Up-regulation of pro-angiogenic pathways and induction of neovascularization by an acute retinal light damage.

The light damage (LD) model was mainly used to study some of the main aspects of age related macular degeneration (AMD), such as oxidative stress and photoreceptor death. Several protocols of light-induced retinal degeneration exist. Acute light damage is characterized by a brief exposure (24 hours) to high intensity light (1000 lux) and leads to focal degeneration of the retina which progresses over time. To date there are not experimental data that relate this model to neovascular events. Therefore, the purpose of this study was to characterize the retina after an acute light damage to assess whether the vascularization was affected. Functional, molecular and morphological investigations were carried out. The electroretinographic response was assessed at all recovery times (7, 60, 120 days after LD). Starting from 7 days after light damage there was a significant decrease in the functional response, which remained low up to 120 days of recovery. At 7 days after light exposure, neo-vessels invaded the photoreceptor layer and retinal neovascularization occurred. Remarkably, neoangiogenesis was associated to the up-regulation of VEGF, bFGF and their respective receptors (VEGFR2 and FGFR1) with the progression of degeneration. These important results indicate that a brief exposure to bright light induces the up-regulation of pro-angiogenic pathways with subsequent neovascularization.

Cytokine modulation in patients with idiopathic pulmonary fibrosis undergoing treatment with steroids, immunosuppressants, and IFN-γ 1b.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of unknown etiology and pathogenic mechanisms. From an etiopathogenic point of view, alveolar macrophages play a key role in accumulation of fibroblasts and deposition of collagen and extracellular matrix by releasing specific cytokines and inflammatory mediators. IPF seems to be also associated with circulating fibrocytes, which might be involved with an abnormal pulmonary vascular repair and remodeling. Based on its hypothesized pathologic mechanisms, anti-inflammatory, anti-fibrotic and immunosuppressive therapies are often used. For these reasons, Interferon-g (IFN-g) has been used to exploit its activity on macrophages and fibroblasts. The aim of this study was to investigate the response to corticosteroids and/or IFN-g 1b treatments based on pulmonary function tests and on inflammatory cytokine patterns of expression on bronchoalveolar lavage (BAL), at baseline and during and after the therapies. Unlike previous studies, we analyzed a period of therapy longer than 1 year. Our results demonstrated the effectiveness of IFN-γ in a group of IPF patients in whom the treatment was prolonged for over a year. These data suggest a positive role of IFN-γ; treatment in patients in the initial stage of the disease.

Effect of low energy light irradiation by light emitting diode on U937 cells.

Photobiomodulation (PBM) can induce a set of different biological modulators either in vitro or in vivo. Experimental evidence has highlighted the role of light effects on the mechanisms related to inflammation, apoptosis and autophagy. The goal of this project was the evaluation of PBM on U937, an established cell line of histiocytic lymphoma origin. Several aspects of modulation of proinflammatory pathways were analyzed and autophagic and proapoptotic mechanisms related to low laser light exposure of cells were studied. As a source of low energy light emission, we used an NIR-LED device, characterized by an 880 nm-wavelength as light source. Flow cytometry analysis was performed on supernatants of controls and treated U937 cells to detect inflammatory cytokine levels. In order to evaluate NF-kB and caspase3 expressions, Western blot analysis was performed according to standard procedures. In this report, we show the effect of PBM on a monocyte/macrophage established tumor cell line (U-937). We demonstrate that LED exposure, in the presence or absence of lipopolysaccharide (LPS), activates cell degranulation, increased expression of Interleukin-8 (IL-8) and modulation of beta galactosidase activity. Evidence shows that the well-known pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and the apoptotic marker (caspase3/cleaved-caspase3 ratio) are up-regulated in response to a proinflammatory biochemical pathway.

A novel biotechnology product for the degradation of biofilm-associated polysaccharides produced by Streptococcus mutans.

In this study we evaluated the activity of ABR preparation, a first-in-class agent obtained through fermentation process by genetically unmodified Bacillus spp., in breaking down polysaccharide produced by Streptococcus mutans, primary coloniser of tooth surface and abundant in dental biofilms. Our results showed that ABR preparation is able in degrading sugars formed by S. mutans, both in broth culture and onto teeth surface. Its activity is not influenced by the presence of saliva, commercial mouthwashes or oral disinfectants. ABR preparation has the potential to remove preformed plaque and counteract its development, thus offering conservative control of gingival and periodontal disease.

