-conditioned medium (NCM; 18 h incubation). B16F10 cells co-cultured with neutrophils or with NCM presented higher invasion, which was abolished if B16F10 cells were previously incubated with FPR antagonists or co-cultured with AnxA1 knockout (AnxA1-/-) neutrophils. The depletion of peripheral neutrophils during lung melanoma metastasis development (anti-Gr1; i.p. every 48 h for 21 days) reduced the number of metastases and AnxA1 serum levels in mice. Our findings show that AnxA1 secreted by neutrophils favors melanoma metastasis evolution via FPR pathways, addressing AnxA1 as a potential biomarker for the detection or progression of melanoma.

Extracellular expression of Saccharomyces cerevisiae's L-asparaginase II in Pichia pastoris results in novel enzyme with better parameters.

L-asparaginase (ASNase) is an efficient inhibitor of tumor development, used in chemotherapy sessions against acute lymphoblastic leukemia (ALL) tumor cells; its use results in 80% complete remission of the disease in treated patients. Saccharomyces cerevisiae's L-asparaginase II (ScASNaseII) has a high potential to substitute bacteria ASNase in patients that developed hypersensitivity, but the endogenous production of it results in hypermannosylated immunogenic enzyme. Here we describe the genetic process to acquire the ScASNaseII expressed in the extracellular medium. Our strategy involved a fusion of mature sequence of protein codified by ASP3 (amino acids 26-362) with the secretion signal sequence of Pichia pastoris acid phosphatase enzyme; in addition, this DNA construction was integrated in P. pastoris Glycoswitch® strain genome, which has the cellular machinery to express and secrete high quantity of enzymes with humanized glycosylation. Our data show that the DNA construction and strain employed can express extracellular asparaginase with specific activity of 218.2 IU mg-1. The resultant enzyme is 40% more stable than commercially available Escherichia coli's ASNase (EcASNaseII) when incubated with human serum. In addition, ScASNaseII presents 50% lower cross-reaction with anti-ASNase antibody produced against EcASNaseII when compared with ASNase from Dickeya chrysanthemi.

Neutrophil depletion in the pre-implantation phase impairs pregnancy index, placenta and fetus development.

Maternal neutrophils cells are players in gestational tolerance and fetus delivery. Nonetheless, their actions in each phase of the pregnancy are unknown. We here investigated the role of maternal neutrophil depletion before the blastocyst implantation phase and outcomes in the pregnancy index, placenta, and fetus development. Neutrophils were pharmacologically depleted by i.p. injection of anti-Gr1 (anti-neutrophils; 200 µg) 24 hours after plug visualization in allogeneic-mated C57BL/6/BALB/c mice. Depletion of peripheral neutrophils lasted until 48 hours after anti-Gr1 injection (gestational day 1.5-3.5). On gestational day 5.5, neutrophil depletion impaired the blastocyst implantation, as 50% of pregnant mice presented reduced implantation sites. On gestational day 18.5, neutrophil depletion reduced the pregnancy rate and index, altered the placenta disposition in the uterine horns, and modified the structure of the placenta, detected by reduced junctional zone, associated with decreased numbers of giant trophoblast cells, spongiotrophoblast. Reduced number of placenta cells labeled for vascular endothelial growth factor (VEGF), platelet-endothelial cell adhesion molecule (PECAM-1), and intercellular cell adhesion molecule (ICAM-1), important markers of angiogenesis and adhesiveness, were detected in neutrophil depleted mice. Furthermore, neutrophil depletion promoted a higher frequency of monocytes, natural killers, and T regulatory cells, and lower frequency of cytotoxic T cells in the blood, and abnormal development of offspring. Associated data obtained herein highlight the pivotal role of neutrophils actions in the early stages of pregnancy, and address further investigations on the imbricating signaling evoked by neutrophils in the trophoblastic interaction with uterine epithelium.

In vivo evaluation of toxicity and anti-inflammatory activity of iron oxide nanoparticles conjugated with ibuprofen.

