Slight activation of nuclear factor kappa-B is associated with increased hepatic stellate cell apoptosis in human schistosomal fibrosis

To investigate the relationship between NF-kappa B activation and hepatic stellate cell (HSC) apoptosis in hepatosplenic schistosomiasis, hepatic biopsies from patients with Schistosoma mansoni-induced periportal fibrosis, hepatitis C virus-induced cirrhosis, and normal liver were submitted to alpha-smooth muscle actin (alpha-SMA) and NF-kappa B p65 immunohistochemistry, as well as to NF-kappa B Southwestern histochemistry and TUNEL assay. The numbers of alpha-SMA-positive cells and NF-kappa B- and NF-kappa B p65-positive HSC nuclei were reduced in schistosomal fibrosis relative to liver cirrhosis. In addition, increased HSC NF-kappa B p65 and TUNEL labeling was observed in schistosomiasis when compared to cirrhosis. These results suggest a possible relationship between the slight activation of the NF-kappa B complex and the increase of apoptotic HSC number in schistosome-induced fibrosis, taking place to a reduced HSC number in schistosomiasis in relation to liver cirrhosis. Therefore, the NF-kappa B pathway may constitute an important down-regulatory mechanism in the pathogenesis of human schistosomiasis mansoni, although further studies are needed to refine the understanding of this process. (C) 2009 Elsevier B.V. All rights reserved.

CCR5 Down-regulates Osteoclast Function in Orthodontic Tooth Movement

During orthodontic tooth movement, there is local production of chemokines and an influx of leukocytes into the periodontium. CCL5 plays an important role in osteoclast recruitment and activation. This study aimed to investigate whether the CCR5-receptor influences these events and, consequently, orthodontic tooth movement. An orthodontic appliance was placed in wild-type mice (WT) and CCR5-deficient mice (CCR5(-/-)). The expression of mediators involved in bone remodeling was evaluated in periodontal tissues by Real-time PCR. The number of TRAP-positive osteoclasts and the expression of cathepsin K, RANKL, and MMP13 were significantly higher in CCR5(-/-). Meanwhile, the expression of two osteoblastic differentiation markers, RUNX2 and osteocalcin, and that of bone resorption regulators, IL-10 and OPG, were lower in CCR5(-/-). Analysis of the data also showed that CCR5(-/-) exhibited a greater amount of tooth movement after 7 days of mechanical loading. The results suggested that CCR5 might be a down-regulator of alveolar bone resorption during orthodontic movement.

Melatonin inhibits LPS-induced NO production in rat endothelial cells

Endothelial cells produce NO by activation of constitutive nitric oxide synthase (NOS) and transcription of inducible NOS (iNOS). We have previously shown that melatonin, in the nanomolar range, inhibits activation of constitutive NOS, and in the present paper, we evaluated whether it could interfere with the expression of iNOS, which is activated by lipopolysaccharide (LPS), a major component of gram-negative bacteria cell walls. Primary cultures of rat endothelial cells were loaded with fluorescent probe for NO detection. Nuclear factor kappa B (NF-kappa B) translocation in endothelial cells elicited by LPS was measured by electromobility shift assay, and the vasodilation of aortic rings was accessed by recording isometric contraction. Melatonin in a micromolar but not in a nanomolar range inhibits the NO production induced by LPS. This effect is not dependent on the activation of G protein-coupled melatonin receptors. The nuclear NF-kappa B translocation is a process necessary for iNOS transcription, and melatonin also inhibits its translocation. LPS induced vasodilation only in endothelium-intact aortic rings, and melatonin (10 mu m) inhibits the vasodilation. Here, we show that concentrations compatible with nocturnal melatonin surge (nm) did not interfere with the activity of iNOS. Considering that micromolar melatonin concentrations could be locally achieved through production by activated immune competent cells, extra-pineal melatonin could have a protective effect against tissue injury. We propose that melatonin blocked the LPS-induced vasodilation by inhibiting the NF-kappa B pathway. Finally, we propose that the effect of melatonin on vascular reactivity is one of the mechanisms that underlies the protective effect of this indolamine against LPS.