Essential amino acids improve insulin activation of AKT/MTOR signaling in soleus muscle of aged rats.

Essential amino acids (EAA) improve basal muscle protein synthesis in the elderly. Nevertheless, in settings of prolonged supplementation, putative signal pathways of EAA are currently unknown. The purpose of this study was to test the effects of prolonged supplementation of EAA enriched mixture (12-L-Amin) on Insulin/Insulin-like Growth Factor-1 (IGF1) pathway by measuring total and phosphorylated Akt (Ser473) and its upstream (IRS1 at Ser636) and downstream (mTOR at Ser2448, p70S6K at Thr389) targets in basal conditions and following acute insulin (0.1 U/L) incubation in vitro. To this aim, soleus muscles were dissected from male Wistar rats divided in three groups of 7 each: adults (AD, 10 mo of age), elderly (EL, 22 mo of age) and elderly supplemented (EL-AA, 12-L-Amin 1.5gr/Kg die in drinking water for 3 mo). EL showed reduced basal and post-insulin mTOR and p70S6K activation and reduced post-insulin IRS1 degradation relative to AD. EL-AA showed an increase of post-insulin Akt activation, no change in basal and post-insulin phospho-mTOR, lower reduction of phospho-p70S6K and increased post-insulin IRS1 degradation relative to AD. These results demonstrate that chronic 12-LAmin administration exerts anti-ageing effects on the activation/inactivation of the Insulin/IGF1/mTOR pathway which is identified as putative target of EAA in the elderly.

Endothelial cell anergy is mediated by bFGF through the sustained activation of p38-MAPK and NF-kappaB inhibition.

Tumors escape from immune surveillance by, among other mechanisms, the down- regulation of endothelial adhesion molecules, such as ICAM-1, and by unresponsiveness to inflammatory signals, a process mediated by angiogenic factors that is called endothelial cell anergy. Here we present the cell biological regulation of these processes. The angiogenic basic fibroblast growth factor (bFGF/FGF-2) was found to inhibit tumor necrosis factor-alpha (TNF-alpha)- induced elevation of ICAM-1, at transcriptional level. Furthermore, we found that bFGF inhibits the TNF-mediated activation of NF-kappaB by blocking phosphorylation and degradation of IkappaBalpha. We also found that bFGF induces hyperphosphorylation of p38 MAPK on endothelial cells, whereas inhibition of such kinase abrogates the effect of bFGF on the TNF-mediated activation of NF-kappaB. Thus, we suggest that bFGF acts as an inhibitor of leukocyte adhesion in tumor vessels by decreasing the ICAM-1 expression through the sustained activation of p38-MAPK and via inhibition of NF-kappaB.

Genomic organization and cytokine-mediated inducibility of the human TRIM-8/Gerp gene.

Cytokine signaling is negatively regulated by a set of SH2 domain-containing proteins, the Suppressors of Cytokine Signaling (SOCS) acting as intracellular modulators. Experimental evidence indicates that SOCS gene expression is induced by cytokines and pro-inflammatory stimuli and is highly controlled both at transcription and translation level. Furthermore, SOCS proteins appear rapidly degraded inside the cells, mostly controlling their stability by interacting with specific molecules such as elongin B and C. It has been shown that SOCS-1/JAB, a member of the SOCS family, interacts with TRIM-8/Gerp, a new ring protein specifically binding SOCS-1 recombitant polypeptide in-vitro and in-vivo. Trim-8/Gerp, transcribes IFN-gamma in epithelial and lymphoid cells and is expressed mostly ubiquitously in murine and human tissues. Here in this report we present the genomic organization of this new SOCS-1 interactor, and we add new tools for extending investigation of the complex mechanism that undergoes negatively regulation of cytokine signaling.

