Contribution by polymorphonucleate granulocytes to elevated gamma-glutamyltransferase in cystic fibrosis sputum

Background

Cystic fibrosis (CF) is an autosomal recessive disorder characterized by a chronic neutrophilic airways inflammation, increasing levels of oxidative stress and reduced levels of antioxidants such as glutathione (GSH). Gamma-glutamyltransferase (GGT), an enzyme induced by oxidative stress and involved in the catabolism of GSH and its derivatives, is increased in the airways of CF patients with inflammation, but the possible implications of its increase have not yet been investigated in detail.

Principal Findings

The present study was aimed to evaluate the origin and the biochemical characteristics of the GGT detectable in CF sputum. We found GGT activity both in neutrophils and in the fluid, the latter significantly correlating with myeloperoxidase expression. In neutrophils, GGT was associated with intracellular granules. In the fluid, gel-filtration chromatography showed the presence of two distinct GGT fractions, the first corresponding to the human plasma b-GGT fraction, the other to the free enzyme. The same fractions were also observed in the supernatant of ionomycin and fMLP-activated neutrophils. Western blot analysis confirmed the presence of a single band of GGT immunoreactive peptide in the CF sputum samples and in isolated neutrophils.

Conclusions

In conclusion, our data indicate that neutrophils are able to transport and release GGT, thus increasing GGT activity in CF sputum. The prompt release of GGT may have consequences on all GGT substrates, including major inflammatory mediators such as S-nitrosoglutathione and leukotrienes, and could participate in early modulation of inflammatory response.     

On the mechanism of action of SJ-172550 in inhibiting the interaction of MDM4 and p53

SJ-172550 (1) was previously discovered in a biochemical high throughput screen for inhibitors of the interaction of MDMX and p53 and characterized as a reversible inhibitor (J. Biol. Chem. 2010; 285:10786). Further study of the biochemical mode of action of 1 has shown that it acts through a complicated mechanism in which the compound forms a covalent but reversible complex with MDMX and locks MDMX into a conformation that is unable to bind p53. The relative stability of this complex is influenced by many factors including the reducing potential of the media, the presence of aggregates, and other factors that influence the conformational stability of the protein. This complex mechanism of action hinders the further development of compound 1 as a selective MDMX inhibitor.     

GDF5 Regulates TGF?-Dependent Angiogenesis in Breast Carcinoma MCF-7 Cells: In Vitro and In Vivo Control by Anti-TGF? Peptides

Background

TGFß overproduction in cancer cells is one of the main characteristics of late tumor progression being implicated in metastasis, tumor growth, angiogenesis and immune response. We investigated the therapeutic efficacy of anti-TGFß peptides in the control of angiogenesis elicited by conditional over-expression of TGFß.

Methods

We have inserted in human MCF7 mammary-cancer cells a mutated TGFß gene in a tetracycline-repressible vector to obtain conditional expression of mature TGFß upon transient transfection, evaluated the signaling pathways involved in TGFß-dependent endothelial cells activation and the efficacy of anti-TGFß peptides in the control of MCF7-TGFß-dependent angiogenesis.

Results

TGFß over-expression induced in MCF7 several markers of the epithelial-to-mesenchymal transition. Conditioned-medium of TGFß-transfected MCF7 stimulated angiogenesis in vivo and in vitro by subsequent activation of SMAD2/3 and SMAD1/5 signaling in endothelial cells, as well as SMAD4 nuclear translocation, resulting in over-expression of the pro-angiogenic growth and differentiation factor-5 (GDF5). Inhibition or silencing of GDF5 in TGFß-stimulated EC resulted in impairment of GDF5 expression and of TGFß-dependent urokinase-plasminogen activator receptor (uPAR) overproduction, leading to angiogenesis impairment. Two different TGFß antagonist peptides inhibited all the angiogenesis-related properties elicited in EC by exogenous and conditionally-expressed TGFß in vivo and in vitro, including SMAD1/5 phosphorylation, SMAD4 nuclear translocation, GDF5 and uPAR overexpression. Antagonist peptides and anti-GDF5 antibodies efficiently inhibited in vitro and in vivo angiogenesis.

Conclusions

TGFß produced by breast cancer cells induces in endothelial cells expression of GDF5, which in turn stimulates angiogenesis both in vitro and in vivo. Angiogenesis activation is rapid and the involved mechanism is totally opposed to the old and controversial dogma about the AKL5/ALK1 balance. The GDF-dependent pro-angiogenic effects of TGFß are controlled by anti-TGFß peptides and anti-GDF5 antibodies, providing a basis to develop targeted clinical studies.     

Food components with anticaries activity

Caries is the most common oral infectious disease in the world. Its development is influenced also by diet components that interfere with pathogen mutans group Streptococci (MGS) activity. A very active research to identify functional foods and their components that are generally recognised as safe has been ongoing, with the aim of developing alternative approaches, to the use of synthetic chlorhexidine, and at the reduction or prevention of caries. Until now convincing evidence exists only for green tea as a functional food for oral health, partly owing to its high content of catechins, especially epigallocatechin-gallate. A number of other foods showed potential anticaries activity. Some other foods able to act against MGS growth and/or their virulence factors in in vitro tests are: apple, red grape seeds, red wine (proanthocyanidins), nutmeg (macelignan), ajowan caraway (nafthalen-derivative), coffee (trigonelline, nicotinic and chlorogenic acids, melanoidins), barley coffee (melanoidins), chicory and mushroom (quinic acid). In vivo anticaries activity has been shown by cranberry (procyanidins), glycyrrhiza root (glycyrrhizol-A), myrtus ethanolic extract, garlic aqueous extract, cocoa extracts (procyanidins), and propolis (apigenin, tt-farnesol).     

