Publications by authors named "Eugenio Scanziani"

88 Publications

Total laryngectomy in a cat with a laryngeal peripheral nerve sheath tumor.

Vet Surg 2021 Apr 29. Epub 2021 Apr 29.

Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.

Objective: To report the surgical technique and outcome of total laryngectomy in a single clinical case.

Study Design: Case report.

Animal: A 5-year-old female spayed domestic shorthair cat.

Methods: A cat presented for acute, severe respiratory distress caused by an invasive laryngeal mass. Incisional biopsy was indicative of sarcoma. Computed tomography of head, neck, and thorax was performed revealing no evidence of metastasis. A total laryngectomy and permanent tracheostomy were performed, and the cat could breathe without difficulties immediately postoperatively. Histopathology confirmed a laryngeal low-grade peripheral nerve sheath tumor (PNST).

Results: Surgical margins were free of tumor cells. Surgical revision of the tracheostomy stoma due to obstructive granulation tissue was necessary 24 days after the initial surgery. Nine days after revision surgery, the cat was discharged from the hospital. No evidence of local recurrence or metastasis was detected on repeat computed tomography of the head, neck, and thorax at 6 months, nor on chest radiographs at 12 months postoperatively. At the time of writing (13 months postoperatively), the cat is still alive with a good quality of life.

Conclusion: Total laryngectomy with permanent tracheostomy allowed the complete removal of an obstructive laryngeal PNST and provided a good quality of life in a cat.

Clinical Significance: To the authors' knowledge, this case report represents the first detailed description of the surgical procedure and clinical outcome for a total laryngectomy in a cat.
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http://dx.doi.org/10.1111/vsu.13646DOI Listing
April 2021

Modeling, optimization, and comparable efficacy of T cell and hematopoietic stem cell gene editing for treating hyper-IgM syndrome.

EMBO Mol Med 2021 Mar 21;13(3):e13545. Epub 2021 Jan 21.

San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Precise correction of the CD40LG gene in T cells and hematopoietic stem/progenitor cells (HSPC) holds promise for treating X-linked hyper-IgM Syndrome (HIGM1), but its actual therapeutic potential remains elusive. Here, we developed a one-size-fits-all editing strategy for effective T-cell correction, selection, and depletion and investigated the therapeutic potential of T-cell and HSPC therapies in the HIGM1 mouse model. Edited patients' derived CD4 T cells restored physiologically regulated CD40L expression and contact-dependent B-cell helper function. Adoptive transfer of wild-type T cells into conditioned HIGM1 mice rescued antigen-specific IgG responses and protected mice from a disease-relevant pathogen. We then obtained ~ 25% CD40LG editing in long-term repopulating human HSPC. Transplanting such proportion of wild-type HSPC in HIGM1 mice rescued immune functions similarly to T-cell therapy. Overall, our findings suggest that autologous edited T cells can provide immediate and substantial benefits to HIGM1 patients and position T-cell ahead of HSPC gene therapy because of easier translation, lower safety concerns and potentially comparable clinical benefits.
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http://dx.doi.org/10.15252/emmm.202013545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933961PMC
March 2021

Ventilation With Argon Improves Survival With Good Neurological Recovery After Prolonged Untreated Cardiac Arrest in Pigs.

J Am Heart Assoc 2020 12 8;9(24):e016494. Epub 2020 Dec 8.

Department of Anesthesiology, Intensive Care and Emergency Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico Milan Italy.

Background Ventilation with the noble gas argon (Ar) has shown neuroprotective and cardioprotective properties in different in vitro and in vivo models. Hence, the neuroprotective effects of Ar were investigated in a severe, preclinically relevant porcine model of cardiac arrest. Methods and Results Cardiac arrest was ischemically induced in 36 pigs and left untreated for 12 minutes before starting cardiopulmonary resuscitation. Animals were randomized to 4-hour post-resuscitation ventilation with: 70% nitrogen-30% oxygen (control); 50% Ar-20% nitrogen-30% oxygen (Ar 50%); and 70% Ar-30% oxygen (Ar 70%). Hemodynamic parameters and myocardial function were monitored and serial blood samples taken. Pigs were observed up to 96 hours for survival and neurological recovery. Heart and brain were harvested for histopathology. Ten animals in each group were successfully resuscitated. Ninety-six-hour survival was 60%, 70%, and 90%, for the control, Ar 50%, and Ar 70% groups, respectively. In the Ar 50% and Ar 70% groups, 60% and 80%, respectively, achieved good neurological recovery, in contrast to only 30% in the control group (<0.0001). Histology showed less neuronal degeneration in the cortex (<0.05) but not in the hippocampus, and less reactive microglia activation in the hippocampus (=0.007), after Ar compared with control treatment. A lower increase in circulating biomarkers of brain injury, together with less kynurenine pathway activation (<0.05), were present in Ar-treated animals compared with controls. Ar 70% pigs also had complete left ventricular function recovery and smaller infarct and cardiac troponin release (<0.01). Conclusions Post-resuscitation ventilation with Ar significantly improves neurologic recovery and ameliorates brain injury after cardiac arrest with long no-flow duration. Benefits are greater after Ar 70% than Ar 50%.
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http://dx.doi.org/10.1161/JAHA.120.016494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955395PMC
December 2020

Trabectedin and Lurbinectedin Extend Survival of Mice Bearing C26 Colon Adenocarcinoma, without Affecting Tumor Growth or Cachexia.

Cancers (Basel) 2020 Aug 17;12(8). Epub 2020 Aug 17.

Department of Neurosciences, Mario Negri Institute for Pharmacological Research IRCCS, 20156 Milan, Italy.

