Center for Preclinical Investigation

Milano, Italy

Center for Preclinical Investigation

Milano, Italy
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Gatti S.,The Surgical Center | Lonati C.,The Surgical Center | Lonati C.,Center for Preclinical Investigation | Acerbi F.,Fondazione IRCCS Instituto Neurologico Carlo Besta | And 10 more authors.
Experimental Neurology | Year: 2012

Subarachnoid hemorrhage (SAH) is still a major cause of morbidity and mortality. α-Melanocyte stimulating hormone (α-MSH) and other melanocortin peptides exert potent neuroprotective action and they might modulate key molecules involved in SAH-induced vasospasm. The aim of this research was to determine whether treatment with the α-MSH analog Nle4,DPhe7-α-MSH (NDP-MSH) exerts protective effects in experimental SAH in the rat. Initial experiments examined effects of NDP-MSH on the basilar artery phenotype in the absence of injury. In these tests intrathecal injection of small concentrations (10. ng) of the peptide induced a tolerant phenotype similar to that observed after ischemic preconditioning. Then the effect of systemic treatment with NDP-MSH (100μg i.v.) on experimental SAH was evaluated. SAH was induced by a single-blood injection into the cisterna magna. The basilar artery phenotype was examined at 4 h and the artery caliber at 5. days following SAH. Expression of 96 genes was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) using Custom Taqman Low-Density Arrays. Four hours after SAH, the transcriptional profile of the basilar artery was deeply disrupted. Transcript alteration included genes involved in inflammation, stress response, apoptosis, and vascular remodeling. Treatment with NDP-MSH prevented most of these transcription changes and decreased phosphorylation of extracellular-signal-regulated kinases (ERK1/2) and inhibitor protein IκBα. Vasospasm on day 5 was significantly reduced by NDP-MSH administration. These results combine with others on CNS inflammation to suggest that the melanocortins could be safe and effective therapeutic candidates to treat SAH-related complications. © 2012 Elsevier Inc.


Grieco P.,University of Naples Federico II | Carotenuto A.,University of Naples Federico II | Auriemma L.,University of Naples Federico II | Limatola A.,University of Naples Federico II | And 10 more authors.
PLoS ONE | Year: 2013

Previous investigations indicate that α-melanocyte-stimulating hormone (α-MSH) and certain synthetic analogues of it exert antimicrobial effects against bacteria and yeasts. However, these molecules have weak activity in standard microbiology conditions and this hampers a realistic clinical use. The aim in the present study was to identify novel peptides with broad-spectrum antimicrobial activity in growth medium. To this purpose, the Gly10 residue in the [DNal(2′)-7, Phe-12]-MSH(6-13) sequence was replaced with conventional and unconventional amino acids with different degrees of conformational rigidity. Two derivatives in which Gly10 was replaced by the residues Aic and Cha, respectively, had substantial activity against Candida strains, including C. albicans, C. glabrata, and C. krusei and against gram-positive and gram-negative bacteria. Conformational analysis indicated that the helical structure along residues 8-13 is a key factor in antimicrobial activity. Synthetic analogues of α-MSH can be valuable agents to treat infections in humans. The structural preferences associated with antimicrobial activity identified in this research can help further development of synthetic melanocortins with enhanced biological activity. © 2013 Grieco et al.


Arnason B.G.,University of Chicago | Berkovich R.,University of Southern California | Catania A.,Center for Preclinical Investigation | Lisak R.P.,Wayne State University | Zaidi M.,Mount Sinai School of Medicine
Multiple Sclerosis Journal | Year: 2013

The therapeutic benefits of adrenocorticotropic hormone in multiple sclerosis are usually ascribed to its corticotropic actions. Evidence is presented that adrenocorticotropic hormone, approved for multiple sclerosis relapses, acts via corticosteroid-independent melanocortin pathways to engender down-modulating actions on immune-system cells and the cytokines they synthesize. Immune response-dampening effects are also brought about by agent-induced neurotransmitters that inhibit immunocytes. The likelihood that adrenocorticotropic hormone promotes microglial quiescence and counteracts glucocorticoid-mediated bone resorption is discussed. © The Author(s) 2012.


