Search

OBJECTIVE: Hereditary periodic fever syndromes (HPFs) develop as a result of uncontrolled activation of the inflammatory response, with a substantial contribution from interleukin-1beta or tumor necrosis factor alpha (TNFalpha). The HPFs include familial Mediterranean fever (FMF), hyperimmunoglobulinemia D with periodic fever syndrome (HIDS), TNF receptor-associated syndrome (TRAPS), and cryopyrinopathies, which are attributable to mutations of the MEFV, MVK, TNFRSF1A, and CIAS1 genes, respectively. However, in many patients, the mutated gene has not been determined; therefore, the condition in these patients with an HPF-like clinical picture is referred to as idiopathic periodic fever (IPF). The aim of this study was to assess involvement of X-linked inhibitor of apoptosis (XIAP), which plays a role in caspase inhibition and NF-kappaB signaling, both of which are processes that influence the development of inflammatory cells. METHODS: The XIAP gene (X-linked) was sequenced in 87 patients with IPF, 46 patients with HPF (13 with HIDS, 17 with TRAPS, and 16 with FMF), and 182 healthy control subjects. The expression of different alleles was evaluated by sequencing XIAP-specific complementary DNA mini-libraries and by real-time polymerase chain reaction and Western blot analyses. The functional effect of XIAP on caspase 9 activity was assessed by a fluorimetric assay, and cytokine secretion was evaluated by enzyme-linked immunosorbent assay. RESULTS: Sequencing disclosed a 1268A>C variation that caused a Q423P amino acid substitution. The frequency of 423Q-homozygous female patients and 423Q-hemizygous male patients was significantly higher in the IPF group than in the control group (69% versus 51%; odds ratio 2.17, 95% confidence interval 1.23-3.87, P = 0.007), whereas no significant difference was detected in the HPF group (59%) compared with controls. In primary lymphocytes and transfected cell lines, 423Q, as compared with 423P, was associated with higher XIAP protein and messenger RNA expression and lower caspase 9 activation. In lipopolysaccharide-activated monocytes, 423Q was associated with higher secretion of TNFalpha. CONCLUSION: These results suggest that 423Q is a predisposing factor for IPF development, possibly through its influence on monocyte function. [ABSTRACT FROM AUTHOR]

In previous studies, we have shown that mice carrying the hypomorphic mutation which reduce the expression of the transcription factor GATA1 in Mk, the GATA1low mutation, develop myelofibrosis, a phenotype which resemble primary myelofibrosis, the most severe of the myeloproliferative neoplasms. The phenotype is driven by high levels of TGF-beta and the bone marrow from Gata1low mice, as those from the patients, is characterized by a strong activation of non-canonical TGFß signaling including high level of expression of the transcription factor c-Jun which regulates myelo-monocytic maturation. More recently, over-expression of c-Jun has been described to be responsible for multi-organ fibrosis in mice [1]. The strong c-Jun signature suggested to us that also Gata1low mice may develop multi-organ fibrosis with age. This hypothesis was tested by histo-pathological analyses of bone marrow, spleen, liver, skin, lungs, heart and kidney of Gata 1low mice and of their wild-type littermates at 1-, 8- and 15-months of age. Cellular organization was detected by hematoxylin-eosin staining while fibrosis was detected by Gomory and Mallory staining. In addition to bone marrow and spleen, fibrosis was detected in skin, lung, heart and kidney but not in liver. At 1-month, fibrosis was detected only in skin and showed a tissue distribution resembling that observed in Scleroderma. At 8-months, fibrosis was detected in bone marrow, lung and heart. In the lung, the alveoli has thickened walls and fibers bundles were observed on the bronchus walls. In the heart, collagen fibers appeared of variable thickness and the cardiomyocytes had abnormal morphology which a strong reduction of intercalar disks. In the kidney, fibrosis was observed at 15-months and was localized in the medullary and nephron region. In conclusion, in addition to myelofibrosis, Gata1low mice may represent models for scleroderma, idiopathic pulmonary fibrosis and heart and kidney fibrosis, depending on age. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Lower levels of selenium and vitamin E have been described in gestational diabetes, a condition similar to type 2 diabetes, but few data are available about zinc (known to be associated with diabetes) and gestational hyperglycemia. This study evaluated the dietary intake of antioxidant vitamins, zinc, selenium, and serum levels of zinc and selenium in women with gestational hyperglycemia and normoglycemia. METHODS: A food-frequency questionnaire was administered to 504 pregnant women (210 with hyperglycemia and 294 with normoglycemia). Serum levels of zinc and selenium were analyzed during pregnancy in a second cohort of 71 hyperglycemic and 123 normoglycemic women, with a mean age and body mass index similar to those in the first cohort. RESULTS: Dietary intakes of zinc and selenium were significantly lower in hyperglycemic patients. In multiple logistic regression analysis, intakes were negatively associated with gestational hyperglycemia (odds ratios of 0.89 for zinc and 0.97 for selenium) after multiple adjustments. There were no significant differences in vitamin intakes. In the second cohort of 194 patients, serum levels of zinc and selenium were significantly lower in patients who had impaired glucose tolerance and negatively associated with gestational hyperglycemia in a multiple logistic regression model (odds ratios of 0.93 for serum zinc and 0.92 for serum selenium). CONCLUSIONS: Our data suggested a significant inverse association of dietary intakes and serum levels of zinc and selenium with gestational hyperglycemia. If future studies confirm these results, it might be a useful interventional approach to appropriate dietary counseling in order to evaluate the possible decrease in gestational metabolic abnormalities and their adverse consequences. [ABSTRACT FROM AUTHOR]