The murine p202 protein, an IFN-inducible modulator of transcription, is activated by the mitogen platelet-derived growth factor.

p202 is a murine interferon (IFN)-inducible protein belonging to a cluster of IFN-inducible genes (the 200 family) located in a segment of chromosome 1. It is a nuclear DNA-binding protein that is able to modulate transcription by interacting with a heterogeneous set of transcription factors, including NF-kappaB, (p50/p65), AP-1, c-fos, c-jun, and RB-1. The p202 protein is believed to attenuate cell growth/proliferation, mainly through the activation of IFN-stimulated of gene factor 3 (ISGF3), which binds IFN-stimulated response elements (ISRE) located in the promoters of type I IFN genes. In this report, we show that the p202 gene can also be induced by platelet-derived growth factor (PDGF), a mitogen known to drive G(0)-arrested cells toward reentry into the cell cycle. PDGF transiently enhances the steady-state mRNA level of p202 and increases the p202 protein level independently from IFN signaling, by acting at the transcriptional level on its promoter. The kinetics of p202 induction by PDGF are faster and more transient than those of IFN. These data identify p202 as a member of the IFN-inducible gene family that can be directly regulated by mitogenic stimuli.

Two gamma-interferon-activation sites (GAS) on the promoter of the human intercellular adhesion molecule (ICAM-1) gene are required for induction of transcription by IFN-gamma.

We describe the molecular features of the interferon (IFN)-gamma-mediated transcription of the human intercellular adhesion molecule (ICAM-1) gene. We identified putative IFN-gamma-activated sites (GAS) distributed throughout a large segment of the ICAM-1 promoter (4.0 kb region). Using computer-assisted search, these sequences were similar to potential IFN-gamma responsive elements that have a core sequence 5'-TTNCNNNAA-3'. In this report we show that in the ICAM-1 promoter a GAS site is located at -115 from the translation initiation site, and binds with strong affinity to IFN-gamma-activated Signal Transducers and Activators of Transcription (STAT1) homodimers. The same sequence is responsible for the IFN-gamma-mediated transcription of the ICAM-1 gene. Moreover, we present evidence that a more distal GAS element that maps at -2787 from the translation initiation site, binds IFN-gamma-activated STAT1 dimers with lower affinity. Multimeric copies of such GAS sequence inserted into a tkCAT minimal promoter can drive transcription, demonstrating that the -2787 bp GAS element has an independent functional activity upon binding of IFN-gamma-activated STAT1 proteins as documented by in vitro binding assays. Furthermore, using recombinant ICAM-CAT mutants, we show that, in vivo, the -2787 GAS, but not a mutagenized -2787 GAS site, when coupled to the more proximal -115 GAS element, has an additive effect in enhancing the IFN-gamma-mediated transcription of ICAM-1 promoter. Nevertheless, using a recombinant construct bearing the wild type -2787 GAS element and a mutagenized -115 GAS element, we could not detect any transcription after transfection of U937 recipient cells, suggesting that the -2787 bp GAS element is not sufficient as such for gene activation, but can cooperate with its cognate proximal sequence to give full function to the ICAM-1 promoter during the IFN-gamma response. Taken together these data provide evidence that two GAS sites are required for the full potential activity in the mechanism of ICAM-1 gene activation by IFN-gamma.

Deficient cytokine signaling in mouse embryo fibroblasts with a targeted deletion in the PKR gene: role of IRF-1 and NF-kappaB.

The interferon (IFN)-induced double-stranded RNA (dsRNA)-activated Ser/Thr protein kinase (PKR) plays a role in the antiviral and antiproliferative effects of IFN. PKR phosphorylates initiation factor eIF2alpha, thereby inhibiting protein synthesis, and also activates the transcription factor, nuclear factor-kappaB (NF-kappaB), by phosphorylating the inhibitor of NF-kappaB, IkappaB. Mice devoid of functional PKR (Pkr(o/o)) derived by targeted gene disruption exhibit a diminished response to IFN-gamma and poly(rI:rC) (pIC). In embryo fibroblasts derived from Pkr(o/o) mice, interferon regulatory factor 1 (IRF-1) or guanylate binding protein (Gbp) promoter-reporter constructs were unresponsive to IFN-gamma or pIC but response could be restored by co-transfection with PKR. The lack of responsiveness could be attributed to a diminished activation of IRF-1 and/or NF-kappaB in response to IFN-gamma or pIC. Thus, PKR acts as a signal transducer for IFN-stimulated genes dependent on the transcription factors IRF-1 and NF-kappaB.

Interferon-alpha-induced phosphorylation and activation of cytosolic phospholipase A2 is required for the formation of interferon-stimulated gene factor three.