Aim: The low solubility and consequent poor bioavailability of ibuprofen (IBU) is a major drawback that can be overcome by anchoring IBU on ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) as effective multifunctional carriers for drug delivery. Methods: USPIONs were conjugated with glycerol phosphate (USPION-GP) and also co-conjugated with IBU (USPION-GP/IBU), and their in vivo toxicity and anti-inflammatory effects investigated. Phosphate buffer saline (control), IBU, USPION-GP and USPION-GP/IBU were intravenously administered 15 min before lipopolysaccharide-induced peritonitis in male Balb/c mice. Results: 4 h later, USPION bioconjugates did not appear to have caused toxicity to blood leukocytes or caused alterations in the spleen, liver or kidneys. Also, they inhibited lipopolysaccharide-induced neutrophil mobilization into the peritoneum. Conclusion: The absence of systemic toxicity and the unexpected anti-inflammatory action of USPION bioconjugates indicates that they could be a novel and effective approach to administer IBU and warrant further investigation.

Intravital Microscopy: A Tool to Investigate the Toxicity in the Immune System, Vessel Rheology, and Xenobiotic Distribution.

Intravital microscopy (IVM) is an essential experimental approach for evaluating, in real time, cell interactions in the blood and rheological parameters in the microcirculation of the living animals. Different tissues are surgically exposed to the visualization of the microvascular network in optical microscopies connected to video cameras and image software. By evaluating in situ microcirculatory network, IVM allows the visualization and quantification of physiological and pathological processes in the blood or in the adjacent tissues considering the whole system. Therefore, IVM has been used to evaluate the effects and mechanisms of actions in the microvascular network caused by pharmacological or toxic chemical agents. In this chapter, different experimental approaches are described to study the toxic effects and mechanisms of xenobiotics in the microcirculatory network.

Influence of lysosomal protease sensitivity in the immunogenicity of the antitumor biopharmaceutical asparaginase.

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.

Annexin A1/Formyl Peptide Receptor Pathway Controls Uterine Receptivity to the Blastocyst.

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.

Antiangiogenic effects of the chemopreventive agent tributyrin, a butyric acid prodrug, during the promotion phase of hepatocarcinogenesis.

Agents that inhibit angiogenic factors may prevent the development of hepatocellular carcinoma (HCC). Thus, the objective of this study was to kinetically evaluate the antiangiogenic activity of tributyrin (TB), a butyric acid prodrug, in the promotion stage of hepatocarcinogenesis. For this purpose, the resistant hepatocyte (RH) model was used for induction of preneoplastic lesions in Wistar rats. During the promotion phase, the animals received TB or maltodextrin (MD) as control daily. The rats were killed at three time-points (P1, P2 and P3). Increased expression of Vegfa and Vegfr2 was observed during promotion phase of hepatocarcinogenesis, which was not reversed by TB treatment. However, TB treatment reduced the expression of cluster of differentiation (CD) 34-positive vessels at P3 and α-smooth muscle actin (α-SMA)-positive vessels at P2 compared with MD. Enhanced levels of hypoxia inducible factor-1α (HIF-1α) and phosphorylated extracellular signal-regulated kinases (pERK) were detected at P3 when compared with P1 and P2 in the MD treatment. TB treatment reduced the levels of HIF-1α and pERK at P3 relative to the MD control. Experiments with human umbilical vein endothelial cells (HUVEC) showed that sodium butyrate (NaBu) inhibited cell migration and tube formation, confirming the antiangiogenic activity of its prodrug TB. In conclusion, antiangiogenic activity of TB is an early event that already occurs in preneoplastic livers, reinforcing its potential chemopreventive effects against HCC.

Beneficial effects of ascorbic acid to treat lung fibrosis induced by paraquat.

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.

Vitamin D treatment abrogates the inflammatory response in paraquat-induced lung fibrosis.

A high incidence of intentional or accidental paraquat (PQ) ingestion is related to irreversible lung fibrosis and no effective therapy is currently available. Vitamin D has emerged with promising results as an immunomodulatory molecule when abrogating the inflammatory responses of lung diseases. Therefore, we have investigated the role of vitamin D treatments on PQ-induced lung fibrosis in male C57/BL6 mice. Lung fibrosis was induced by a single injection of PQ (10 mg/kg; i.p.). The control group received PQ vehicle. Seven days later, after the PQ injection or the vehicle injection, the mice received vitamin D (5 µg/kg, i.p., once a day) or vehicle, for a further 7 days. Twenty-four hours after the last dose of vitamin D or the vehicle, the analysis were performed. The vitamin D treatments reduced the number of leukocytes in their BALF and they decreased the IL-6, IL-17, TGF-beta and MMP-9 levels and the abrogated collagenase deposits in their lung tissues. Conversely, the vitamin D treatments increased the resolvin D levels in their BALF. Moreover, their tracheal contractility was also significantly reduced by the vitamin D treatments. Altogether, the data that was obtained showed a promising use of vitamin D, in treating the lung fibrosis that had been induced by the PQ intoxications. This may improve its prognostic use for a non-invasive and low cost therapy.