The need to provide accurate predictions in the evolution of the COVID-19 epidemic has motivated the development of different epidemiological models. These models require a careful calibration of their parameters to capture the dynamics of the phenomena and the uncertainty in the data. This work analyzes different parameters related to the personal evolution of COVID-19 (i.e., time of recovery, length of stay in hospital and delay in hospitalization). A Bayesian Survival Analysis is performed considering the age factor and period of the epidemic as fixed predictors to understand how these features influence the evolution of the epidemic. These results can be easily included in the epidemiological SIR model to make prediction results more stable.

Lockdown and social distancing, as well as testing and contact tracing, are the main measures assumed by the governments to control and limit the spread of COVID-19 infection. In reason of that, special attention was recently paid by the scientific community to the mathematical modeling of infection spreading by including in classical models the effects of the distribution of contacts between individuals. Among other approaches, the coupling of the classical SIR model with a statistical study of the distribution of social contacts among the population, led some of the present authors to build a Social SIR model, able to accurately follow the effect of the decrease in contacts resulting from the lockdown measures adopted in various European countries in the first phase of the epidemic. The Social SIR has been recently tested and improved through a fruitful collaboration with the Health Protection Agency (ATS) of the province of Pavia (Italy), that made it possible to have at disposal all the relevant data relative to the spreading of COVID-19 infection in the province (half a million of people), starting from February 2020. The statistical analysis of the data was relevant to fit at best the parameters of the mathematical model, and to make short-term predictions of the spreading evolution in order to optimize the response of the local health system.

In 2002, we discovered that mice carrying the hypomorphic Gata1 low mutation that reduces expression of the transcription factor GATA1 in megakaryocytes (Gata1 low mice) develop myelofibrosis, a phenotype that recapitulates the features of primary myelofibrosis (PMF), the most severe of the Philadelphia-negative myeloproliferative neoplasms (MPNs). At that time, this discovery had a great impact on the field because mutations driving the development of PMF had yet to be discovered. Later studies identified that PMF, as the others MPNs, is associated with mutations activating the thrombopoietin/JAK2 axis raising great hope that JAK inhibitors may be effective to treat the disease. Unfortunately, ruxolitinib, the JAK1/2 inhibitor approved by FDA and EMEA for PMF, ameliorates symptoms but does not improve the natural course of the disease, and the cure of PMF is still an unmet clinical need. Although GATA1 is not mutated in PMF, reduced GATA1 content in megakaryocytes as a consequence of ribosomal deficiency is a hallmark of myelofibrosis (both in humans and mouse models) and, in fact, a driving event in the disease. Conversely, mice carrying the hypomorphic Gata1 low mutation express an activated TPO/JAK2 pathway and partially respond to JAK inhibitors in a fashion similar to PMF patients (reduction of spleen size but limited improvement of the natural history of the disease). These observations cross-validated Gata1 low mice as a bona fide animal model for PMF and prompted the use of this model to identify abnormalities that might be targeted to cure the disease. We will summarize here data generated in Gata1 low mice indicating that the TGF-β/P-selectin axis is abnormal in PMF and represents a novel target for its treatment., (© 2019 International Union of Biochemistry and Molecular Biology.)