Augmentation of insulin secretion by leucine supplementation in malnourished rats: possible involvement of the phosphatidylinositol 3-phosphate kinase/mammalian target protein of rapamycin pathway

A regimen of low-protein diet induces a reduction of pancreatic islet function that is associated with development of metabolic disorders including diabetes and obesity afterward. In the present study, the influence of leucine supplementation on metabolic parameters, insulin secretion to glucose and to amino acids, as well as the levels of proteins that participate in the phosphatidylinositol 3-phosphate kinase (PI3K) pathway was investigated in malnourished rats. Four groups were fed with different diets for 12 weeks: a normal protein diet (17%) without (NP) or with leucine supplementation (NPL) or a low (6%)-protein diet without (LP) or with leucine supplementation (LPL). Leucine was given in the drinking water during the last 4 weeks. As indicated by the intraperitoneal glucose tolerance test, LPL rats exhibited increased glucose tolerance as compared with NPL group. Both NPL and LPL rats had higher circulating insulin levels than controls. The LPL rats also showed increased insulin secretion by pancreatic islets in response to glucose or arginine compared with those observed in islets from LP animals. Glucose oxidation was significantly reduced in NPL, LP, and LPL isolated islets as compared with NP; but no alteration was observed for leucine and glutamate oxidation among the 4 groups. Western blotting analysis demonstrated increased PI3K and mammalian target protein of rapamycin protein contents in LPL compared with LP islets. A significant increase in insulin-induced insulin receptor substrate I associated PI3K activation was also observed in LPL compared with LP islets. These findings indicate that leucine supplementation can augment islet function in malnourished rats and that activation of the PI3K/maminalian target protein of rapamycin pathway may play a role in this process. (C) 2010 Elsevier Inc. All rights reserved.

Matrix metalloproteinases, TIMPs and growth factors regulating ameloblastoma behaviour

Aims: Ameloblastoma is an odontogenic neoplasm with local invasiveness and recurrence. We have previously suggested that growth factors and matrix metalloproteinases (MMPs) influence ameloblastoma invasiveness(1). The aim was to study expression of MMPs, tissue inhibitor of metalloproteinases (TIMPs) and growth factors in ameloblastoma. Methods and results: Thirteen cases of solid/multicystic ameloblastoma were examined. As a control, calcifying cystic odontogenic tumour (CCOT), a non-invasive odontogenic neoplasm with ameloblastomatous epithelium was also studied. Immunohistochemistry detected MMPs, TIMPs and growth factors in ameloblastoma and CCOT. The labelling index (LI) of MMP-9 and TIMP-2 was significantly higher in ameloblastoma compared with CCOT. The LI of epidermal growth factor (EGF), transforming growth factor (TGF)-alpha and epidermal growth factor receptor (EGFR) was also increased in ameloblastoma. This neoplasm showed greater expression of MMPs, TIMPs and growth factors compared with CCOT. We then analysed these molecules in ameloblastoma cells and stroma. Ameloblastoma cells exhibited increased LI of MMP-1, -2 and EGFR. We found a positive correlation between EGF and TIMP-1, and between TGF-alpha and TIMP-2. It is known that signals generated by growth factors are transduced by the ERK pathway. Ameloblastoma stroma exhibited the phosphorylated (activated) form of ERK. Conclusions: These results suggest an interplay involving growth factors MMPs and TIMPs that may contribute to ameloblastoma behaviour. Signals generated by this molecular network would be transduced by ERK 1/2 pathway.