Capsoni F.,University of Milan | Ongari A.M.,University of Milan | Lonati C.,Center for Preclinical Investigation | Accetta R.,Instituto Ortopedico Galeazzi | And 2 more authors.
BMC Musculoskeletal Disorders | Year: 2015

Background: Alpha-melanocyte-stimulating-hormone (α-MSH) has marked anti-inflammatory potential. Proinflammatory cytokines are critical mediators of the disturbed cartilage homeostasis in osteoarthritis, inhibiting anabolic activities and increasing catabolic activities in chondrocytes. Since human chondrocytes express α-MSH receptors, we evaluated the role of the peptide in modulating chondrocyte production of pro-inflammatory cytokines, matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in response to interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Methods: Human articular chondrocytes were obtained from osteoarthritic joint cartilage from subjects undergoing hip routine arthroplasty procedures. The cells were cultured with or without α-MSH in the presence of IL-1β or TNF-α. Cell-free supernatants were collected and cells immediately lysed for RNA purification. Expression of cytokines, MMPs, TIMPs, iNOS was determined by Reverse Transcription Real-time Polymerase Chain Reaction and enzyme-linked immunosorbent assay. Griess reaction was used for NO quantification. Results: Gene expression and secretion of IL-6, IL-8, MMP-3, MMP-13 were significantly increased in IL-1β or TNF-α-stimulated chondrocytes; α-MSH did not modify the release of IL-6 or IL-8 while the peptide significantly reduced their gene expression on TNF-α-stimulated cells. A significant inhibition of MMP3 gene expression and secretion from IL-1β or TNFα-stimulated chondrocytes was induced by α-MSH. On the other hand, α-MSH did not modify the release of MMP-13 by cytokine-stimulated chondrocyte but significantly decreased gene expression of the molecule on TNF-α-stimulated cells. Detectable amount of TIMP-3 and TIMP-4 were present in the supernatants of resting chondrocytes and a significant increase of TIMP-3 gene expression and release was induced by α-MSH on unstimulated cells. TIMP-3 secretion and gene expression were significantly increased in IL-1β-stimulated chondrocytes and α-MSH down-regulated gene expression but not secretion of the molecule. TIMP-4 gene expression (but not secretion) was moderately induced in IL-1β-stimulated chondrocytes with a down-regulation exerted by α-MSH. IL-1β and TNF-α were potent stimuli for NO production and iNOS gene expression by chondrocytes; no inhibition was induced by α-MSH on cytokine-stimulated NO production, while the peptide significantly reduced gene expression of iNOS. Conclusions: Our results underscore a potential anti-inflammatory and chondroprotective activity exerted by α-MSH, increasing TIMP-3 gene expression and release on resting cells and down- modulating TNF-α-induced activation of human chondrocytes. However, the discrepancy between the influences exerted by α-MSH on gene expression and protein release as well as the difference in the inhibitory pattern exerted by α-MSH in TNF-α- or IL-1β-stimulated cells leave some uncertainty on the role of the peptide on chondrocyte modulation. © 2015 Capsoni et al.


PubMed | Center for Preclinical Investigation, Instituto Ortopedico Galeazzi and University of Milan
Type: | Journal: BMC musculoskeletal disorders | Year: 2015