Myelofibrosis is the advanced stage of the Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), characterized by systemic inflammation, hematopoietic failure in the bone marrow, and development of extramedullary hematopoiesis, mainly in the spleen. The only potentially curative therapy for this disease is hematopoietic stem cell transplantation, an option that may be offered only to those patients with a compatible donor and with an age and functional status that may face its toxicity. By contrast, with the Philadelphia-positive MPNs that can be dramatically modified by inhibitors of the novel BCR-ABL fusion-protein generated by its genetic lesion, the identification of the molecular lesions that lead to the development of myelofibrosis has not yet translated into a treatment that can modify the natural history of the disease. Therefore, the cure of myelofibrosis remains an unmet clinical need. However, the excitement raised by the discovery of the genetic lesions has inspired additional studies aimed at elucidating the mechanisms driving these neoplasms towards their final stage. These studies have generated the feeling that the cure of myelofibrosis will require targeting both the malignant stem cell clone and its supportive microenvironment. We will summarize here some of the biochemical alterations recently identified in MPNs and the novel therapeutic approaches currently under investigation inspired by these discoveries., Competing Interests: No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed., (Copyright: © 2019 Eran Z et al.)

Cultures of stem cells from discarded sources supplemented with dexamethasone, a synthetic glucocorticoid receptor agonist, generate cultured red blood cells (cRBCs) in numbers sufficient for transfusion. According to the literature, however, erythroblasts generated with dexamethasone exhibit low enucleation rates giving rise to cRBCs that survive poorly in vivo . The knowledge that the glucocorticoid receptor regulates lipid metabolism and that lipid composition dictates the fragility of the plasma membrane suggests that insufficient lipid bioavailability restrains generation of cRBCs. To test this hypothesis, we first compared the expression profiling of erythroblasts generated with or without dexamethasone. This analysis revealed differences in expression of 55 genes, 6 of which encoding proteins involved in lipid metabolism. These were represented by genes encoding the mitochondrial proteins 3-Hydroxymethyl-3-Methylglutaryl-CoA lyase, upregulated, and 3-Oxoacid CoA-Transferase1 and glycerol-3-phosphate acyltransferase1, both downregulated, and the proteins ATP-binding cassette transporter1 and Hydroxysteroid-17-Beta-Dehydrogenase7, upregulated, and cAMP-dependent protein kinase catalytic subunit beta, downregulated. This profiling predicts that dexamethasone, possibly by interfering with mitochondrial functions, impairs the intrinsic lipid metabolism making the synthesis of the plasma membrane of erythroid cells depend on lipid-uptake from external sources. Optical and electron microscopy analyses confirmed that the mitochondria of erythroblasts generated with dexamethasone are abnormal and that their plasma membranes present pebbles associated with membrane ruptures releasing exosomes and micro-vesicles. These results indicate that the lipid supplements of media currently available are not adequate for cRBCs. To identify better lipid supplements, we determined the number of erythroblasts generated in synthetic media supplemented with either currently used liposomes or with lipoproteins purified from human plasma [the total lipoprotein fraction (TL) or its high (HDL), low (LDL) and very low (VLDL) density lipoprotein components]. Both LDL and VLDL generated numbers of erythroid cells 3-2-fold greater than that observed in controls. These greater numbers were associated with 2-3-fold greater amplification of erythroid cells due both to increased proliferation and to resistance to stress-induced death. In conclusion, dexamethasone impairs lipid metabolism making ex vivo expansion of erythroid cells highly dependent on lipid absorbed from external sources and the use of LDL and VLDL as lipid supplements improves the generation of cRBCs.

The physiological effects of acetylcholine on keratinocytes depend on the presence of nicotinic and muscarinic receptors. The role of nonneuronal acetylcholine in keratinocytes could have important clinical implications for patients with various skin disorders such as nonhealing wounds. In order to evaluate the efficacy of highly diluted acetylcholine solutions obtained by sequential kinetic activation, we aimed to investigate the effects of these solutions on normal human keratinocytes. Two different concentrations (10 fg/mL and 1 pg/mL) and formulations (kinetically activated and nonkinetically activated) of acetylcholine were used to verify keratinocyte viability, proliferation, and migration and the intracellular pathways involved using MTT, crystal violet, wound healing, and Western blot compared to 147 ng/mL acetylcholine. The activated formulations (1 pg/mL and 10 fg/mL) revealed a significant capacity to increase migration, cell viability, and cell proliferation compared to 147 ng/mL acetylcholine, and these effects were more evident after a single administration. Sequential kinetic activation resulted in a statistically significant decrease in reactive oxygen species production accompanied by an increase in mitochondrial membrane potential and a decrease in oxygen consumption compared to 147 ng/mL acetylcholine. The M1 muscarinic receptor was involved in these effects. Finally, the involvement of ERK/mitogen-activated protein kinases (MAPK) and KI67 confirmed the effectiveness of the single treatment on cell proliferation. The intracellular pathways of calcium were investigated as well. Our results indicate for the first time that highly diluted and kinetically activated acetylcholine seems to play an active role in an in vitro model of wound healing. Moreover, the administration of acetylcholine within the physiological range may not only be effective but is also likely to be safe., (© 2016 S. Karger AG, Basel.)

