Pharmacological effects of 3-iodothyronamine (T1AM) in mice include facilitation of memory acquisition and retention and reduction of pain threshold

BACKGROUND AND PURPOSE: 3-Iodothyronamine (T1AM), an endogenous derivative of thyroid hormones, is regarded as a rapid modulator of behaviour and metabolism. To determine whether brain thyroid hormone levels contribute to these effects, we investigated the effect of central administration of T1AM on learning and pain threshold of mice either untreated or pretreated with clorgyline (2.5 mg•kg-1, i.p.), an inhibitor of amine oxidative metabolism. EXPERIMENTAL APPROACH: T1AM (0.13, 0.4, 1.32 and 4 mg•kg-1) or vehicle was injected i.c.v. into male mice, and after 30 min their effects on memory acquisition capacity, pain threshold and curiosity were evaluated by the following tests: passive avoidance, licking latency on the hot plate and movements on the hole-board platform. Plasma glycaemia was measured using a glucorefractometer. Brain levels of triiodothyroxine (T3), thyroxine (T4) and T1AM were measured by HPLC coupled to tandem MS. ERK1/2 activation and c-fos expression in different brain regions were evaluated by Western blot analysis. RESULTS: T1AM improved learning capacity, decreased pain threshold to hot stimuli, enhanced curiosity and raised plasma glycaemia in a dose-dependent way, without modifying T3 and T4 brain concentrations. T1AM effects on learning and pain were abolished or significantly affected by clorgyline, suggesting a role for some metabolite(s), or that T1AM interacts at the rapid desensitizing target(s). T1AM activated ERK in different brain areas at lower doses than those effective on behaviour. CONCLUSIONS AND IMPLICATIONS: T1AM is a novel memory enhancer. This feature might have important implications for the treatment of endocrine and neurodegenerative-induced memory disorders

The pro-healing effect of exendin-4 on wounds produced by abrasion in normoglycemic mice

Experimental evidence suggested that Exendin-4 (Exe4), an agonist at glucagon like receptor-1 (GLP-1R), promoted tissue regeneration. We aimed to verify the effect of Exe4, in the absence or in the presence of Exendin-4(9-39), an antagonist at GLP-1R, on the healing of abraded skin. Two wounds (approximately 1.1×1.1 cm; namely “upper” and “lower” in respect of the head) were produced by abrasion on the back of 12 mice, which were then randomly assigned to receive an intradermal injection (20 μl) of : Group 1: saline (NT) or Exe4 (62 ng) in the upper and lower wound respectively; Group 2: Exendin-4 (9-39)(70 ng) in the upper and Exendin-4 (9-39)(70 ng) and, after 15 min, Exe4 (62 ng) in the lower wound. Wounds were measured at the time of abrasion (T0) and 144 h (T3) afterward taking pictures with a ruler and by using a software. The inflammatory cell infiltrate, fibroblasts/myofibroblasts, endothelial cells and GLP-1R expression, were each labeled by immunofluorescence in each wound, pERK1/2 was evaluated by Western-blot in wound lysates. At T3, the percentage of healing surface was 53% and 92% for NT and Exe4 wounds respectively and 68% and 79% for those treated with Exendin-4 (9-39) and Exendin-4 (9-39) + Exe4 respectively. Exe4, but not Exendin-4(9-39) induced quantitative increase in fibroblasts/myofibroblasts and vessel density when compared to NT wounds. This increase was not evident in wounds treated with Exendin-4 (9-39) + Exe4. Exe4 promotes wound healing opening to the possible dermatological use of this incretin analogue

Stimulatory Interactions between Human Coronary Smooth Muscle Cells and Dendritic Cells