Alpha-melanocyte-stimulating-hormone (-MSH) has marked anti-inflammatory potential. Proinflammatory cytokines are critical mediators of the disturbed cartilage homeostasis in osteoarthritis, inhibiting anabolic activities and increasing catabolic activities in chondrocytes. Since human chondrocytes express -MSH receptors, we evaluated the role of the peptide in modulating chondrocyte production of pro-inflammatory cytokines, matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in response to interleukin-1 (IL-1) and tumor necrosis factor- (TNF-).Human articular chondrocytes were obtained from osteoarthritic joint cartilage from subjects undergoing hip routine arthroplasty procedures. The cells were cultured with or without -MSH in the presence of IL-1 or TNF-. Cell-free supernatants were collected and cells immediately lysed for RNA purification. Expression of cytokines, MMPs, TIMPs, iNOS was determined by Reverse Transcription Real-time Polymerase Chain Reaction and enzyme-linked immunosorbent assay. Griess reaction was used for NO quantification.Gene expression and secretion of IL-6, IL-8, MMP-3, MMP-13 were significantly increased in IL-1 or TNF--stimulated chondrocytes; -MSH did not modify the release of IL-6 or IL-8 while the peptide significantly reduced their gene expression on TNF--stimulated cells. A significant inhibition of MMP3 gene expression and secretion from IL-1 or TNF-stimulated chondrocytes was induced by -MSH. On the other hand, -MSH did not modify the release of MMP-13 by cytokine-stimulated chondrocyte but significantly decreased gene expression of the molecule on TNF--stimulated cells. Detectable amount of TIMP-3 and TIMP-4 were present in the supernatants of resting chondrocytes and a significant increase of TIMP-3 gene expression and release was induced by -MSH on unstimulated cells. TIMP-3 secretion and gene expression were significantly increased in IL-1-stimulated chondrocytes and -MSH down-regulated gene expression but not secretion of the molecule. TIMP-4 gene expression (but not secretion) was moderately induced in IL-1-stimulated chondrocytes with a down-regulation exerted by -MSH. IL-1 and TNF- were potent stimuli for NO production and iNOS gene expression by chondrocytes; no inhibition was induced by -MSH on cytokine-stimulated NO production, while the peptide significantly reduced gene expression of iNOS.Our results underscore a potential anti-inflammatory and chondroprotective activity exerted by -MSH, increasing TIMP-3 gene expression and release on resting cells and down- modulating TNF--induced activation of human chondrocytes. However, the discrepancy between the influences exerted by -MSH on gene expression and protein release as well as the difference in the inhibitory pattern exerted by -MSH in TNF-- or IL-1-stimulated cells leave some uncertainty on the role of the peptide on chondrocyte modulation.


Catania A.,Center for Preclinical Investigation | Lonati C.,Center for Preclinical Investigation | Sordi A.,Center for Preclinical Investigation | Leonardi P.,Center for Preclinical Investigation | And 4 more authors.
Peptides | Year: 2010

α-Melanocyte-stimulating hormone (α-MSH) is a pro-opiomelanocortin (POMC)-derived peptide that exerts multiple protective effects on host cells. Previous investigations showed that treatment with α-MSH or synthetic melanocortin agonists reduces heart damage in reperfusion injury and transplantation. The aim of this preclinical research was to determine whether melanocortin treatment induces preconditioning-like cardioprotection. In particular, the plan was to assess whether melanocortin administration causes phenotype changes similar to those induced by repetitive ischemic events. The idea was conceived because both ischemic preconditioning and melanocortin signaling largely depend on cAMP response element binding protein (CREB) phosphorylation. Rats received single i.v. injections of 750 μg/kg of the α-MSH analogue Nle4,DPhe7-α-MSH (NDP-MSH) or saline and were sacrificed at 0.5, 1, 3, or 5 h. Western blot analysis showed that rat hearts expressed melanocortin 1 receptor (MC1R) protein. Treatment with NDP-MSH was associated with early and marked increase in interleukin 6 (IL-6) mRNA. This was followed by signal transducer and activator of transcription 3 (STAT3) phosphorylation and induction of suppressor of cytokine signaling 3 (SOCS3). There were no changes in expression of other cytokines of the IL-6 family. Expression of IL-10, IL-1β, and TNF-α was likewise unaltered. In hearts of rats treated with NDP-MSH there was increased expression of the orphan nuclear receptor Nur77. The data indicate that NDP-MSH induces phenotype changes that closely resemble ischemic preconditioning and likely contribute to its established protection against reperfusion injury. In addition, the increased expression of Nur77 and SOCS3 could be part of a broader anti-inflammatory effect. © 2009 Elsevier Inc. All rights reserved.