Background: Gastric diseases are a worldwide problem in modern society, as reported in the USA, in the range of 0.5-2 episodes/year/person and an incidence of 5-100 episodes/1000/week according to seasons and age. There is convincing evidence that oxidative stress is involved in the pathogenesis of acute gastric injury. Acid secreted from gastric parietal cells determines mucosal injuries which in turn cause inflammation and oxidative stress. Consequent inflammation produces free radicals by mitochondria thus causing lipid peroxidation, oxidative and acidic stress, which can lead to cell apoptosis. Vitamin D 3, the active form of vitamin D, may counteract intracellular cell death and improve epithelial regeneration., Methods: This study was planned to assess whether vitamin D 3 is a protective factor against acid injury and oxidative stress in gastric epithelial cells. Primary epithelial cells and GTL-16 cells have been used to test the effects of Grisù® alone or in combination with vitamin D 3 during oxidative stress or high acid exposition measuring cell viability, ROS production, cellular adhesion time along with apoptotic, autophagic and survival pathways. The combined effect of Grisù® and vitamin D 3 was found more effective in counteracting the negative consequences of oxidative stress and acidity conditions than some other gastroprotective agents, such as Maalox® or Gaviscon®., Results: In case of oxidative stress or acidity condition the stimulation with Grisù® alone caused an improvement of cell viability and a reduction of ROS production on epithelial gastric cells. In addition, the adhesion time of the cells was improved. All these effects were increased by the presence of vitamin D 3 . Similar data were also observed in primary gastric epithelial cells confirming the results obtained in GTL-16 cells., Conclusions: These results suggest that Grisù® in combination with vitamin D 3 may exert a gastroprotective effect to maintain or restore the integrity of gastric epithelium through an antioxidant pathway, inhibiting apoptosis and activating survival kinases. Moreover, the combination of Grisù® and vitamin D 3 improves cell viability and decreases ROS production compared to other gastroprotective agents combined with vitamin D 3 . All these data were validated using primary cells isolated from gastric tissue.