Trabectedin (ET743) and lurbinectedin (PM01183) limit the production of inflammatory cytokines that are elevated during cancer cachexia. Mice carrying C26 colon adenocarcinoma display cachexia (i.e., premature death and body wasting with muscle, fat and cardiac tissue depletion), high levels of inflammatory cytokines and subsequent splenomegaly. We tested whether such drugs protected these mice from cachexia. Ten-week-old mice were inoculated with C26 cells and three days later randomized to receive intravenously vehicle or 0.05 mg/kg ET743 or 0.07 mg/kg PM01183, three times a week for three weeks. ET743 or PM01183 extended the lifespan of C26-mice by 30% or 85%, respectively, without affecting tumor growth or food intake. Within 13 days from C26 implant, both drugs did not protect fat, muscle and heart from cachexia. Since PM01183 extended the animal survival more than ET743, we analyzed PM01183 further. In tibialis anterior of C26-mice, but not in atrophying myotubes, PM01183 restrained the NF-κB/PAX7/myogenin axis, possibly reducing the pro-inflammatory milieu, and failed to limit the C/EBP/atrogin-1 axis. Inflammation-mediated splenomegaly of C26-mice was inhibited by PM01183 for as long as the treatment lasted, without reducing IL-6, M-CSF or IL-1β in plasma. ET743 and PM01183 extend the survival of C26-bearing mice unchanging tumor growth or cachexia but possibly restrain muscle-related inflammation and C26-induced splenomegaly.
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http://dx.doi.org/10.3390/cancers12082312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463843PMC
August 2020

LKB1 Deficiency Renders NSCLC Cells Sensitive to ERK Inhibitors.

J Thorac Oncol 2020 03 18;15(3):360-370. Epub 2019 Oct 18.

Laboratory of Molecular Pharmacology, Department of Oncology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milan, Italy.

Introduction: Serine/threonine kinase 11 (LKB1/STK11) is one of the most mutated genes in NSCLC accounting for approximately one-third of cases and its activity is impaired in approximately half of KRAS-mutated NSCLC. At present, these patients cannot benefit from any specific therapy.

Methods: Through CRISPR/Cas9 technology, we systematically deleted LKB1 in both wild-type (WT) and KRAS-mutated human NSCLC cells. By using these isogenic systems together with genetically engineered mouse models we investigated the cell response to ERK inhibitors both in vitro and in vivo.

Results: In all the systems used here, the loss of LKB1 creates vulnerability and renders these cells particularly sensitive to ERK inhibitors both in vitro and in vivo. The same cells expressing a WT LKB1 poorly respond to these drugs. At the molecular level, in the absence of LKB1, ERK inhibitors induced a marked inhibition of p90 ribosomal S6 kinase activation, which in turn abolished S6 protein activation, promoting the cytotoxic effect.

Conclusions: This work shows that ERK inhibitors are effective in LKB1 and LKB1/KRAS-mutated tumors, thus offering a therapeutic strategy for this prognostically unfavorable subgroup of patients. Because ERK inhibitors are already in clinical development, our findings could be easily translatable to the clinic. Importantly, the lack of effect in cells expressing WT LKB1, predicts that treatment of LKB1-mutated tumors with ERK inhibitors should have a favorable toxicity profile.
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http://dx.doi.org/10.1016/j.jtho.2019.10.009DOI Listing
March 2020

Fenretinide treatment accelerates atherosclerosis development in apoE-deficient mice in spite of beneficial metabolic effects.

Br J Pharmacol 2020 01 14;177(2):328-345. Epub 2019 Nov 14.

Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy.

Background And Purpose: Fenretinide, a synthetic retinoid derivative first investigated for cancer prevention and treatment, has been shown to ameliorate glucose tolerance, improve plasma lipid profile and reduce body fat mass. These effects, together with its ability to inhibit ceramide synthesis, suggest that fenretinide may have an anti-atherosclerotic action.

Experimental Approach: To this aim, nine-week-old apoE-knockout (EKO) female mice were fed for twelve weeks a Western diet, without (control) or with (0.1% w/w) fenretinide. As a reference, wild-type (WT) mice were treated similarly. Growth and metabolic parameters were monitored throughout the study. Atherosclerosis development was evaluated in the aorta and at the aortic sinus. Blood and lymphoid organs were further characterized with thorough cytological/histological and immunocytofluorimetric analyses.

Key Results: Fenretinide treatment significantly lowered body weight, glucose levels and plasma levels of total cholesterol, triglycerides, and phospholipids. In the liver, fenretinide remarkably reduced hepatic glycogenosis and steatosis driven by the Western diet. Treated spleens were abnormally enlarged, with severe follicular atrophy and massive extramedullary haematopoiesis. Severe renal hemosiderin deposition was observed in treated EKO mice. Treatment resulted in a threefold increase of total leukocytes (WT and EKO) and raised the activated/resting monocyte ratio in EKO mice. Finally, atherosclerosis development was markedly increased at the aortic arch, thoracic and abdominal aorta of fenretinide-treated mice.

Conclusions And Implications: We provide the first evidence that, despite beneficial metabolic effects, fenretinide treatment may enhance the development of atherosclerosis.
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http://dx.doi.org/10.1111/bph.14869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6989955PMC
January 2020

Abrogation of EMILIN1-β1 integrin interaction promotes experimental colitis and colon carcinogenesis.

Matrix Biol 2019 10 31;83:97-115. Epub 2019 Aug 31.

Unit of Molecular Oncology, Centro di Riferimento Oncologico di Aviano (CRO), IRCCS, Aviano, Italy. Electronic address:

Colon cancer is one of the first tumor types where a functional link between inflammation and tumor onset has been described; however, the microenvironmental cues affecting colon cancer progression are poorly understood. Here we demonstrate that the expression of the ECM molecule EMILIN-1 halts the development of AOM-DSS induced tumors. In fact, upon AOM-DSS treatment the Emilin1 (E1) mice were characterized by a higher tumor incidence, bigger adenomas and less survival. Similar results were obtained with the E933A EMILIN-1 (E1-E933A) transgenic mouse model, expressing a mutant EMILIN-1 unable to interact with α4/α9β1 integrins. Interestingly, upon chronic treatment with DSS, E1 and E1-E933A mice were characterized by the presence of increased inflammatory infiltrates, higher colitis scores and more severe mucosal injury respect to the wild type (E1) mice. Since alterations of the intestinal lymphatic network are a well-established feature of human inflammatory bowel disease and EMILIN-1 is a key structural element in the maintenance of the integrity of lymphatic vessels, we assessed the lymphatic vasculature in this context. The analyses revealed that both E1 and E1-E933A mice displayed a higher density of LYVE-1 positive vessels; however, their functionality was severely compromised after colitis induction. Taken together, these results suggest that the loss of EMILIN-1 expression may cause the reduction of the inflammatory resolution during colon cancer progression due to a decreased lymph flow and impaired inflammatory cell drainage.
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http://dx.doi.org/10.1016/j.matbio.2019.08.006DOI Listing
October 2019

Multiplex staining depicts the immune infiltrate in colitis-induced colon cancer model.

Sci Rep 2019 09 2;9(1):12645. Epub 2019 Sep 2.