Endogenous annexin A1 (AnxA1) modulates early-phase gestation and offspring sex-ratio skewing.

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.

Anacardic acids from cashew nuts ameliorate lung damage induced by exposure to diesel exhaust particles in mice.

Anacardic acids from cashew nut shell liquid, a Brazilian natural substance, have antimicrobial and antioxidant activities and modulate immune responses and angiogenesis. As inflammatory lung diseases have been correlated to environmental pollutants exposure and no reports addressing the effects of dietary supplementation with anacardic acids on lung inflammation in vivo have been evidenced, we investigated the effects of supplementation with anacardic acids in a model of diesel exhaust particle- (DEP-) induced lung inflammation. BALB/c mice received an intranasal instillation of 50 µ g of DEP for 20 days. Ten days prior to DEP instillation, animals were pretreated orally with 50, 150, or 250 mg/kg of anacardic acids or vehicle (100 µ L of cashew nut oil) for 30 days. The biomarkers of inflammatory and antioxidant responses in the alveolar parenchyma, bronchoalveolar lavage fluid (BALF), and pulmonary vessels were investigated. All doses of anacardic acids ameliorated antioxidant enzyme activities and decreased vascular adhesion molecule in vessels. Animals that received 50 mg/kg of anacardic acids showed decreased levels of neutrophils and tumor necrosis factor in the lungs and BALF, respectively. In summary, we demonstrated that AAs supplementation has a potential protective role on oxidative and inflammatory mechanisms in the lungs.

Intracellular mechanisms of hydroquinone toxicity on endotoxin-activated neutrophils.

Circulating neutrophils promptly react to different substances in the blood and orchestrate the beginning of the innate inflammatory response. We have shown that in vivo exposure to hydroquinone (HQ), the most oxidative compound of cigarette smoke and a toxic benzene metabolite, affects circulating neutrophils, making them unresponsive to a subsequent bacterial infection. In order to understand the action of toxic molecular mechanisms on neutrophil functions, in vitro HQ actions on pro-inflammatory mediator secretions evoked by Escherichia coli lipopolysaccharide (LPS) were investigated. Neutrophils from male Wistar rats were cultured with vehicle or HQ (5 or 10 µM; 2 h) and subsequently incubated with LPS (5 µg/ml; 18 h). Hydroquinone treatment impaired LPS-induced nitric oxide (NO), tumour necrosis factor α (TNF-α), interleukin (IL)-1ß and IL-6 secretions by neutrophils. The toxic effect was not dependent on cell death, reduced expression of the LPS receptor or toll-like receptor-4 (TLR-4) or cell priming, as HQ did not induce reactive oxygen species generation or ß(2)integrin membrane expression. The action of toxic mechanisms on cytokine secretion was dependent on reduced gene synthesis, which may be due to decreased nuclear factor κB (NF-κB) nuclear translocation. Conversely, this intracellular pathway was not involved in impaired NO production because HQ treatments only affected inducible nitric oxide synthase protein expression and activity, suggesting posttranscriptional and/or posttranslational mechanisms of action. Altogether, our data show that HQ alters the action of different LPS-activated pathways on neutrophils, which may contribute to the impaired triggering of the host innate immune reaction detected during in vivo HQ exposure.

In vivo hydroquinone exposure impairs MCP-1 secretion and monocyte recruitment into the inflamed lung.