Recent studies have postulated a role for vitamin D and its receptor on cerebral function, and anti-inflammatory, immunomodulatory and neuroprotective effects have been described; vitamin D can inhibit proinflammatory cytokines and nitric oxide synthesis during various neurodegenerative insults, and may be considered as a potential drug for the treatment of these disorders. In addition, iron is crucial for neuronal development and neurotransmitter production in the brain, but its accumulation as catalytic form (Fe(3+)) impairs brain function and causes the dysregulation of iron metabolism leading to tissue damage due to the formation of toxic free radicals (ROS). This research was planned to study the role of vitamin D to prevent iron damage in neuroblastoma BE(2)M17 cells. Mechanisms involved in neurodegeneration, including cell viability, ROS production, and the most common intracellular pathways were studied. Pretreatment with calcitriol (the active form of vitamin D) reduced cellular injury induced by exposure to catalytic iron., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Background: Neurokinin 1 receptors (NK-1R) have been involved in several psychiatric disorders including major depression, but less is known for bipolar disorder (BD)., Method: We compared NK-1R expression and Substance P (SP) ability to induce NF-κB activation in monocytes from BD patients and healthy donors (HD), also looking for the effects of tobacco smoke. After informed written consent, 20 euthymic BD patients, either bipolar type 1 (BDI) or type 2 (BDII), and 14 age-matched healthy donors (HD) were enrolled. NK-1R expression in monocytes was evaluated by Western blot and expressed as the ratio between NK-1R and Na(+)/K(+)-ATPase protein expressions. NF-κB activation was assessed by measuring the nuclear content of the p50 subunit (ELISA kit)., Results: NK-1R expression was significantly reduced (P<0.001) in monocytes from BD patients as compared to HD, with no major differences between BDI and BDII patients. Tobacco smoke enhanced NK-1R expression in HD, but not in BD patients. Un-stimulated monocytes from BD patients presented a constitutively higher (P<0.05) content of nuclear p50 subunit as compared to HD. SP and an NK-1R agonist induced NF-κB activation, with a higher effect in HD: this effect was receptor-mediated as it was abrogated by an NK-1R antagonist., Limitations: As a pilot study enrolling 20 BD patients, an obvious limitation is the sample size., Conclusions: Our results show the existence of a relevant alteration in NK-1R expression in BD patients and further suggest SP involvement in BD, so improving our understanding of the underlying mechanisms of this disease., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Aims: Cyclooxygenase (COX)-inhibiting nitric oxide donors (CINODs) are a new class of drugs that structurally combine a COX inhibitor with a nitric oxide (NO) donating moiety. This combination reduces potential toxicity of the non-steroidal anti-inflammatory drugs (NSAIDs) whilst maintaining the analgesic and anti-inflammatory effects. The present study was undertaken to investigate the anti-inflammatory effects of NCX 429, a naproxen-based CINOD, and to assess the additional properties of NO donation beyond those related to naproxen., Main Methods: We evaluated the in vitro effects of NCX 429 on oxy-radical production, phagocytosis, cytokine release, MMP-9, PPARγ expression and NF-κB activation in human monocytes/MDM and compared to naproxen. Moreover, we compared the in vivo efficacy of NCX 429 and naproxen in a murine model of peritonitis., Key Findings: In all the experiments performed in vitro, NCX 429 reduced the inflammatory responses with equal or higher efficacy compared to naproxen. Moreover, in in vivo experiments, NCX 429, at the lowest dose tested, was able to significantly inhibit cell influx in response to IL-1β administration although naproxen was found to be more potent than NCX 429 at reducing PGE2 in inflammatory exudates., Significance: These results demonstrate that both in vitro and in vivo--in a murine model of peritonitis--NCX 429 elicits significant anti-inflammatory activity, beyond the simple COX inhibition or pure NO release. Therefore, NO donation along with COX inhibition may represent a strategy for investigating inflammatory diseases in which pain and function are not fully resolved by analgesics/anti-inflammatory drugs., (© 2015.)

Microparticles (MPs) are membrane fragments that may play a role in the pathogenesis of chronic respiratory diseases. We aimed to investigate whether human monocytes/macrophage-derived MPs could induce a pro-inflammatory phenotype in human bronchial smooth muscle cells (BSMC) and the effect of montelukast in this setting. Experimental methods included isolation of human monocytes/macrophages and generation of monocyte-derived MPs, RT-PCR analysis of gene expression, immunoenzymatic determination of pro-inflammatory factor release, bioluminescent assay of intracellular cAMP levels and electromobility shift assay analysis of NF-κB nuclear translocation. Stimulation of human BSMC with monocyte-derived MPs induced a pro-inflammatory switch in human BSMC by inducing gene expression (COX-2 and IL-8), protein release in the supernatant (PGE2 and IL-8), and heterologous β2-adrenoceptor desensitization. The latter effect was most likely related to autocrine PGE2 since pre-treatment with COX inhibitors restored the ability of salbutamol to induce cAMP synthesis in desensitized cells. Challenge with MPs induced nuclear translocation of NF-κB and selective NF-κB inhibition decreased MP-induced cytokine release in the supernatant. Montelukast treatment prevented IL-8 release and heterologous β2-adrenoceptor desensitization in human BSMC exposed to monocyte-derived MPs by blocking NF-κB nuclear translocation. These findings provide evidence on the role of human monocyte-derived MPs in the airway smooth muscle phenotype switch as a novel potential mechanism in the progression of chronic respiratory diseases and on the protective effects by montelukast in this setting., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Increasing evidence suggests that tachykinins are involved in the control of different pathological conditions, including psychiatric disorders. In this study we evaluated the expression of NK(1) and NK(2) receptors (NK-1R and NK-2R), as well as the effects of substance P (SP) and neurokinin A (NKA), in monocytes isolated from 15 healthy subjects and 15 patients with recurrent major depressive disorder (RMDD), under stable antidepressant therapy. NK-1R expression in monocytes from RMDD patients was significantly decreased as compared to healthy subjects, whereas NK-2R expression was markedly increased. Both NK-1R and NK-2R expression correlated with HAM-D, but not HAM-A, score. SP, NKA and selective NK-1R and NK-2R agonists stimulated TNF-α release in monocytes of both groups, with a significant higher effect observed in RMDD. Moreover they induced NF-κB activation, which was reversed by selective NK-1R and NK-2R antagonists, so demonstrating that it was receptor-mediated. The occurrence of a profound alteration in NK receptor expression in RMDD is a novel finding that suggests NK-1R and NK-2R pathways as possible relevant players in major depressive disorder, so improving our understanding of the complex pathogenesis of the disease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Context: Mutations within the PROP1 gene represent one of the main causes of familial combined pituitary hormone deficiency (CPHD). However, most of the cases are sporadic with an unknown genetic cause., Objective: The aim of this study was the search for low penetrance variations within and around a conserved regulatory element in the intron 1 of PROP1, contributing to a multifactorial form of the disease in sporadic patients., Methods and Patients: A fragment of 570 bp encompassing the conserved region was sequenced in 107 CPHD patients and 294 controls, and an association study was performed with the four identified variants, namely c.109+435G>A (rs73346254), c.109+463C>T (rs4498267), c.109+768C>G (rs4431364), and c.109+915&#95;917ins/delTAG (rs148607624). The functional role of the associated polymorphisms was evaluated by luciferase reporter gene expression analyses and EMSA., Results: A statistically significant increased frequency was observed in the patients for rs73346254A (P = 5 × 10(-4)) and rs148607624delTAG (P = 0.01) alleles. Among all the possible allele combinations, only the haplotype bearing both risk alleles showed a significantly higher frequency in the patients vs. controls (P = 4.7 × 10(-4)) and conferred a carrier risk of 4.19 (P = 1.2 × 10(-4)). This haplotype determined a significant decrease of the luciferase activity in comparison with a basal promoter and the other allelic combinations in GH4C and MCF7 cells (P = 4.6 × 10(-6); P = 5.5 × 10(-4), respectively). The EMSA showed a differential affinity for nuclear proteins for the alternative alleles of the two associated variations., Conclusions: Variations with a functional significance conferring susceptibility to CPHD have been identified in the PROP1 gene, indicating a multifactorial origin of this disorder in sporadic cases.