Treatment of cells with interferon (IFN)-alpha caused phosphorylation and activation of cytosolic phospholipase A2 (cPLA2). The protein tyrosine kinase Jak1 was found to be necessary for the activation of cPLA2. Jak1 could be co-immunoprecipitated with cPLA2 from cell extracts, indicating that a close physical interaction occurs between these two proteins. The induction of IFN-stimulated gene factor three (ISGF3) by IFN-alpha, is blocked by cPLA2 inhibitors in cell cultures and in cell-free reconstituted systems. However, these inhibitors do not block IFN-alpha or gamma-induced binding of STAT1 to the inverted repeat (IR) element of the IFN regulatory factor 1 (IRF-1) gene. Thus, cPLA2 activations occurs as an early event in the IFN-alpha response and is selectively involved in ISGF3-dependent gene activation.

Involvement of an arachidonic-acid-dependent pathway in the interferon-beta-mediated expression of C202 gene in Ehrlich-ascites-tumor cells.

We have investigated the signal transduction mechanism of the expression of the C202 gene mediated by interferon beta (IFN-beta) in the murine Ehrlich's ascites tumor cell line. We have shown that treatment of cells with IFN-beta transiently enhances within minutes the release of free arachidonic acid through membrane phospholipase activity. Furthermore, prior treatment with either p-bromophenacyl bromide, an antagonist of both cytosolic and secretory phospholipase A2, or neomycin, which blocks phospholipase C activity, significantly decreased the activation of the murine IFN-beta-inducible gene, C202. Moreover, an increase of the expression of the C202 gene was observed after blocking of both the cyclooxygenase and lipoxygenase pathways. This suggests that further metabolism of arachidonic acid to epoxides via epoxygenase-catalysed pathways may be a mechanism by which second messengers for IFN-beta-mediated effects on C202 gene expression are generated. Taken together, these results indicate that lipids as second messengers may be important mediators in the IFN-beta-based activation of C202 gene expression.

Roles of protein-tyrosine phosphatases in Stat1 alpha-mediated cell signaling.

Different Stat proteins are activated through phosphorylation of unique tyrosine residues in response to different cytokines and growth factors. Interferon-gamma activates Stat1 molecules that form homodimers and bind cognate DNA elements. Here we show that treatment of permeabilized cells with 200-500 microM peroxo-derivatives of vanadium, molybdenum, and tungsten results in the accumulation of constitutively phosphorylated Stat1 alpha molecules. In contrast, treatment of permeabilized cells with orthovanadate, vanadyl sulfate, molybdate, and tungstate at the same range of concentrations does not result in the accumulation of activated Stat1 alpha molecules in the absence of ligand. However, these compounds inhibit the inactivation of interferon-gamma-induced DNA-binding activity of Stat1 alpha. A 4-6-h exposure of the permeabilized cells to orthovanadate, molybdate, and tungstate, but not vanadyl sulfate, results in a ligand-independent activation of Stat1 alpha, which is blocked by the inhibition or depletion of NADPH oxidase activity in the cells, indicating that NADPH oxidase-catalyzed superoxide formation is required for the bioconversion of these metal oxides to the corresponding peroxo-compounds. Interestingly, ligand-independent Stat1 alpha activation by peroxo-derivatives of these transition metals does not require Jak1, Jak2, or Tyk2 kinase activity, suggesting that other kinases can phosphorylate Stat1 alpha on tyrosine 701.

Enzymatic synthesis of S-aminoethyl-L-cysteine from pantetheine.

The recently characterized compound S-aminoethylcysteine ketimine can be synthesized from purified S-aminoethylcysteine by enzymatic systems (transaminases or L-amino acid oxidase) present in mammalian tissues. S-Aminoethylcysteine, which could be considered as the natural precursor of the ketimine, is produced from L-serine and cysteamine by the action of the enzyme cystathionine-beta-synthase. We demonstrate in this paper that pantetheine, a normal cellular component, is an efficient cysteamine donor for the synthesis of S-aminoethylcysteine and of S-aminoethylcysteine ketimine in the place of free cysteamine, and we describe the enzymatic system, composed of partially purified enzymes, for the in vitro synthesis of S-aminoethylcysteine ketimine from pantetheine. This seems to indicate a new biological role for pantetheine.