Reduced maximal oxygen consumption and overproduction of proinflammatory cytokines in athletes

Objective: It was the aim of this study to evaluate whether chronic pain in athletes is related to performance, measured by the maximum oxygen consumption and production of hormones and cytokines. Methods: Fifty-five athletes with a mean age of 31.9 +/- 4.2 years engaged in regular competition and showing no symptoms of acute inflammation, particularly fever, were studied. They were divided into 2 subgroups according to the occurrence of pain. Plasma concentrations of adrenaline, noradrenaline, cortisol, prolactin, growth hormone and dopamine were measured by radioimmunoassay, and the production of the cytokines interleukin (IL)-1, IL-2, IL-4, IL-6, tumor necrosis factor-alpha, interferon-alpha and prostaglandin E-2 by whole-blood culture. Maximal oxygen consumption was determined during an incremental treadmill test. Results: There was no change in the concentration of stress hormones, but the athletes with chronic pain showed a reduction in maximum oxygen consumption (22%) and total consumption at the anaerobic threshold (25%), as well as increased cytokine production. Increases of 2.7-, 8.1-, 1.7- and 3.7-fold were observed for IL-1, IL-2, tumor necrosis factor-alpha and interferon-alpha, respectively. Conclusions: Our data show that athletes with chronic pain have enhanced production of proinflammatory cytokines and lipid mediators and reduced performance in the ergospirometric test. Copyright (c) 2008 S. Karger AG, Basel.

Effects of isoproterenol treatment for 7 days on inflammatory mediators in the rat aorta

The aim of the present study was to evaluate the effect of overstimulation of beta-adrenoceptors on vascular inflammatory mediators. Wistar rats were treated with the beta-adrenoceptor agonist isoproterenol (0.3 mg(.)kg(-1.)day(-1) sc) or vehicle (control) for 7 days. At the end of treatment, the right carotid artery was catheterized for arterial and left ventricular (LV) hemodynamic evaluation. Isoproterenol treatment increased LV weight but did not change hemodynamic parameters. Aortic mRNA and protein expression were quantified by real-time RT-PCR and Western blot analysis, respectively. Isoproterenol enhanced aortic mRNA and protein expression of IL-1 beta (124% and 125%) and IL-6 (231% and 40%) compared with controls but did not change TNF-alpha expression. The nuclear-to-cytoplasmatic protein expression ration of the NF-beta B p65 subunit was increased by isoproterenol treatment (51%); in addition, it reduced the cytoplasmatic expression of I kappa B-alpha (52%) in aortas. An electrophoretic mobility shift assay was performed using the aorta, and increased NF-kappa B DNA binding (31%) was observed in isoproterenol-treated rats compared with controls (P < 0.05). Isoproterenol treatment increased phenylephrine-induced contraction in aortic rigs (P < 0.05), which was significantly reduced by superoxide dismutase (150 U/ml) and sodium salicylate (5 mM). Cotreatment with thalidomide (150 mg(.)kg(-1.)day(-1) for 7 days) also reduced hyperreactivity to phenylephrine induced by isoproterenol. In conclusion, overstimulation of beta-adrenoceptors increased proinflammatory cytokines and upregulated NF-kappa B in the rat aorta. Moreover, local oxidative stress and the proinflammatory state seem to play key roles in the altered vascular reactivity of the rat aorta induced by chronic beta-adrenergic stimulation.

Amlodipine reduces the antimigratory effect of diclofenac in spontaneously hypertensive rats

Amlodipine, an antihypertensive drug, and diclofenac, an anti inflammatory drug, may generally be combined, particularly in elderly patients; therefore, the potential for their interaction is high. We aim to determine if amlodipine interferes with the antimigratory effect of diclofenac. For this, male spontaneously hypertensive rats (SHRs) were treated with either diclofenac (1 mg.kg(-1).d(-1), 15 d) alone or combined with amlodipine (10 mg.kg(-1).d(-1), 15 d). Leukocyte rolling, adherence, and migration were studied by intravital microscopy. Diclofenac did not change (180.0 +/- 2.3), whereas amlodipine combined (163.4 +/- 5.1) or not (156.3 +/- 4.3) with diclofienac reduced the blood pressure (BP) levels in SHR (183.1 +/- 4.4). Diclofenac and amlodipine reduced leukocyte adherence, migration, and ICAM-I expression, whereas only diclofenac reduced rolling leukocytes as well. Combined with amlodipine, the effect of the diclofenac was reduced. Neither treatment tested increased the venular shear rate or modified the venular diameters, number of circulating leukocytes, P-selectin, PECAM-1, L-selectin, or CD-18 expressions. No difference could be found in plasma concentrations of both drugs given alone or in association. In conclusion, amlodipine reduces leukocyte migration in SHR, reducing endothelial cell ICAM-1 expression. Amlodipine reduces the effect of the diclofenac, possibly by the same mechanism. A pharmacokinetic interaction as well as an effect on the other adhesion molecules tested could be discarded.