Microparticles (MP) are phospholipid vesicles shed by cells upon activation or apoptosis. Monocyte-derived MP upregulate the synthesis of proinflammatory mediators by lung epithelial cells; the molecular bases of such activity are unknown. Peroxisome proliferator-activated receptors (PPAR) have been demonstrated to be involved in the modulation of nuclear factor (NF)-κB transcriptional activity and inflammation. We investigated whether the upregulation of the synthesis of proinflammatory cytokines by human lung epithelial cells induced by monocyte/macrophage-derived MP involves NF-κB activation and is modulated by PPAR-γ. MP were generated by stimulation of human monocytes/macrophages with the calcium ionophore, A23187. MP were incubated with human lung epithelial cells. NF-κB translocation was assessed by electrophoretic mobility shift assay. Interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 synthesis was assessed by ELISA and RT-PCR. Stimulation of A549 alveolar cells with monocyte/macrophage-derived MP caused an increase in NF-κB activation and IL-8 and MCP-1 synthesis that was inhibited by pre-incubation with the PPAR-γ agonists, rosiglitazone and 15-deoxy-Δ12,14-prostaglandin-J2. Parallel experiments with normal human bronchial epithelial cells largely confirmed the results. The effects of PPAR-γ agonists were reversed by the specific antagonist, GW9662. Upregulation of the synthesis of proinflammatory mediators by human lung epithelial cells induced by monocyte/macrophage-derived MP is mediated by NF-κB activation through a PPAR-γ dependent pathway.

There is a great interest in the potential health benefits of biologically active phenolic compounds in cocoa (Theobroma cacao) and dark chocolate. We investigated the anti-inflammatory potential of clovamide (a N-phenylpropenoyl-L-amino acid amide present in cocoa beans) and two phenolic extracts from unroasted and roasted cocoa beans, by evaluating superoxide anion (O(2)(-)) production, cytokine release, and NF-κB activation in human monocytes stimulated by phorbol 12-myristate 13-acetate (PMA). The effects of rosmarinic acid are shown for comparison. Clovamide and rosmarinic acid inhibited PMA-induced O(2)(-) production and cytokine release (with a bell-shaped curve and maximal inhibition at 10-100 nM), as well as PMA-induced NF-κB activation; the two cocoa extracts were less effective. In all tests, clovamide was the most potent compound and also enhanced peroxisome proliferator-activated receptor-γ (PPARγ) activity, which may exert anti-inflammatory effects. These findings indicate clovamide as a possible bioactive compound with anti-inflammatory activity in human cells.