Alveolar macrophages (AMs) are important cells in the resolution of the inflammatory process and they come into direct contact with inhaled pollutants. Hydroquinone (HQ) is an environmental pollutant and a component of cigarette smoke that causes immunosuppressive effects. In the present work, we showed that mice exposed to low levels of aerosolized HQ (25 ppm; 1 h/day/5 days) presented impaired mononuclear cell migration to the lipopolysaccharide (LPS)-inflamed lung. This may have been due to reduced monocyte chemoattractant protein-1 (MCP-1) secretion into bronchoalveolar lavage fluid (BALF), and it was not related to alterations to mononuclear cell mobilization into the blood or adhesion molecules expression on mononuclear cell membranes. Corroborating the actions of HQ on MCP-1 secretion, reduced MCP-1 concentrations were also found in the supernatant of ex vivo AM and tracheal tissue collected from HQ-exposed mice. A direct action of HQ on MCP-1 secretion, resulting from impaired gene synthesis, was verified by in vitro incubation of naive AMs or tracheal tissue with HQ. The role of reduced levels of MCP-1 in the BALF on monocyte migration was analysed in the human monocytic lineage THP-1 in in vitro chemotaxis assays, which showed that the reduced concentrations of MCP-1 found in the BALF or cell supernatants from HQ-exposed mice impaired cell migration. Considering the fact that MCP-1 presents a broad spectrum of actions on pathophysiological conditions and that resident mononuclear cells are involved in lung tissue homeostasis and in immune host defence, the mechanism of HQ toxicity presented herein might be relevant to the genesis of infectious lung diseases in smokers and in inhabitants of polluted areas.

Endothelial dysfunction in the pulmonary artery induced by concentrated fine particulate matter exposure is associated with local but not systemic inflammation.

Clinical evidence has identified the pulmonary circulation as an important target of air pollution. It was previously demonstrated that in vitro exposure to fine particulate matter (aerodynamic diameter≤2.5 µm, PM2.5) induces endothelial dysfunction in isolated pulmonary arteries. We aimed to investigate the effects of in vivo exposure to urban concentrated PM2.5 on rat pulmonary artery reactivity and the mechanisms involved. For this, adult Wistar rats were exposed to 2 weeks of concentrated São Paulo city air PM2.5 at an accumulated daily dose of approximately 600 µg/m3. Pulmonary arteries isolated from PM2.5-exposed animals exhibited impaired endothelium-dependent relaxation to acetylcholine without significant changes in nitric oxide donor response compared to control rats. PM2.5 caused vascular oxidative stress and enhanced protein expression of Cu/Zn- and Mn-superoxide dismutase in the pulmonary artery. Protein expression of endothelial nitric oxide synthase (eNOS) was reduced, while tumor necrosis factor (TNF)-α was enhanced by PM2.5 inhalation in pulmonary artery. There was a significant positive correlation between eNOS expression and maximal relaxation response (Emax) to acetylcholine. A negative correlation was found between vascular TNF-α expression and Emax to acetylcholine. Plasma cytokine levels, blood cells count and coagulation parameters were similar between control and PM2.5-exposed rats. The present findings showed that in vivo daily exposure to concentrated urban PM2.5 could decrease endothelium-dependent relaxation and eNOS expression on pulmonary arteries associated with local high TNF-α level but not systemic pro-inflammatory factors. Taken together, the present results elucidate the mechanisms underlying the trigger of cardiopulmonary diseases induced by urban ambient levels of PM2.5.

Synthesis, anti-inflammatory activity and molecular docking studies of 2,5-diarylfuran amino acid derivatives.

A series of 2,5-diaryl substituted furans functionalized with several amino acids were synthesized and evaluated as the cyclooxygenases COX-1 and COX-2 enzymes inhibitors. The proline-substituted compound inhibited PGE(2) secretion by LPS-stimulated neutrophils, suggesting selectivity for COX-2. Molecular docking studies in the binding site of COX-2 were performed.

In vivo hydroquinone exposure alters circulating neutrophil activities and impairs LPS-induced lung inflammation in mice.