Lonati C.,The Surgical Center | Sordi A.,The Surgical Center | Giuliani D.,University of Modena and Reggio Emilia | Spaccapelo L.,University of Modena and Reggio Emilia | And 7 more authors.
Anesthesiology | Year: 2012

Background: Melanocortin peptides improve hemodynamic parameters and prevent death during severe hemorrhagic shock. In the present research we determined influences of a synthetic melanocortin 1/4 receptor agonist on the molecular changes that occur in rat liver during hemorrhage. Methods: Controlled-volume hemorrhage was performed in adult rats under general anesthesia by a stepwise blood withdrawal until mean arterial pressure fell to 40 mmHg. Then rats received either saline or the synthetic melanocortin 1/4 receptor agonist Butir-His-D-Phe-Arg-Trp-Sar- NH 2 (Ro27-3225; n = 6-8 per group). Hemogasanalysis was performed throughout a 60-min period. Gene expression in liver samples was determined at 1 or 3 h using quantitative real-time polymerase chain reaction. Results: At 1 h, in saline-treated shocked rats, there were significant increases in activating transcription factor 3 (Atf3), early growth response 1 (Egr1), heme oxygenase (decycling) 1 (Hmox1), FBJ murine osteosarcoma viral oncogene homolog (Fos), and jun oncogene (Jun). These changes were prevented by Ro27-3225 (mean ± SEM: Atf3 152.83 ± 58.62 vs. 579.00 ± 124.13, P = 0.002; Egr1 13.21 ± 1.28 vs. 26.63 ± 1.02, P = 0.001; Hmox1 3.28 ± 0.31 vs. 166.54 ± 35.03, P = 0.002; Fos 4.36 ± 1.03 vs. 14.90±3.44, P<0.001; Jun 6.62±1.93 vs. 15.07±2.09, P = 0.005; respectively). Increases in alpha-2-macroglobulin (A2m), heat shock 70kD protein 1A (Hspa1a), erythropoietin (Epo), and interleukin-6 (Il6) occurred at 3 h in shocked rats and were prevented by Ro27-3225 treatment (A2m 6.90 ± 0.82 vs. 36.73 ± 4.00, P < 0.001; Hspa1a 10.34 ±3.28 vs. 25.72 ± 3.64, P = 0.001; Epo 0.49 ± 0.13 vs. 2.37 ± 0.73, P = 0.002; Il6 1.05 ± 0.15 vs. 1.88 ± 0.23, P < 0.001; respectively). Further, at 3 h in shocked rats treated with Ro27-3225 there were significant increases in tight junction protein 1 (Tjp1; 27.30 ± 2.43 vs. 5.03 ±1.68, P < 0.001) and nuclear receptor subfamily 4, group A, member 1 (Nr4a1; 91.03 ± 16.20 vs. 30.43 ± 11.0, P = 0.01) relative to sham animals. Treatment with Ro27-3225 rapidly restored blood pressure, hemogasanalysis parameters, and lactate blood levels. Conclusions: Melanocortin treatment significantly prevents most of the systemic and hepatic detrimental changes induced by hemorrhage. Copyright © 2012, the American Society of Anesthesiologists, Inc.


Catania A.,Center for Preclinical Investigation | Lonati C.,Center for Preclinical Investigation | Sordi A.,Center for Preclinical Investigation | Carlin A.,Center for Preclinical Investigation | And 4 more authors.
TheScientificWorldJournal | Year: 2010