<div><p>Despite inflammatory and immune mechanisms participating to atherogenesis and dendritic cells (DCs) driving immune and non-immune tissue injury response, the interactions between DCs and vascular smooth muscle cells (VSMCs) possibly relevant to vascular pathology including atherogenesis are still unclear. To address this issue, immature DCs (iDCs) generated from CD14⁺ cells isolated from healthy donors were matured either with cytokines (mDCs), or co-cultured (ccDCs) with human coronary artery VSMCs (CASMCs) using transwell chambers. Co-culture induced DC immunophenotypical and functional maturation similar to cytokines, as demonstrated by flow cytometry and mixed lymphocyte reaction. In turn, factors from mDCs and ccDCs induced CASMC migration. MCP-1 and TNFα, secreted from DCs, and IL-6 and MCP-1, secreted from CASMCs, were primarily involved. mDCs adhesion to CASMCs was enhanced by CASMC pre-treatment with IFNγ and TNFα ICAM-1 and VCAM-1 were involved, since the expression of specific mRNAs for these molecules increased and adhesion was inhibited by neutralizing antibodies to the counter-receptors CD11c and CD18. Adhesion was also inhibited by CASMC pre-treatment with the HMG-CoA-reductase inhibitor atorvastatin and the PPARγ agonist rosiglitazone, which suggests a further mechanism for the anti-inflammatory action of these drugs. Adhesion of DCs to VSMCs was shown also <i>in vivo</i> in rat carotid 7 to 21 days after crush and incision injury. The findings indicate that DCs and VSMCs can interact with reciprocal stimulation, possibly leading to perpetuate inflammation and vascular wall remodelling, and that the interaction is enhanced by a cytokine-rich inflammatory environment and down-regulated by HMGCoA-reductase inhibitors and PPARγ agonists.</p></div

Primers and conditions for real time-PCR.

<p>Primers and conditions for real time-PCR.</p

Immunophenotypical and functional maturation of DCs.

<p><b>A</b>) Flow cytometry for DC markers in immature DCs (iDCs), DCs matured with a standard protocol (mDCs), DCs co-cultured with CASMCs (ccDCs) and DC co-cultured with CASMC pre-treated with 50 ng/mL TNFα and IFNγ (ccDCs + cytokines); all co-cultures were inside transwells. Mean ±SE of median fluorescence intensity (MFI), n = 12; *P<0.05 vs. iDC, ANOVA. <b>B</b>) Proliferation of lymphocyte alone (0) and in mixed reaction with iDCs, mDCs, ccDC or ccDCs + cytokines. Lymphocyte proliferation is expressed as mean ±SE of counts-per-minute/well (cpm/well; 4 experiments, each in triplicate); *P<0.05 vs. iDCs, ANOVA. <b>C</b>) IL-6, MCP-1, IL-1β and TNFα release from CASMC, iDCs, mDCs, ccDCs as assayed by Milliplex method. Atorvastatin (ator; 1 µmol/L) and rosiglitazone (rosig; 20 µmol/L) did not influence the release of cytokines in co-cultures. The release of cytokines was measured from 1×10⁶ cells DCs and 5×10⁴ CASMCs. Results are expressed as pg/mL. Mean ±SE of 4 experiments. *P<0.05 vs iDC, ANOVA. <b>D</b>) Flow cytometry analysis of DC maturation and of the effect of neutralizing antibodies against TNFα, MCP-1 and IL-6 or of a cocktail of all those antibodies. Representative histograms of ccDCs in the absence (filled histogram) and in the presence of antibodies (open histogram, black lines) are shown. The histogram for the isotype control is included (open histogram, faint line). The effect of non immune IgG is not shown.</p

Effects of atorvastatin and rosiglitazone on mDC adhesion to cytokine-stimulated CASMCs.