Molecular Oncology Unit, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy.

Assessment of the host immune response pattern is of increasing importance as highly prognostic and diagnostic, in immune-related diseases and in some types of cancer. Chronic inflammation is a major hallmark in colon cancer formation, but, despite the extent of local inflammatory infiltrate has been demonstrated to be extremely informative, its evaluation is not routinely assessed due to the complexity and limitations of classical immunohistochemistry (IHC). In the last years, technological advance helped in bypassing technical limits, setting up multiplex IHC (mIHC) based on tyramide signal amplification (TSA) method and designing software suited to aid pathologists in cell scoring analysis. Several studies verified the efficacy of this method, but they were restricted to the analysis of human samples. In the era of translational medicine the use of animal models to depict human pathologies, in a more complete and complex approach, is really crucial. Nevertheless, the optimization and validation of this method to species other than human is still poor. We took advantage of Multispectral Imaging System to identify the immunoprofile of Dextran Sulphate Sodium (DSS)-treated mouse colon. We optimized a protocol to sequentially stain formalin fixed paraffin embedded murine colon samples for CD3, CD8a, CD4, and CD4R5B0 antigens. With this approach we obtained a detailed lymphocyte profile, while preserving the morphological tissue context, generally lost with techniques like gene expression profiling or flow cytometry. This study, comparing the results obtained by mIHC with immunophenotyping performed with cytofluorimetric and standard IHC methods validates the potentiality and the applicability of this innovative approach.
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http://dx.doi.org/10.1038/s41598-019-49164-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6718623PMC
September 2019

Identification of potent triazolylpyridine nicotinamide phosphoribosyltransferase (NAMPT) inhibitors bearing a 1,2,3-triazole tail group.

Eur J Med Chem 2019 Nov 1;181:111576. Epub 2019 Aug 1.

Dipartimento di Scienze del Farmaco, Università del Piemonte Orientale, Largo Donegani 2, 28100, Novara, Italy. Electronic address:

The enzyme nicotinamide phosphoribosyltransferase is both a key intracellular enzyme for NAD biosynthesis (iNAMPT) and an extracellular cytokine (eNAMPT). The relationship between this latter role and the catalytic activity of the enzyme is at present unknown. With the intent of discovering inhibitors specifically able to target eNAMPT, we increased the polarity of MV78 (EC = 5.8 nM; IC = 3.1 nM), a NAMPT inhibitor previously discovered by us. The replacement of a phenyl ring with a 1,2,3-triazole bearing a protonable N,N-dialkyl methanamine group gave a series of molecules which maintained the inhibition of the enzymatic activity but were unable to cross the plasma membrane and affect cell viability in vitro. Compounds 30b and 30f can therefore be considered as the first experimental/pharmacological tools for scientists that wish to understand the role of the catalytic activity of eNAMPT. Serendipitously, we also discovered a compound (25) which, notwithstanding its high polarity, was able to cross the plasma membrane being cytotoxic, a potent NAMPT inhibitor and effective in reducing growth of triple negative mammary carcinoma in mice. In our hands, 25 lacked retinal and cardiac toxicity, although we observed a lesser toxicity of NAMPT inhibitors in general compared to other reports.
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http://dx.doi.org/10.1016/j.ejmech.2019.111576DOI Listing
November 2019

Long-Term Study on the Effects of Housing C57BL/6NCrl Mice in Cages Equipped With Wireless Technology Generating Extremely Low-Intensity Electromagnetic Fields.

Toxicol Pathol 2019 07 22;47(5):598-611. Epub 2019 May 22.

1 Mouse and Animal Pathology Laboratory (MAPLab), Fondazione Unimi, Milano, Italy.

The recent development of mouse cages equipped with monitoring wireless technology raised questions on the potential effects on animals induced by electromagnetic fields (EMFs) generated by electronic boards positioned underneath the cages. The aims of this study were to characterize the EMF produced by digitally ventilated cages (DVC) and perform a clinicopathological study on mice maintained in DVC for up to 1 year. The EMFs were measured in empty individually ventilated cages (IVC) and DVC. Male (n = 160) and female (n = 160) C57BL/6NCrl mice were randomly housed in IVC and DVC in a single rack, 4 mice per cage. Body weight and food and water consumption were recorded at 14-day intervals. At sacrifice (days 60, 120, 180, and 365), body and testes weight was measured, and necropsy, hematology, bone marrow cytology, histology, and immunohistochemistry for cleaved-caspase 3 on the testes were performed. Digitally ventilated cages produced extremely low-intensity electric fields ranging from 5 Hz to 3 GHz. No exposure-related clinical signs and mortality occurred. Occasional statistical differences in body weight, food and water consumption, hematology, bone marrow, and histopathology were recorded, but considered without biological or clinical relevance. In conclusion, long-term maintenance in DVC had no definite effects on C57BL/6NCrl mice.
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http://dx.doi.org/10.1177/0192623319852353DOI Listing
July 2019

Efficacy of a Selective Binder of αβ Integrin Linked to the Tyrosine Kinase Inhibitor Sunitinib in Ovarian Carcinoma Preclinical Models.

Cancers (Basel) 2019 Apr 13;11(4). Epub 2019 Apr 13.

Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo 42, 20133 Milan, Italy.

Ovarian carcinoma, the most lethal gynecological cancer, is characterized by late diagnosis, with drug resistance limiting the efficacy of platinum-based therapy. Since some integrins are upregulated in cancer, including ovarian carcinoma, they represent a potential target for drug delivery. Receptor tyrosine kinases are also deregulated in cancer and their expression has been associated with drug resistance. Here, the antitumor effects of three conjugates possessing a selective binder of the extracellular portion of integrin αβ covalently linked to the tyrosine kinase inhibitor sunitinib were investigated in cisplatin-sensitive and -resistant ovarian carcinoma cells expressing both tyrosine kinase VEGFR2 and αβ at different levels. We found that one of the three compounds was active in inhibiting the growth of both drug-sensitive and -resistant cells in the micromolar range with a slightly increased potency in resistant cells as compared to sunitinib. The same compound markedly impaired cell migratory and invasive abilities and reduced paxillin phosphorylation. Antitumor activity studies in IGROV-1/Pt1 cells xenografted in nude mice revealed a striking activity of this conjugate versus sunitinib. Taken together, our results support the interest of integrin-targeted sunitinib conjugates for the treatment of drug-resistant tumors.
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http://dx.doi.org/10.3390/cancers11040531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6521192PMC
April 2019

Chromosome Transplantation: Correction of the Chronic Granulomatous Disease Defect in Mouse Induced Pluripotent Stem Cells.