Melanocortin peptides, the collective term for α-, β-, and γ-melanocyte-stimulating hormone (α-, β-, γ-MSH) and adrenocorticotropic hormone (ACTH), are elements of an ancient modulatory system. Natural melanocortins derive from the common precursor pro-opiomelanocortin (POMC). Five receptor subtypes for melanocortins (MC 1-MC5) are widely distributed in brain regions and in peripheral cells. Melanocortin receptor activation by natural or synthetic ligands exerts marked anti-inflammatory and immunomodulatory effects. The anticytokine action and the inhibitory influences on inflammatory cell migration make melanocortins potential new drugs for treatment of inflammatory disorders. Effectiveness in treatment of acute, chronic, and systemic inflammatory disorders is well documented in preclinical studies. Further, melanocortins are promising compounds in neuroprotection. This review examines the main signaling circuits in anti-inflammatory and immunomodulatory actions of melanocortins, and the potential therapeutic use of these molecules. ©2010 with author. Published by TheScientificWorld.


PubMed | Center for Preclinical Investigation, The Surgical Center and University of Milan
Type: Journal Article | Journal: PloS one | Year: 2016

Ex vivo lung perfusion (EVLP) is a promising procedure for evaluation, reconditioning, and treatment of marginal lungs before transplantation. Small animal models can contribute to improve clinical development of this technique and represent a substantial platform for bio-molecular investigations. However, to accomplish this purpose, EVLP models must sustain a prolonged reperfusion without pharmacological interventions. Currently available protocols only partly satisfy this need. The aim of the present research was accomplishment and optimization of a reproducible model for a protracted rat EVLP in the absence of anti-inflammatory treatment. A 180 min, uninjured and untreated perfusion was achieved through a stepwise implementation of the protocol. Flow rate, temperature, and tidal volume were gradually increased during the initial reperfusion phase to reduce hemodynamic and oxidative stress. Low flow rate combined with open atrium and protective ventilation strategy were applied to prevent lung damage. The videos enclosed show management of the most critical technical steps. The stability and reproducibility of the present procedure were confirmed by lung function evaluation and edema assessment. The meticulous description of the protocol provided in this paper can enable other laboratories to reproduce it effortlessly, supporting research in the EVLP field.


Valenza F.,Instituto Of Ricovero E Cura A Carattere Scientifico Irccs | Valenza F.,University of Milan | Rosso L.,Unit Operativa di Chirurgia Toracica | Coppola S.,Instituto Of Ricovero E Cura A Carattere Scientifico Irccs | And 11 more authors.
Journal of Heart and Lung Transplantation | Year: 2012

Background: We recently showed in a pig model of ex vivo lung perfusion (EVLP) that lung edema correlates with glucose consumption. We investigated whether salbutamol, a β-adrenergic receptor agonist known to upregulate fluid transport in the lung, modulates glucose concentration in the perfusate during EVLP. Methods: Lungs from domestic pigs underwent normothermic EVLP. At the end of controlled reperfusion, lungs were ventilated and perfused for 60 minutes, then randomized to salbutamol (β-Agonist) infusion or placebo (Control) for 180 minutes. Functional parameters were assessed. Results: In the β-Agonist group, glucose concentration decreased over time more than corresponding Control values (analysis of variance [ANOVA], p = 0.05). Mean pulmonary artery pressure (mPAP) was 16 ± 1 mm Hg in the β-Agonist group vs 21 ± 1 mm Hg in the Controls (ANOVA p < 0.05). Baseline mPAP was correlated with the drop of mPAP after the β-agonist infusion (R 2 = 0.856, p < 0.05). Dynamic compliance dropped from 51 ± 10 to 31 ± 6 ml/cm H 2O in the β-Agonist group and from 60 ± 4 to 21 ± 3 ml/cm H 2O in the Control group (ANOVA, p < 0.05 β-agonist vs Control). The Δ partial pressure of oxygen/fraction of inspired oxygen was 418 ± 15 and 393 ± 12 mm Hg in the β-Agonist and Control groups, respectively (t-test p = 0.106). Conclusions: Glucose concentration in the perfusate was affected by salbutamol. Salbutamol was associated with lower pulmonary pressures and better lung mechanics. These data suggest a possible role for salbutamol as a pharmacologic adjunct during EVLP before transplantation. © 2012 International Society for Heart and Lung Transplantation. All rights reserved.

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