Administration of Neural Precursor Cells Ameliorates Renal Ischemia-Reperfusion Injury

In this study we evaluated whether administration of stem cells of neural origin (neural precursor cells, NPCs) could be protective against renal ischemia-reperfusion injury (IRI). We hypothesized that stem cell outcomes are not tissue-specific and that NPCs can improve tissue damage through paracrine mechanisms, especially due to immunomodulation. To this end, Wistar rats (200-250 g) were submitted to 1-hour ischemia and treated with NPCs (4 x 10(6) cells/animal) at 4 h of reperfusion. To serve as controls, ischemic animals were treated with cerebellum homogenate harvested from adult rat brain. All groups were sacrificed at 24 h of reperfusion. NPCs were isolated from rat fetus telencephalon and cultured until neurosphere formation (7 days). Before administration, NPCs were labeled with carboxyfluorescein diacetate succinimydylester (CFSE). Kidneys were harvested for analysis of cytokine profile and macrophage infiltration. At 24 h, NPC treatment resulted in a significant reduction in serum creatinine (IRI + NPC 1.21 + 0.18 vs. IRI 3.33 + 0.14 and IRI + cerebellum 2.95 + 0.78mg/dl, p < 0.05) and acute tubular necrosis (IRI + NPC 46.0 + 2.4% vs. IRI 79.7 + 14.2%, p < 0.05). NPC-CFSE and glial fibrillary acidic protein (GFAP)-positive cells (astrocyte marker) were found exclusively in renal parenchyma, which also presented GFAP and SOX-2 (an embryonic neural stem cell marker) mRNA expression. NPC treatment resulted in lower renal proinflammatory IL1-beta and TNF-alpha expression and higher anti-inflammatory IL-4 and IL-10 transcription. NPC-treated animals also had less macrophage infiltration and decreased serum proinflammatory cytokines (IL-1 beta, TNF-alpha and INF-gamma). Our data suggested that NPC therapy improved renal function by influencing immunological responses. Copyright (C) 2009 S. Karger AG, Basel

Evidences of the cooperative role of the chemokines CCL3, CCL4 and CCL5 and its receptors CCR1+and CCR5+in RANKL+ cell migration throughout experimental periodontitis in mice

Periodontal disease (PD) is characterized by the inflammatory bone resorption in response to the bacterial challenge, in a host response that involves a series of chemokines supposed to control cell influx into periodontal tissues and determine disease outcome. In this study, we investigated the role of chemokines and its receptors in the immunoregulation of experimental PD in mice. Aggregatibacter actinomycetemcomitans-infected C57BI/6 (WT) mice developed an intense inflammatory reaction and severe alveolar bone resorption, associated with a high expression of CCL3 and the migration of CCR5+, CCR1+ and RANKL+ cells to periodontal tissues. However, CCL3KO-infected mice developed a similar disease phenotype than WT strain, characterized by the similar expression of cytokines (TNF-alpha, IFN-gamma and IL-10), osteoclastogenic factors (RANKL and OPG) and MMPs (MMP-1, MMP-2, MMP-3, TIMP-1 and TIMP-3), and similar patterns of CCR1+, CCR5+ and RANKL+ cell migration. The apparent lack of function for CCL3 is possible due the relative redundancy of chemokine system, since chemokines such as CCL4 and CCL5, which share the receptors CCR1 and CCR5 with CCL3, present a similar kinetics of expression than CCL3. Accordingly, CCL4 and CCL5 kinetics of expression after experimental periodontal infection remain unaltered regardless the presence/absence of CCL3. Conversely, the individual absence of CCR1 and CCR5 resulted in a decrease of leukocyte infiltration and alveolar bone loss. When CCR1 and CCR5 were simultaneously inhibited by met-RANTES treatment a significantly more effective attenuation of periodontitis progression was verified, associated with lower values of bone loss and decreased counts of leukocytes in periodontal tissues. Our results suggest that the absence of CCL3 does not affect the development of experimental PD in mice, probably due to the presence of homologous chemokines CCL4 and CCL5 that overcome the absence of this chemokine. In addition, our data demonstrate that the absence of chemokine receptors CCR1+ and CCR5+ attenuate of inflammatory bone resorption. Finally, our data shows data the simultaneous blockade of CCR1 and CCR5 with MetRANTEs presents a more pronounced effect in the arrest of disease progression, demonstrating the cooperative role of such receptors in the inflammatory bone resorption process throughout experimental PD. (C) 2009 Elsevier Inc. All rights reserved.