Background and Purpose: β₂-Adrenoceptor agonists are important therapeutic agents in the treatment of asthma and chronic obstructive pulmonary disease. The regular use of these drugs has been associated with proasthmatic-like changes that limit their efficacy and increase the risk of severe adverse reactions. We investigated whether the peroxisome-proliferator-activated receptor (PPAR)γ agonist rosiglitazone modulated salbutamol-induced β₂-adrenoceptor desensitization in vivo and in vitro., Experimental Approach: An in vivo model of homologous β₂-adrenoceptor desensitization, established in guinea-pigs by administering salbutamol continuously, was used to study the ability of rosiglitazone to prevent β₂-adrenoceptor tolerance. In vitro experiments on human bronchial smooth muscle cells were performed to increase the clinical relevance of the study., Key Results: In tracheal smooth muscle tissues from desensitized animals, we observed a decrease in the protective effect of salbutamol on carbachol-induced contraction, a hyperresponsiveness to cholinergic stimuli, a modest underexpression of β₂-adrenoceptor gene and a marked decrease in β-adrenoceptor number, relative to control values. Treatment with rosiglitazone preserved salbutamol relaxant activity, mitigated carbachol hyperresponsiveness and partially restored β₂-adrenoceptor binding sites in tracheal tissues from homologously desensitized animals. The highly selective PPARγ agonist, GW1929, reproduced the effect of rosiglitazone, in vivo. In vitro β₂-adrenoceptor desensitization decreased salbutamol-mediated cAMP production, without affecting forskolin responses and β₂-adrenoceptor expression. Rosiglitazone and 15-deoxy-Δ¹²(,)¹⁴-prostaglandin J₂ restored salbutamol sensitivity in homologously desensitized cells., Conclusions and Implications: These data suggest a potential pharmacodynamic interaction between PPARγ agonists and salbutamol on airway smooth muscle responsiveness, supporting the therapeutic potential of this combination in chronic airway disease., (© 2010 The Authors. British Journal of Pharmacology © 2010 The British Pharmacological Society.)