<p>CASMCs were pre-treated with (<b>A</b>) atorvastatin (0.01–1 µmol/L) or (<b>B</b>) rosiglitazone (1–20 µmol/L) before stimulation with 50 ng/mL TNFα or IFNγ for 24 h followed by assay of DC adhesion as indicated for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099652#pone-0099652-g004" target="_blank">Figure 4</a>. Mevalonate (300 µM), added to the maximal atorvastatin concentration, reverted statin effect (A). Adhesion is reported as percent of that induced by TNFα or IFNγ (control). Mean ±SE of 6–8 experiments, each in triplicate. *P<0.05 vs. control, ANOVA. (C) Representative experiment of DC to CASMC adhesion. Calcein-labelled mDC were incubated for 45 min with control CASMCs (0) or with CASMCs stimulated with TNFα or IFNγ (50 ng/mL either cytokine) and pre-treated, or not pre-treated, with 1 µmol/L atorvastatin (atorv) or 20 µmol/L rosiglitazone (rosigl). Mevalonate (300 µmol/L) was used to confirm atorvastatin selectivity.</p

Characterization of co-cultured DCs.

<p><b>A–D</b>) Electron microscopy of DCs matured in transwell culture with CASMC (A, B) or with cytokines (C, D). Asterisks indicate lysosomes; hashes indicate smooth endoplasmic reticulum; arrowheads indicate occasional lipid droplets. Bars  = 2 µm (A, C) or 0.5 µm (B, D). <b>E–H</b>) Phase contrast (E, G) and immunofluorescence microscopy (F, H) for DC-SIGN in immature (iDCs; E, F) and mature DCs (mDCs; G, H). The exposure time was the same for both F and H photomicrographs, to show that the labelling of iDCs was lighter than that of mDCs. Note the labelling on cell extensions, indicating membrane expression of the antigen. Bar  = 30 µm. <b>I</b>) Flow cytometry for DC-SIGN expression in iDCs, mDCs and ccDCs. One representative histograms out of 3 performed is shown. The isotype control is included (open histogram).</p

Adhesion molecules and counter-receptors involved in cytokine-induced mDC adhesion to CASMCs.

<p><b>A</b>) real time RT-PCR of ICAM-1 or VCAM-1 mRNA expression by CASMCs stimulated with TNFα or INFγ (50 ng/mL each cytokine) for 24 h. Data are expressed as fold increase over unstimulated CASMCs. Mean ±SE of 3 experiments. *P<0.05, ***P<0.001 vs. untreated CASMCs, Student's <i>t</i> test. <b>B–D</b>) Effect of neutralizing antibodies on mDC adhesion to CASMCs. CASMCs were untreated (0) or pre-treated with: B) anti-CD11c (0.5 µg/well), C) anti-CD18 (0.5 µg/well), or D) anti-DC-SIGN (0.5 µg/well) neutralizing antibodies. They were pretreated with IFNγ or TNFα for 24 h and then washed before the assay. NI-IgG: non immune IgG. Mean ±SE of 5-7 experiments, each in triplicate. *P<0.05 vs. respective control, Student's <i>t</i> test.</p

Adhesion of mDC to vascular smooth muscle cells.

<p><b>A</b>) Human mature calcein-labeled DC adhesion to CASMC pre-treated for 12-36 h with 50 ng/mL IFNγ or with 50 ng/mL TNFα. After washing, a suspension of calcein-labeled DCs was added and let to adhere for 45 m. Mean ±SE of 6 experiments, each in triplicate. Results are expressed as percentage of mDC adhesion over that on untreated CASMCs (control). *P<0.05, ANOVA. <b>B, C</b>) Electron microscopy of mDCs adherent to citokyne-treated CASMCs; SMC: smooth muscle cells. Bars = 4 µm (B), or 0.5 µm (C). <b>D-G</b>) Electron microscopy of rat carotid repair tissue (neointima) at 7 (D, E), 14 (F) and 21 d (G) upon crush and incision injury; panel H is an enlargement of the boxed part of panel G. Dendritic cells (D) are seen in contact with smooth muscle cells with secretory phenotype (asterisks). Bar  = 2 µm (D), 500 nm (E), or 1 µm (F, G).</p