Inflammation of the Hypothalamus Leads to Defective Pancreatic Islet Function

Type 2 diabetes mellitus results from the complex association of insulin resistance and pancreatic beta-cell failure. Obesity is the main risk factor for type 2 diabetes mellitus, and recent studies have shown that, in diet-induced obesity, the hypothalamus becomes inflamed and dysfunctional, resulting in the loss of the perfect coupling between caloric intake and energy expenditure. Because pancreatic beta-cell function is, in part, under the control of the autonomic nervous system, we evaluated the role of hypothalamic inflammation in pancreatic islet function. In diet-induced obesity, the earliest markers of hypothalamic inflammation are present at 8 weeks after the beginning of the high fat diet; similarly, the loss of the first phase of insulin secretion is detected at the same time point and is restored following sympathectomy. Intracerebroventricular injection of a low dose of tumor necrosis factor a leads to a dysfunctional increase in insulin secretion and activates the expression of a number of markers of apoptosis in pancreatic islets. In addition, the injection of stearic acid intracerebroventricularly, which leads to hypothalamic inflammation through the activation of tau-like receptor-4 and endoplasmic reticulum stress, produces an impairment of insulin secretion, accompanied by increased expression of markers of apoptosis. The defective insulin secretion, in this case, is partially dependent on sympathetic signal-induced peroxisome proliferator receptor-gamma coactivator Delta a and uncoupling protein-2 expression and is restored after sympathectomy or following PGC1 alpha expression inhibition by an antisense oligonucleotide. Thus, the autonomic signals generated in concert with hypothalamic inflammation can impair pancreatic islet function, a phenomenon that may explain the early link between obesity and defective insulin secretion.

Exhaustive exercise causes an anti-inflammatory effect in skeletal muscle and a pro-inflammatory effect in adipose tissue in rats

It is well known that exhaustive exercise increases serum and skeletal muscle IL-6 concentrations. However, the effect of exhaustive exercise on the concentrations of other cytokines in the muscle and in the adipose tissue is controversial. The purpose of this study was to evaluate the effect of exhaustive exercise on mRNA and protein expression of IL-10, TNF-alpha and IL-6 in different types of skeletal muscle (EDL, soleus) and in two different depots of white adipose tissue (mesenteric-MEAT and retroperitoneal-RPAT). Rats were killed by decapitation immediately (E0 group, n = 6), 2 (E2 group, n = 6) and 6 (E6 group, n = 6) hours after the exhaustion protocol, which consisted of running on a treadmill (approximately 70% VO(2max) for 50 min and then subsequently at an elevated rate that increased at 1 m/min every minute, until exhaustion). The control group (C group, n = 6) was not subjected to exercise. Cytokine protein expression increased in EDL, soleus, MEAT and RPAT from all exercised groups, as detected by ELISA. EDL IL-10 and TNF-alpha expression was higher than that of the soleus. The IL-10/TNF-alpha ratio was increased in the skeletal muscle, especially in EDL, but it was found to be decreased in the adipose tissue. These results show that exhaustive exercise presents a different effect depending on the tissue which is analysed: in the muscle, it induces an anti-inflammatory effect, especially in type 2 fibres, while the pro-inflammatory effect prevails in adipose tissue, possibly contributing to increased lipolysis to provide energy for the exercising muscle.