Hydroquinone (HQ) is an environmental contaminant which causes immune toxicity. In this study, the effects of exposure to low doses of HQ on neutrophil mobilization into the LPS-inflamed lung were investigated. Male Swiss mice were exposed to aerosolized vehicle (control) or 12.5, 25 or 50ppm HQ (1h/day for 5 days). One hour later, oxidative burst, cell cycle, DNA fragmentation and adhesion molecules expressions in circulating neutrophils were determined by flow cytometry, and plasma malondialdehyde (MDA) levels were measured by HPLC. Also, 1h later the last exposures, inflammation was induced by LPS inhalation (0.1mg/ml/10min) and 3h later, the numbers of leukocytes in peripheral blood and in the bronchoalveolar lavage fluid (BALF) were determined using a Neubauer chamber and stained smears; adhesion molecules expressed on lung microvessel endothelial cells were quantified by immunohistochemistry; myeloperoxidase (MPO) activity was measured in the lung tissue by colorimetric assay; and cytokines in the BALF were determined by ELISA. In vivo HQ exposure augmented plasma MDA levels and oxidative activity of neutrophils, but did not cause alterations in cell cycle and DNA fragmentation. Under these conditions, the number of circulating leukocytes was not altered, but HQ exposure reduced LPS-induced neutrophil migration into the alveolar space, as these cells remained in the lung tissue. The impaired neutrophil migration into BALF may not be dependent on reduced cytokines secretions in the BALF and lung endothelial adhesion molecules expressions. However, HQ exposure increased the expression of ß(2) and ß(3) integrins and platelet-endothelial cell adhesion molecule-1 (PECAM-1) in neutrophils, which were not further enhanced by fMLP in vitro stimulation, indicating that HQ exposure activates circulating neutrophils, impairing further stimulatory responses. Therefore, it has been shown, for the first time, that neutrophils are target of lower levels of in vivo HQ exposure, which may be considered in host defense in infectious diseases.

Hydroquinone stimulates inflammatory functions in microvascular endothelial cells via NF-κB nuclear activation.

Hydroquinone impairs several leucocyte cell functions, which alter the immune response. Although endothelial cell functions are important for the development of immune responses, hydroquinone actions on endothelial cell have not been shown. Therefore, the effect of hydroquinone exposure (10 or 100 µM for 2 hr) on primary culture of microvascular endothelial cells (PMECs) obtained from the cremaster muscle of Wistar rats incubated in the presence or absence of lipopolysaccharide (LPS, 2 µg/mL) was investigated. Hydroquinone treatment induced the membrane expression of cell adhesion molecules (CAMs) from the immunoglobulin superfamilies ICAM-1 (intercellular), VCAM-1(vascular) and PECAM-1 (platelet endothelial) and induced the secretion of cytokines interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNF-α). The effects were dependent on transcriptional modifications because enhanced CAM mRNA expression as well as both cytokines and nuclear factor κB (NF-κB) nuclear activation was found. These effects may be due to the direct action of hydroquinone rather than its quinone metabolites, because endothelial cells do not present myeloperoxidase enzyme and hydroquinone incubation did not induce the expression of cytochrome P450 2E1 (CYP2E1) or prostaglandin H synthase 1. In addition, the incubation of endothelial cells with benzoquinone (10 µM, 2 hr) impaired PECAM-1 expression and did not modify NF-κB nuclear activation. Taken together, the data herein presented reveal that hydroquinone evokes pro-inflammatory properties in endothelial cells that are triggered by the enhancement of NF-κB nuclear translocation-dependent gene transcription.

Amphetamine modulates cellular recruitment and airway reactivity in a rat model of allergic lung inflammation.

Asthma is characterized by pulmonary cellular infiltration, vascular exudation and airway hyperresponsiveness. Several drugs that modify central nervous system (CNS) activity can modulate the course of asthma. Amphetamine (AMPH) is a highly abused drug that presents potent stimulating effects on the CNS and has been shown to induce behavioral, biochemical and immunological effects. The purpose of this study was to investigate the effects of AMPH on pulmonary cellular influx, vascular permeability and airway reactivity. AMPH effects on adhesion molecule expression, IL-10 and IL-4 release and mast cell degranulation were also studied. Male Wistar rats were sensitized with ovalbumin (OVA) plus alum via subcutaneous injection. One week later, the rats received another injection of OVA-alum (booster). Two weeks after this booster, the rats were subjected to AMPH treatment 12 h prior to the OVA airway challenge. In rats treated with AMPH, the OVA challenge reduced cell recruitment into the lung, the vascular permeability and the cellular expression of ICAM-1 and Mac-1. Additionally, elevated levels of IL-10 and IL-4 were found in samples of lung explants from allergic rats. AMPH treatment, in comparison, increased IL-10 levels but reduced those of IL-4 in the lung explants. Moreover, the tracheal responsiveness to methacholine (MCh), as well as to an in vitro OVA challenge, was reduced by AMPH treatment, and levels of PCA titers were not modified by the drug. Our findings suggest that single AMPH treatment down-regulates several parameters of lung inflammation, such as cellular migration, vascular permeability and tracheal responsiveness. These results also indicate that AMPH actions on allergic lung inflammation include endothelium-leukocyte interaction mechanisms, cytokine release and mast cell degranulation.