Testosterone and farnesoid X receptor agonist INT-747 counteract high fat diet-induced bladder alterations in a rabbit model of metabolic syndrome

In the male, metabolic syndrome (MetS) is associated to an increased risk of benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS). A recently established rabbit model of high fat diet (HFD)-induced MetS showed hypogonadism and the presence of prostate gland alterations, including inflammation, hypoxia and fibrosis. The present study investigated whether HFD-induced MetS might also alter bladder structure and function. Testosterone and the farnesoid X receptor (FXR) agonist INT-747, were evaluated for possible effects on HFD bladder. MetS rabbits develop bladder alterations, including fibrosis (reduced muscle/fiber ratio), hypoxia [2-fold increase as compared to regular diet (RD) group], low-grade inflammation (increased leukocyte infiltration and inflammatory markers) and RhoA/ROCK hyperactivity. Bladder strips from HFD rabbits, pre-contracted with carbachol, showed an overactive response to the selective ROCK inhibitor Y-27632. All these HFD-induced bladder alterations were partially blunted by testosterone and almost completely reverted by INT-747. Both treatments prevented some MetS features (glucose intolerance and visceral fat increase), thus suggesting that their effects on bladder could be ascribed to an improvement of the metabolic and/or hypogonadal state. However, a pathogenetic role for hypogonadism has been ruled out as GnRH analog-induced hypogonadal rabbits, fed a regular diet, did not show any detectable bladder alterations. In addition, INT-747 did not revert the MetS-induced hypogonadal state. FXR mRNA was highly expressed in rabbit bladder and positively associated with visceral fat increase. A direct effect of INT-747 on bladder smooth muscle was further suggested by inhibition of RhoA/ROCK-mediated activity by in vitro experiments on isolated cells. In conclusion, HFD-related MetS features are associated to bladder derangements, which are ameliorated by testosterone or INT-747 administration. INT-747 showed the most marked effects in counteracting MetS-related RhoA/ROCK overactivity, thus opening novel therapeutic opportunities for this drug.     

When art moves the eyes: a behavioral and eye-tracking study

The aim of this study was to investigate, using eye-tracking technique, the influence of bottom-up and top-down processes on visual behavior while subjects, na?ve to art criticism, were presented with representational paintings. Forty-two subjects viewed color and black and white paintings (Color) categorized as dynamic or static (Dynamism) (bottom-up processes). Half of the images represented natural environments and half human subjects (Content); all stimuli were displayed under aesthetic and movement judgment conditions (Task) (top-down processes). Results on gazing behavior showed that content-related top-down processes prevailed over low-level visually-driven bottom-up processes when a human subject is represented in the painting. On the contrary, bottom-up processes, mediated by low-level visual features, particularly affected gazing behavior when looking at nature-content images. We discuss our results proposing a reconsideration of the definition of content-related top-down processes in accordance with the concept of embodied simulation in art perception.     

Progressive Increase of Matrix Metalloprotease-9 and Interleukin-8 Serum Levels during Carcinogenic Process in Human Colorectal Tract

Background

Inflammatory reactions, known to promote tumor growth and invasion, have been found associated with colorectal carcinoma (CRC). Macrophages are the chief component of the inflammatory infiltration that occurs early in the progression from non-invasive to malignant tumor, with a switch from the pro-inflammatory phenotype to the tumor-promoting phenotype. Tumor and stroma are additional sources of inflammation-related molecules. The study aimed to evaluate, during colorectal carcinogenesis from benign to malignant phases: i) the trend of serum levels of IL-8, IL-6, TGFβ1, VEGF and MMPs; ii) the parallel trend of CRP serum levels; iii) derangement of the principal TGFβ1 receptors (TGFβ1RI/RII) in tumor tissues.

Methodology/Principal findings

96 patients with colon adenomas or CRC at different stages of progression, and 17 controls, were recruited. Serum IL-8, IL-6, TGFβ1, VEGF, MMPs and CRP levels were analyzed before endoscopy or surgery. TGFβ1 receptors were evaluated in adenoma biopsies and surgically-removed colorectal adenocarcinomas. Serum levels of IL-8 in adenocarcinoma patients were increased from stage II, when also the enzymatic activity of MMP-9 increased. Of note, the increasing trend of the two serum markers was found significantly correlated. Trend of serum CRP was also very similar to that of IL-8 and MMP-9, but just below statistical significance. TGFβ1 levels were lower at stage III CRC, while IL-6 and VEGF levels had no significant variations. In tissue specimens, TGFβ1 receptors were already absent in about 50% of adenomas, and this percentage of missing receptors markedly increased in CRC stages III and IV.

Conclusions

Combined quantification of serum IL-8, MMP-9 and CRP, appears a reliable and advanced index of inflammation-related processes during malignant phase of colorectal carcinogenesis, since these molecules remain within normal range in colorectal adenoma bearing patients, while consistently increase in the blood of CRC patients, even if from stage II only.