Heat and chemical stress modulate the expression of the alpha-RYR gene in broiler chickens

The biological cause of Pork Stress syndrome, which leads to PSE (pale, soft, exudative) meat, is excessive release of Ca(2+) ions, which is promoted by a genetic mutation in the ryanodine receptors (RyR) located in the sarcoplasmic reticulum of the skeletal muscle cells. We examined the relationship between the formation of PSE meat under halothane treatment and heat stress exposure in chicken alpha RYR hot spot fragments. Four test groups were compared: 1) birds slaughtered without any treatment, i.e., the control group (C); 2) birds slaughtered immediately after halothane treatment (H); 3) birds slaughtered immediately after heat stress treatment (HS), and 4) birds exposed to halothane and to heat stress (H+HS), before slaughtering. Breast muscle mRNA was extracted, amplified by RT-PCR, and sequenced. PSE meat was evaluated using color determination (L*value). The most common alteration was deletion of a single nucleotide, which generated a premature stop codon, resulting in the production of truncated proteins. The highest incidence of nonsense transcripts came with exposure to halothane; 80% of these abnormal transcripts were detected in H and H+HS groups. As a consequence, the incidence of abnormal meat was highest in the H+HS group (66%). In HS, H, and C groups, PSE meat developed in 60, 50, and 33% of the samples, respectively. Thus, halothane apparently modulates alpha RYR gene expression in this region, and synergically with exposure to heat stress, causes Avian Stress syndrome, resulting in PSE meat in broiler chickens.

RNA interference-mediated knockdown of CD49e (alpha 5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesion

Background: The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin alpha 5 beta 1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody. Results: Herein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets. Conclusion: Conceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation.

Identification of the Schistosoma mansoni TNF-Alpha Receptor Gene and the Effect of Human TNF-Alpha on the Parasite Gene Expression Profile

Background: Schistosoma mansoni is the major causative agent of schistosomiasis. The parasite takes advantage of host signals to complete its development in the human body. Tumor necrosis factor-alpha (TNF-alpha) is a human cytokine involved in skin inflammatory responses, and although its effect on the adult parasite's metabolism and egg-laying process has been previously described, a comprehensive assessment of the TNF-alpha pathway and its downstream molecular effects is lacking. Methodology/Principal Findings: In the present work we describe a possible TNF-alpha receptor (TNFR) homolog gene in S. mansoni (SmTNFR). SmTNFR encodes a complete receptor sequence composed of 599 amino acids, and contains four cysteine-rich domains as described for TNFR members. Real-time RT-PCR experiments revealed that SmTNFR highest expression level is in cercariae, 3.5 (+/- 0.7) times higher than in adult worms. Downstream members of the known human TNF-alpha pathway were identified by an in silico analysis, revealing a possible TNF-alpha signaling pathway in the parasite. In order to simulate parasite's exposure to human cytokine during penetration of the skin, schistosomula were exposed to human TNF-alpha just 3 h after cercariae-to-schistosomula in vitro transformation, and large-scale gene expression measurements were performed with microarrays. A total of 548 genes with significantly altered expression were detected, when compared to control parasites. In addition, treatment of adult worms with TNF-alpha caused a significantly altered expression of 1857 genes. Interestingly, the set of genes altered in adults is different from that of schistosomula, with 58 genes in common, representing 3% of altered genes in adults and 11% in 3 h-old early schistosomula. Conclusions/Significance: We describe the possible molecular elements and targets involved in human TNF-alpha effect on S. mansoni, highlighting the mechanism by which recently transformed schistosomula may sense and respond to this host mediator at the site of cercarial penetration into the skin.