Stem Cells 2019 07 2;37(7):876-887. Epub 2019 Apr 2.

National Research Council (CNR)-IRGB/UOS of Milan, Milan, Italy.

In spite of the progress in gene editing achieved in recent years, a subset of genetic diseases involving structural chromosome abnormalities, including aneuploidies, large deletions and complex rearrangements, cannot be treated with conventional gene therapy approaches. We have previously devised a strategy, dubbed chromosome transplantation (CT), to replace an endogenous mutated chromosome with an exogenous normal one. To establish a proof of principle for our approach, we chose as disease model the chronic granulomatous disease (CGD), an X-linked severe immunodeficiency due to abnormalities in CYBB (GP91) gene, including large genomic deletions. We corrected the gene defect by CT in induced pluripotent stem cells (iPSCs) from a CGD male mouse model. The Hprt gene of the endogenous X chromosome was inactivated by CRISPR/Cas9 technology thus allowing the exploitation of the hypoxanthine-aminopterin-thymidine selection system to introduce a normal donor X chromosome by microcell-mediated chromosome transfer. X-transplanted clones were obtained, and diploid XY clones which spontaneously lost the endogenous X chromosome were isolated. These cells were differentiated toward the myeloid lineage, and functional granulocytes producing GP91 protein were obtained. We propose the CT approach to correct iPSCs from patients affected by other X-linked diseases with large deletions, whose treatment is still unsatisfactory. Stem Cells 2019;37:876-887.
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http://dx.doi.org/10.1002/stem.3006DOI Listing
July 2019

Inhibition of SIRT1 deacetylase and p53 activation uncouples the anti-inflammatory and chemopreventive actions of NSAIDs.

Br J Cancer 2019 03 11;120(5):537-546. Epub 2019 Feb 11.

Department of Oncology and Hemato-Oncology, University of Milan, 20133, Milan, Italy.

Background: Nonsteroidal anti-inflammatory drugs (NSAIDs) have been proposed as chemopreventive agents for many tumours; however, the mechanism responsible for their anti-neoplastic activity remains elusive and the side effects due to cyclooxygenase (COX) inhibition prevent this clinical application.

Methods: Molecular biology, in silico, cellular and in vivo tools, including innovative in vivo imaging and classical biochemical assays, were applied to identify and characterise the COX-independent anti-cancer mechanism of NSAIDs.

Results: Here, we show that tumour-protective functions of NSAIDs and exisulind (a sulindac metabolite lacking anti-inflammatory activity) occur through a COX-independent mechanism. We demonstrate these NSAIDs counteract carcinogen-induced proliferation by inhibiting the sirtuin 1 (SIRT1) deacetylase activity, augmenting acetylation and activity of the tumour suppressor p53 and increasing the expression of the antiproliferative gene p21. These properties are shared by all NSAIDs except for ketoprofen lacking anti-cancer properties. The clinical interest of the mechanism identified is underlined by our finding that p53 is activated in mastectomy patients undergoing intraoperative ketorolac, a treatment associated with decreased relapse risk and increased survival.

Conclusion: Our study, for the first-time, links NSAID chemopreventive activity with direct SIRT1 inhibition and activation of the p53/p21 anti-oncogenic pathway, suggesting a novel strategy for the design of tumour-protective drugs.
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http://dx.doi.org/10.1038/s41416-018-0372-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461760PMC
March 2019

Effect of mild hypercapnia on outcome and histological injury in a porcine post cardiac arrest model.

Resuscitation 2019 02 26;135:110-117. Epub 2018 Oct 26.

Division of Intensive Care, Department of Anaesthesiology, Intensive Care and Pain Medicine, Helsinki University Hospital and University of Helsinki, Finland; Emergency Medicine and Services, Helsinki University Hospital and Department of Emergency Medicine, University of Helsinki, Finland. Electronic address:

Aim Of The Study: To evaluate in an established porcine post cardiac arrest model the effect of a mild hypercapnic ventilatory strategy on outcome.

Methods: The left anterior descending coronary artery was occluded in 14 pigs and ventricular fibrillation induced and left untreated for 12 min. Cardiopulmonary resuscitation was performed for 5 min prior to defibrillation. After resuscitation, pigs were assigned to either normocapnic (end-tidal carbon dioxide (EtCO) target: 35-40 mmHg) or hypercapnic ventilation (EtCO 45-50 mmHg). Hemodynamics was invasively measured and EtCO was monitored with an infrared capnometer. Blood gas analysis, serum neuron-specific enolase (NSE) and high sensitive cardiac troponin T (hs-cTnT) were assessed. Survival and functional recovery were evaluated up to 96 h.

Results: Twelve pigs were successfully resuscitated and eight survived up to 96 h, with animals in the hypercapnic group showing trend towards a longer survival. EtCO and arterial partial pressure of CO were higher in the hypercapnic group compared to the normocapnic one (p < 0.01), during the 4-hour intervention. Hypercapnia was associated with higher mean arterial pressure compared to normocapnia (p < 0.05). No significant differences were observed in hs-cTnT and in NSE between groups, although the values tended to be lower in the hypercapnic one. Neuronal degeneration was lesser in the frontal cortex of hypercapnic animals compared to the normocapnic ones (p < 0.05). Neurological recovery was equivalent in the two groups.

Conclusion: Mild hypercapnia after resuscitation was associated with better arterial pressure and lesser neuronal degeneration in this model. Nevertheless, no corresponding improvements in neurological recovery were observed.
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http://dx.doi.org/10.1016/j.resuscitation.2018.10.024DOI Listing
February 2019

Topiramate protects apoE-deficient mice from kidney damage without affecting plasma lipids.

Pharmacol Res 2019 03 26;141:189-200. Epub 2018 Dec 26.

Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, via Balzaretti 9, 20133 Milano, Italy. Electronic address:

Topiramate is an anticonvulsant drug also prescribed for migraine prophylaxis that acts through several mechanisms of action. Several studies indicate that topiramate induces weight loss and a moderate reduction of plasma lipids and glucose. Based on these favourable metabolic effects, aim of this study was to evaluate if topiramate could modulate atherosclerosis development and protect target organs of dysmetabolic conditions. Thirty apoE-deficient mice were divided into three groups and fed for 12 weeks a high fat diet (Control) or the same diet containing topiramate at 0.125% and 0.250%. Body weight, water and food intake were monitored throughout the study. Plasma lipids and glucose levels were measured and a glucose tolerance test was performed. Atherosclerosis development was evaluated in the whole aorta and at the aortic sinus. Histological analysis of liver, kidney and adipose tissue was performed. Topiramate did not affect weight gain and food intake. Glucose tolerance and plasma lipids were not changed and, in turn, atherosclerosis development was not different among groups. Topiramate did not modify liver and adipose tissue histology. Conversely, in the kidneys, the treatment reduced the occurrence of glomerular lipidosis by decreasing foam cells accumulation and reducing the expression of inflammatory markers. Blood urea nitrogen levels were also reduced by treatment. Our results indicate that topiramate does not affect atherosclerosis development, but preserves kidney structure and function. The study suggests that topiramate could be investigated in drug repurposing studies for the treatment of glomerular lipidosis.
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http://dx.doi.org/10.1016/j.phrs.2018.12.022DOI Listing
March 2019

A novel endovenous scaffold for the treatment of chronic venous obstruction in a porcine model: Histological and ultrastructural assessment.

Phlebology 2019 Jun 18;34(5):336-346. Epub 2018 Oct 18.

2 Unit of Vascular and Transplantation Surgery, University of Catania, Catania, Italy.

Objective: To investigate the biological effects of a novel endovenous scaffold in a porcine model.

Methods: Petalo is a compliant venous scaffold implanted into the internal jugular veins of 12 healthy pigs. The pigs were sacrificed at one, two, three, and six months, respectively. Microscopic investigations were performed at two blinded laboratories.

Results: Neo-intima formation progressively covering up the stent metallic bars was observed. The inflammatory response of the venous wall showed a peak after three months by the implant, followed by marked reduction after six months. The device induced a significant ( p < 0.01) increase of the thickness respect to the control regions, but was comparable in sections obtained after three and six months.

Conclusions: The implant of Petalo compliant venous scaffold in the venous wall of this porcine model is characterized by neointima formation and by an inflammatory reaction which tends to decrease after six months. Our data point against the induction of smooth muscle cells proliferation and migration as confirmed by electronic transmission microscopy analyses.
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http://dx.doi.org/10.1177/0268355518805686DOI Listing
June 2019

Tumor progression and metastatic dissemination in ovarian cancer after dose-dense or conventional paclitaxel and cisplatin plus bevacizumab.

Int J Cancer 2018 11 7;143(9):2187-2199. Epub 2018 Aug 7.

Laboratory of Biology and Treatment of Metastasis, Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.

The efficacy of therapeutic regimens incorporating weekly or every-3-weeks paclitaxel (PTX) for ovarian cancer is debated. We investigated the addition of bevacizumab in regimens of chemotherapy with different PTX doses and schedules in preclinical models. Treatments were cisplatin (DDP) with weekly PTX (conventional), or dose-dense-equi (every other day to the conventional cumulative dose), or dose-dense-high (total dose 1.5 times higher), with or without bevacizumab. Treatment efficacy was evaluated analyzing tumor growth in different time-windows in two patient-derived ovarian cancer xenografts with different sensitivity to cisplatin. Tumor progression, metastasis and survival were studied in ovarian cancer models growing orthotopically and disseminating in the mouse peritoneal cavity. Short-term effects on cell cycle, tumor cell proliferation/apoptosis and vasculature were evaluated by flow cytometry and immunohistochemistry. PTX dose-dense (with/without DDP) was superior to the conventional scheme in a dose-dependent manner; the high efficacy was confirmed by the lower ratio of tumor to normal cells. All schemes benefited from bevacizumab, which reduced tumor vessels. However, DDP/PTX dose-dense-high (only chemotherapy) was at least as active as DDP/PTX conventional plus bevacizumab. DDP/PTX dose-dense-high plus bevacizumab was the most effective in delaying tumor progression, though it did not prolong mouse survival and the continuous treatment with bevacizumab was associated with a malignant disease. These findings indicate that the effect of bevacizumab in combination with chemotherapy may depend on the schedule-dose of the treatment and help to explain the unclear benefits after bevacizumab.
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http://dx.doi.org/10.1002/ijc.31596DOI Listing
November 2018

Novel Compliant Scaffold with Specific Design for Venous System: Results of a Porcine Model Study.

Biomed Res Int 2018 31;2018:7312315. Epub 2018 Jan 31.

Vascular Surgery and Organ Transplant Unit, Department of Surgery, University Hospital of Catania, Catania, Italy.

Background: Stenting has become the first-line treatment of obstructive venous disease because of poor results of balloon angioplasty. This preclinical study aimed to investigate the safety and efficacy profile of a novel compliant venous scaffold (CVS) denominated Petalo CVS, specifically designed for venous diseases.

Materials And Methods: Twelve healthy pigs weighing 90 kg were used to test Petalo CVS. The devices were implanted into the internal jugular veins (IJVs) using a femoral vein percutaneous approach. The safety profile including the success rate of device releasing, anchoring, and positioning was evaluated immediately. Fracture, migration, primary patency, and endothelial response were assessed at 1, 2, 3, and 6 months after the study procedure.

Results: A total of 32 devices were successfully released in both IJVs. No procedure- or device-related complications were reported, and all pigs successfully completed the different scheduled follow-up periods. The primary patency rate was 100%, and no fracture or migration of the device into the brachiocephalic trunk was reported. Histological examination revealed only minimal lesions with minimal or absent inflammatory reaction surrounding the incorporated metallic rods.

Conclusions: This porcine model study showed a promising safety and efficacy profile of Petalo CVS, a novel endovenous device based on specific concepts.
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http://dx.doi.org/10.1155/2018/7312315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5832035PMC
September 2018

Supersulfated low-molecular weight heparin synergizes with IGF1R/IR inhibitor to suppress synovial sarcoma growth and metastases.

Cancer Lett 2018 02 7;415:187-197. Epub 2017 Dec 7.

Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo 42, 20133 Milan, Italy. Electronic address:

Synovial sarcoma (SS) is an aggressive tumor with propensity for lung metastases which significantly impact patients' prognosis. New therapeutic approaches are needed to improve treatment outcome. Targeting the heparanase/heparan sulfate proteoglycan system by heparin derivatives which act as heparanase inhibitors/heparan sulfate mimetics is emerging as a therapeutic approach that can sensitize the tumor response to chemotherapy. We investigated the therapeutic potential of a supersulfated low molecular weight heparin (ssLMWH) in preclinical models of SS. ssLMWH showed a potent anti-heparanase activity, dose-dependently inhibited SS colony growth and cell invasion, and downregulated the activation of receptor tyrosine kinases including IGF1R and IR. The combination of ssLMWH and the IGF1R/IR inhibitor BMS754807 synergistically inhibited proliferation of cells exhibiting IGF1R hyperactivation, also abrogating cell motility and promoting apoptosis in association with PI3K/AKT pathway inhibition. The drug combination strongly enhanced the antitumor effect against the CME-1 model, as compared to single agent treatment, abrogating orthotopic tumor growth and significantly repressing spontaneous lung metastatic dissemination in treated mice. These findings provide a strong preclinical rationale for developing drug regimens combining heparanase inhibitors/HS mimetics with IGF1R antagonists for treatment of metastatic SS.
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http://dx.doi.org/10.1016/j.canlet.2017.12.009DOI Listing
February 2018

FoxO-1 contributes to the efficacy of the combination of the XPO1 inhibitor selinexor and cisplatin in ovarian carcinoma preclinical models.

Biochem Pharmacol 2018 01 16;147:93-103. Epub 2017 Nov 16.

Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, 20133 Milan, Italy. Electronic address:

The XPO1/CRM1 inhibitor selinexor (KPT-330), is currently being evaluated in multiple clinical trials as an anticancer agent. XPO1 participates in the nuclear export of FoxO-1, which we previously found to be decreased in platinum-resistant ovarian carcinoma. The aim of this study was to determine whether enriching FoxO-1 nuclear localization using selinexor would increase ovarian cancer cell sensitivity to cisplatin. Selinexor, as a single agent, displayed a striking antiproliferative effect in different ovarian carcinoma cell lines. A schedule-dependent synergistic effect of selinexor in combination with cisplatin was found in cisplatin-sensitive IGROV-1, the combination efficacy being more evident in sensitive than in the resistant cells. In IGROV-1 cells, the combination was more effective when selinexor followed cisplatin exposure. A modulation of proteins involved in apoptosis (p53, Bax) and in cell cycle progression (p21) was found by Western blotting. Selinexor-treated cells exhibited enriched FoxO-1 nuclear staining. Knock-down experiments with RNA interference indicated that FOXO1-silenced cells displayed a reduced sensitivity to selinexor. FOXO1 silencing also tended to reduce the efficacy of the drug combination at selected cisplatin concentrations. Selinexor significantly inhibited tumor growth, induced FoxO-1 nuclear localization and improved the efficacy of cisplatin in IGROV-1 xenografts. Taken together, our results support FoxO-1 as one of the key factors promoting sensitivity towards selinexor and the synergistic interaction between cisplatin and selinexor in ovarian carcinoma cells with selected molecular backgrounds, highlighting the need for treatment regimens tailored to the molecular tumor features.
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http://dx.doi.org/10.1016/j.bcp.2017.11.009DOI Listing
January 2018

The atypical receptor CCRL2 is required for CXCR2-dependent neutrophil recruitment and tissue damage.

Blood 2017 09 25;130(10):1223-1234. Epub 2017 Jul 25.

Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.

CCRL2 is a 7-transmembrane domain receptor that shares structural and functional similarities with the family of atypical chemokine receptors (ACKRs). CCRL2 is upregulated by inflammatory signals and, unlike other ACKRs, it is not a chemoattractant-scavenging receptor, does not activate β-arrestins, and is widely expressed by many leukocyte subsets. Therefore, the biological role of CCRL2 in immunity is still unclear. We report that CCRL2-deficient mice have a defect in neutrophil recruitment and are protected in 2 models of inflammatory arthritis. In vitro, CCRL2 was found to constitutively form homodimers and heterodimers with CXCR2, a main neutrophil chemotactic receptor. By heterodimerization, CCRL2 could regulate membrane expression and promote CXCR2 functions, including the activation of β2-integrins. Therefore, upregulation of CCRL2 observed under inflammatory conditions is functional to finely tune CXCR2-mediated neutrophil recruitment at sites of inflammation.
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http://dx.doi.org/10.1182/blood-2017-04-777680DOI Listing
September 2017

Estimating canine cancer incidence: findings from a population-based tumour registry in northwestern Italy.

BMC Vet Res 2017 Jun 28;13(1):203. Epub 2017 Jun 28.

Biostatistics, Epidemiology and Risk Analysis Unit, Istituto Zooprofillattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Via Bologna 148, 10154, Torino, Italy.

Background: Canine cancer registry data can be put to good use in epidemiological studies. Quantitative comparison of tumour types may reveal unusual cancer frequencies, providing directions for research and generation of hypotheses of cancer causation in a specific area, and suggest leads for identifying risk factors. Here we report canine cancer incidence rates calculated from a population-based registry in an area without any known specific environmental hazard.

Results: In its 90 months of operation from 2001 to 2008 (the observation period in this study), the population-based Piedmont Canine Cancer Registry collected data on 1175 tumours confirmed by histopathological diagnosis. The incidence rate was 804 per 100,000 dog-years for malignant tumours and 897 per 100,000 dog-years for benign tumours. Higher rates for all cancers were observed in purebred dogs, particularly in Yorkshire terrier and Boxer. The most prevalent malignant neoplasms were cutaneous mastocytoma and hemangiopericytoma, and mammary gland complex carcinoma and simplex carcinoma.

Conclusions: The Piedmont canine cancer registry is one of few of its kind whose operations have been consistently supported by long-term public funding. The registry-based cancer incidence rates were estimated with particular attention to the validity of data collection, thus minimizing the potential for bias. The findings on cancer incidence rates may provide a reliable reference for comparison studies. Researches conducted on dogs, used as sentinels for community exposure to environmental carcinogens, can be useful to detect excess risks in the incidence of malignant tumours in the human population.
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http://dx.doi.org/10.1186/s12917-017-1126-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5490209PMC
June 2017

Duration of Untreated Cardiac Arrest and Clinical Relevance of Animal Experiments: The Relationship Between the "No-Flow" Duration and the Severity of Post-Cardiac Arrest Syndrome in a Porcine Model.