Previous studies have shown that NCX 6550 (NCX), a nitric oxide (NO)-donating pravastatin, induces anti-inflammatory effects in murine macrophage cell lines. Here, we have studied its activity in human monocyte/macrophages, by investigating cytokine release, NF-κB translocation and peroxisome proliferator-activated receptor γ (PPARγ) expression and function. For comparison, pravastatin, isosorbide-5-mononitrate (ISMN), sodium nitroprusside (SNP) and the PPARγ ligand 15-deoxy-Δ(12,14)-prostaglandin J(2) (PGJ) were also tested. Monocytes and macrophages (MDM: monocyte-derived macrophages) were isolated from healthy donors; cytokine release was measured by ELISA, NF-κB by electrophoretic mobility shift assay and PPARγ by Western blot and Real-Time PCR. NCX (1 nM-50 μM) dose-dependently inhibited phorbol 12-myristate 13-acetate (PMA)-induced TNF-α release from monocytes (IC(50)=240 nM) and MDM (IC(50)=52 nM). At 50 μM, it was more effective than pravastatin, ISMN and SNP (P<0.05), but less efficient than PGJ. Similar results were obtained for IL-6. Likewise, NCX was more effective than pravastatin and the other NO donors in inhibiting PMA-induced NF-κB translocation in both cell types, and, at the highest concentration, significantly (P<0.05) enhanced PPARγ protein expression in monocytes. We conclude that NCX 6550 exerts a significant anti-inflammatory activity in human monocyte/macrophages, that is also contributed by its NO donating properties, as the effects exerted by NCX are significantly higher than those evoked by pravastatin in many experimental assays. These data further indicate that the incorporation of a NO-donating moiety into a statin structure confers pharmacological properties which may translate into useful therapeutic benefits., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Objective: Atherosclerosis and restenosis are largely ruled by inflammation. The aim of this study was to test the effects of a short-course, high-dose oral prednisone on the release of interleukin-6 (IL-6) and tumour necrosis factor (TNF)-alpha from circulating monocytes and on the neointimal growth that follows bare metal stent (BMS) implantation. In a sub-group of patients activated NF-kappaB was also evaluated., Methods: Out of 40 patients with coronary artery disease treated with BMS implantation, 20 were randomly assigned to receive oral prednisone during 40 days according to a standardized protocol. In non-stimulated and stimulated (LPS and PMA) monocytes we evaluated the release of IL-6 and TNF-alpha, and NF-kappaB p50 subunit translocation at baseline, at 10 and 30 days. Late luminal loss (LLL) 9 months after angioplasty was calculated by quantitative coronary angiography., Results: Plasma concentrations of prednisone correlated inversely with IL-6 and TNF-alpha release (R2=0.45, p=0.04 and R2=0.69, p=0.005, respectively) and NF-kappaB activation from monocytes (R2=0.58, p=0.01). The reduction of TNF-alpha release and NF-kappaB activation were significantly related (R2=0.56, p=0.01). Prednisone patients showed a significantly larger reduction of cytokine release and NF-kappaB activation compared to non-treated patients, at 10 days and 30 days. LLL was lower in the prednisone group (0.44+/-0.35 mm versus 0.80+/-0.53 mm, p=0.02) and correlated with reduction of TNF-alpha (R2=0.41, p=0.01)., Conclusions: High doses of oral prednisone reduce NF-kappaB pathway activation and pro-inflammatory cytokine release in circulating activated monocytes of patients treated with coronary stenting. TNF-alpha release reduction correlates with decreased LLL., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Background and Purpose: Tobacco smoke represents a relevant risk factor for coronary heart disease (CHD). Although peroxisome proliferator-activated receptor (PPAR)gamma activation reduces inflammation and atherosclerosis, expression of PPARgamma in cells and its modulation by smoking are poorly investigated. We previously reported that monocyte/macrophages from healthy smokers exhibited an enhanced constitutive expression of PPARgamma. Here, we evaluated PPARgamma expression and basal cytokine release in monocytes and monocyte-derived macrophages (MDMs) from 85 CHD patients, classified by their smoking habit (smokers, non-smokers and ex-smokers), and assessed the role of PPARgamma ligands in this context., Experimental Approach: PPARgamma protein was detected by Western blot and semi-quantified by PPARgamma/beta-actin ratio; cytokine release was measured by elisa and nuclear factor-kappaB (NF-kappaB) translocation by electrophoretic mobility shift assays., Key Results: As compared to the other groups, MDMs from smoker CHD patients exhibited a reduced PPARgamma/beta-actin ratio and an increased spontaneous release of tumour necrosis factor-alpha (TNF-alpha) and interleukin-6, but with no major variations in monocytes. In cells from selected CHD patients, rosiglitazone inhibited TNF-alpha release and NF-kappaB translocation induced by phorbol-12-myristate 13-acetate. The selective PPARgamma antagonist GW9662 reversed these effects, with some variations related to smoking habit., Conclusions and Implications: In CHD patients, exposure to tobacco smoke profoundly affected PPARgamma expression, and this was related to levels of secretion of pro-inflammatory cytokines. MDMs from CHD smokers showed the lowest PPARgamma expression and released more inflammatory cytokines. Moreover, rosiglitazone's ability to inhibit cytokine release and its reversal by GW9662 clearly indicated PPARgamma involvement in these changes in CHD patients.

Peroxisome proliferator-activated receptor (PPAR) activation reduces inflammation and atherosclerosis, but recent evidence raised concerns about its beneficial clinical effects. However, the effects of gender on PPAR expression and basal cytokine release have not been investigated. In the present study, we evaluated PPAR-gamma and -alpha expression, as well as cytokine release, in monocyte/macrophages from 15 male and 15 female patients with coronary artery disease (CAD) in comparison with healthy controls. Both expression and activation of PPAR-alpha and PPAR-gamma proteins were evaluated by Western blot and electrophoretic mobility shift assay. Gene expression was evaluated by real-time polymerase chain reaction; cytokine release was measured by enzyme-linked immunosorbent assay. Monocyte/macrophages of CAD patients yielded a constitutively enhanced (approximately 10-fold; p < 0.001) protein expression of PPAR-gamma, but not PPAR-alpha, compared with healthy controls. Evaluation of PPAR-gamma gene expression showed a 60-fold increase in monocytes from CAD patients, compared with healthy donors. Moreover, monocytes spontaneously released higher amounts of proinflammatory cytokines than macrophages. It is interesting that monocytes from CAD females expressed significantly higher levels of PPAR-gamma protein compared with male patients (p < 0.05) and showed the lowest basal release of tumor necrosis factor-alpha. These results indicate that the expression of PPAR-gamma is significantly higher in CAD patients than in healthy donors and that, together with cytokine release, it seems to be gender-related. In fact, CAD women demonstrated the highest PPAR-gamma expression and the lowest cytokine release. Such differences may, in part, modulate the response to PPAR-gamma activators.