Shock 2018 02;49(2):205-212

IRCCS-Istituto di Ricerche Farmacologiche "Mario Negri," Milan, Italy.

Introduction: The study investigated the effect of untreated cardiac arrest (CA), that is, "no-flow" time, on postresuscitation myocardial and neurological injury, and survival in a pig model to identify an optimal duration that adequately reflects the most frequent clinical scenario.

Methods: An established model of myocardial infarction followed by CA and cardiopulmonary resuscitation was used. Twenty-two pigs were subjected to three no-flow durations: short (8-10 min), intermediate (12-13 min), and long (14-15 min). Left ventricular ejection fraction (LVEF) was assessed together with thermodilution cardiac output (CO) and high sensitivity cardiac troponin T (hs-cTnT). Neurological impairment was evaluated by neurological scores, serum neuron specific enolase (NSE), and histopathology.

Results: More than 60% of animals survived when the duration of CA was ≤13 min, compared to only 20% for a duration ≥14 min. Neuronal degeneration and neurological scores showed a trend toward a worse recovery for longer no-flow durations. No animals achieved a good neurological recovery for a no-flow ≥14 min, in comparison to a 56% for a duration ≤13 min (P = 0.043). Serum NSE levels significantly correlated with the no-flow duration (r = 0.892). Longer durations of CA were characterized by lower LVEF and CO compared to shorter durations (P < 0.05). The longer was the no-flow time, the higher was the number of defibrillations delivered (P = 0.043). The defibrillations delivered significantly correlated with LVEF and plasma hs-cTnT.

Conclusions: Longer no-flow durations caused greater postresuscitation myocardial and neurological dysfunction and reduced survival. An untreated CA of 12-13 min may be an optimal choice for a clinically relevant model.
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http://dx.doi.org/10.1097/SHK.0000000000000914DOI Listing
February 2018

An explant of heifer mammary gland to study the immune response of the organ.

Res Vet Sci 2017 Oct 6;114:44-50. Epub 2017 Mar 6.

Department of Veterinary Medicine, University of Milan, via Celoria 10, 20133 Milano, Italy. Electronic address:

Continuous or primary epithelial cell lines have been extensively used to study the mammary gland immune response, but they are constituted by a single cell population. Our aim was to test whether an explant of heifer gland, where the tissue structure is maintained, might be a valid model to investigate the innate immune response to infection. The study was carried out on 2mm-sections of heifer udders, in 2 consecutive trials, using LPS or LTA in the first trial and two different concentrations of Staphylococcus aureus (Staph. aureus) in the second. Treated and untreated sections were collected after 1h, 3h and 6h incubation; in the first trial, a final time-point at 18h was considered. The mRNA expression of TNFα, IL-1β, IL-6, IL-8 and LAP was analyzed by quantitative real-time PCR. Histological examination showed well-preserved morphology of the tissue, and apoptosis only showed a slight, not significant increase throughout the experiment. IL-1β and IL-6 were significantly up-regulated, in response to LPS or Staph. aureus, while TNF-α and IL-8 significantly increased only under LPS treatment. LAP expression showed a significant late increase when stimulated by LPS. The immunochemical staining of the sections demonstrated a higher number of T lymphocytes within the alveolar epithelium, in comparison with interstitial localization. Since the explants belonged to pubertal non-pregnant heifers, T cells may be regarded as resident cells, suggesting their participation in the regulation of mammary homeostasis. Therefore, applying our model would give new insights in the investigation of udder pathophysiology.
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http://dx.doi.org/10.1016/j.rvsc.2017.03.002DOI Listing
October 2017

Functional Single-Chain Polymer Nanoparticles: Targeting and Imaging Pancreatic Tumors in Vivo.

Biomacromolecules 2016 10 21;17(10):3213-3221. Epub 2016 Sep 21.

IK4-CIDETEC, P° Miramón 196, 20014 Donostia-San Sebastián, Spain.

The development of tools for the early diagnosis of pancreatic adenocarcinoma is an urgent need in order to increase treatment success rate and reduce patient mortality. Here, we present a modular nanosystem platform integrating soft nanoparticles with a targeting peptide and an active imaging agent for diagnostics. Biocompatible single-chain polymer nanoparticles (SCPNs) based on poly(methacrylic acid) were prepared and functionalized with the somatostatin analogue PTR86 as the targeting moiety, since somatostatin receptors are overexpressed in pancreatic cancer. The gamma emitter Ga was incorporated by chelation and allowed in vivo investigation of the pharmacokinetic properties of the nanoparticles using single photon emission computerized tomography (SPECT). The resulting engineered nanosystem was tested in a xenograph mouse model of human pancreatic adenocarcinoma. Imaging results demonstrate that accumulation of targeted SCPNs in the tumor is higher than that observed for nontargeted nanoparticles due to improved retention in this tissue.
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http://dx.doi.org/10.1021/acs.biomac.6b00941DOI Listing
October 2016

Bioluminescence imaging of estrogen receptor activity during breast cancer progression.

Am J Nucl Med Mol Imaging 2016 28;6(1):32-41. Epub 2016 Jan 28.

Department of Pharmacological and Biomolecular Sciences, Center of Excellence on Neurodegenerative Diseases, University of Milan 20133, Milan, Italy.

Estrogen receptors (ER) are known to play an important regulatory role in mammary gland development as well as in its neoplastic transformation. Although several studies highlighted the contribution of ER signaling in the breast transformation, little is known about the dynamics of ER state of activity during carcinogenesis due to the lack of appropriate models for measuring the extent of receptor signaling in time, in the same animal. To this aim, we have developed a reporter mouse model for the non-invasive in vivo imaging of ER activity: the ERE-Luc reporter mouse. ERE-Luc is a transgenic mouse generated with a firefly luciferase (Luc) reporter gene driven by a minimal promoter containing an estrogen responsive element (ERE). This model allows to measure receptor signaling in longitudinal studies by bioluminescence imaging (BLI). Here, we have induced sporadic mammary cancers by treating systemically ERE-Luc reporter mice with DMBA (9,10-dimethyl 1,2-benzanthracene) and measured receptor signaling by in vivo imaging in individual animals from early stage until a clinically palpable tumor appeared in the mouse breast. We showed that DMBA administration induces an increase of bioluminescence in the whole abdominal area 6 h after treatment, the signal rapidly disappears. Several weeks later, strong bioluminescence is observed in the area corresponding to the mammary glands. In vivo and ex vivo imaging analysis demonstrated that this bioluminescent signal is localized in the breast area undergoing neoplastic transformation. We conclude that this non-invasive assay is a novel relevant tool to identify the activation of the ER signaling prior the morphological detection of the neoplastic transformation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4749503PMC
April 2016

Human malignant mesothelioma is recapitulated in immunocompetent BALB/c mice injected with murine AB cells.

Sci Rep 2016 Mar 10;6:22850. Epub 2016 Mar 10.

Chromatin Dynamics Unit, Division of Genetics and Cell Biology, San Raffaele Hospital, Milano, Italy.

Malignant Mesothelioma is a highly aggressive cancer, which is difficult to diagnose and treat. Here we describe the molecular, cellular and morphological characterization of a syngeneic system consisting of murine AB1, AB12 and AB22 mesothelioma cells injected in immunocompetent BALB/c mice, which allows the study of the interplay of tumor cells with the immune system. Murine mesothelioma cells, like human ones, respond to exogenous High Mobility Group Box 1 protein, a Damage-Associated Molecular Pattern that acts as a chemoattractant for leukocytes and as a proinflammatory mediator. The tumors derived from AB cells are morphologically and histologically similar to human MM tumors, and respond to treatments used for MM patients. Our system largely recapitulates human mesothelioma, and we advocate its use for the study of MM development and treatment.
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http://dx.doi.org/10.1038/srep22850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4785401PMC
March 2016

Tissue distribution and acute toxicity of silver after single intravenous administration in mice: nano-specific and size-dependent effects.

Part Fibre Toxicol 2016 Feb 29;13:12. Epub 2016 Feb 29.

Fondazione Filarete, 20139, Milan, Italy.

Background: Silver nanoparticles (AgNPs) are an important class of nanomaterials used as antimicrobial agents for a wide range of medical and industrial applications. However toxicity of AgNPs and impact of their physicochemical characteristics in in vivo models still need to be comprehensively characterized. The aim of this study was to investigate the effect of size and coating on tissue distribution and toxicity of AgNPs after intravenous administration in mice, and compare the results with those obtained after silver acetate administration.

Methods: Male CD-1(ICR) mice were intravenously injected with AgNPs of different sizes (10 nm, 40 nm, 100 nm), citrate-or polyvinylpyrrolidone-coated, at a single dose of 10 mg/kg bw. An equivalent dose of silver ions was administered as silver acetate. Mice were euthanized 24 h after the treatment, and silver quantification by ICP-MS and histopathology were performed on spleen, liver, lungs, kidneys, brain, and blood.

Results: For all particle sizes, regardless of their coating, the highest silver concentrations were found in the spleen and liver, followed by lung, kidney, and brain. Silver concentrations were significantly higher in the spleen, lung, kidney, brain, and blood of mice treated with 10 nm AgNPs than those treated with larger particles. Relevant toxic effects (midzonal hepatocellular necrosis, gall bladder hemorrhage) were found in mice treated with 10 nm AgNPs, while in mice treated with 40 nm and 100 nm AgNPs lesions were milder or negligible, respectively. In mice treated with silver acetate, silver concentrations were significantly lower in the spleen and lung, and higher in the kidney than in mice treated with 10 nm AgNPs, and a different target organ of toxicity was identified (kidney).

Conclusions: Administration of the smallest (10 nm) nanoparticles resulted in enhanced silver tissue distribution and overt hepatobiliary toxicity compared to larger ones (40 and 100 nm), while coating had no relevant impact. Distinct patterns of tissue distribution and toxicity were observed after silver acetate administration. It is concluded that if AgNPs become systemically available, they behave differently from ionic silver, exerting distinct and size-dependent effects, strictly related to the nanoparticulate form.
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http://dx.doi.org/10.1186/s12989-016-0124-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4772516PMC
February 2016

Chromosome transplantation as a novel approach for correcting complex genomic disorders.

Oncotarget 2015 Nov;6(34):35218-30

Milan Unit, Istituto di Ricerca Genetica e Biomedica, CNR, Milan, Italy.

Genomic disorders resulting from large rearrangements of the genome remain an important unsolved issue in gene therapy. Chromosome transplantation, defined as the perfect replacement of an endogenous chromosome with a homologous one, has the potential of curing this kind of disorders. Here we report the first successful case of chromosome transplantation by replacement of an endogenous X chromosome carrying a mutation in the Hprt genewith a normal one in mouse embryonic stem cells (ESCs), correcting the genetic defect. The defect was also corrected by replacing the Y chromosome with an X chromosome. Chromosome transplanted clones maintained in vitro and in vivo features of stemness and contributed to chimera formation. Genome integrity was confirmed by cytogenetic and molecular genome analysis. The approach here proposed, with some modifications, might be used to cure various disorders due to other X chromosome aberrations in induced pluripotent stem (iPS) cells derived from affected patients.
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http://dx.doi.org/10.18632/oncotarget.6143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742100PMC
November 2015

Targeted Gene Correction in Osteopetrotic-Induced Pluripotent Stem Cells for the Generation of Functional Osteoclasts.

Stem Cell Reports 2015 Oct 3;5(4):558-68. Epub 2015 Sep 3.

Milan Unit, Istituto di Ricerca Genetica e Biomedica, CNR, 20138 Milan, Italy; Humanitas Clinical and Research Center, via Manzoni 56, 20089 Rozzano (Mi), Italy.

Autosomal recessive osteopetrosis is a human bone disease mainly caused by TCIRG1 gene mutations that prevent osteoclasts resorbing activity, recapitulated by the oc/oc mouse model. Bone marrow transplantation is the only available treatment, limited by the need for a matched donor. The use of induced pluripotent stem cells (iPSCs) as an unlimited source of autologous cells to generate gene corrected osteoclasts might represent a powerful alternative. We generated iPSCs from oc/oc mice, corrected the mutation using a BAC carrying the entire Tcirg1 gene locus as a template for homologous recombination, and induced hematopoietic differentiation. Similarly to physiologic fetal hematopoiesis, iPSC-derived CD41(+) cells gradually gave rise to CD45(+) cells, which comprised both mature myeloid cells and high proliferative potential colony-forming cells. Finally, we differentiated the gene corrected iPSC-derived myeloid cells into osteoclasts with rescued bone resorbing activity. These results are promising for a future translation into the human clinical setting.
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http://dx.doi.org/10.1016/j.stemcr.2015.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624934PMC
October 2015