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[PhD Students Meeting] [2015]

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Introduction

Since its first edition in 2007, the PhD Students Meeting organized by Italian PhD students affiliated to The Open University has established itself as an exciting event very useful for students as it brings together companies and experts in numerous research fields. The 2015 PhD Students Meeting program will feature captivating talks from both academics and industry representatives on new trends in various scientific areas. This year, oral and poster presentations will be held under a new track: PhD as an opportunity to enjoy Science which is not to be missed! Many experts from excellent Italian research institute have already confirmed their participation and will be glad to share their opinions on the most controversial scientific topics. The 2015 PhD Student Meeting includes five full-time plenary sessions on the following subjects: Cancer Biology, Molecular Bases of Diseases, Immunology, Neuroscience and New frontiers in Biotechnology. We invite you to join us at the 2015 PhD Students Meeting, on July 9-10, 2015 at the Istituto di Ricerche Farmacologiche Mario Negri in Milan.

The PhD meeting committee

Emanuela Fina, Andrea Tomirotti, Federico Moro, Serena Bettoni and Giovanni Castino


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Scientific program

Thursday July 9, 2015

h. 9.00 – 9.30 REGISTRATION

h. 9.30 – 9.45 OPENING REMARKS Silvio Garattini, MD

CANCER BIOLOGY SESSION Moderators: Emanuela Fina and Giovanni Castino

h. 9.45 – 10.30

OPENING LECTURE Cristiano Simone, MD, PhD PhD: a one-way ticket to science

h. 10.30 – 10.50

ORAL PRESENTATION Valentina Celestini, PhD candidate SIRT3-dependent deacetylation of mitochondrial FoxO3A regulates colorectal

cancer cell fate

h. 10.50 – 11.10

ORAL PRESENTATION Martina Magni, PhD candidate DBC1 is required for Chk2-dependent KAP1 phosphorylation and repair of

heterochromatic DNA lesions

h. 11.10 – 11.30 COFFEE BREAK (offered by Zeiss)

h. 11.30 – 11.50

ORAL PRESENTATION Matteo Dugo, PhD candidate Dissecting melanoma heterogeneity by integrative genomic analysis for tailored

anti-cancer therapy

h. 11.50 – 12.10

ORAL PRESENTATION Giulia Bottai, PhD candidate AXL sustains tumor-associated inflammation and is a prognostic indicator of

poor outcome in triple-negative breast cancers

h. 12.10 – 13.00 POSTER SESSION I

h. 13.00 – 14.00 LUNCH

MOLECULAR BASES OF DISEASES SESSION Moderators: Serena Bettoni and Emanuela Fina

h. 14.00 – 14.45

OPENING LECTURE Marina Noris, PhD Next generation sequencing for identification of molecular basis of rare diseases

h. 14.45 – 15.05

ORAL PRESENTATION Paraskevas Iatropoulos, PhD candidate Complement gene variants determine the risk of immune- complex mediated

MPGN and C3 glomerulopathy and predict long-term renal outcome

h. 15.05 – 15.25

ORAL PRESENTATION Carolina Greco, PhD candidate The Epigenetic cofactor UHRF1 controls the metabolic re-programming of

hypertrophic cardiomyocytes


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h. 15.25 – 16.45 POSTER SESSION II


h. 17.05 – 17.25

ORAL PRESENTATION Montserrat Climent, PhD candidate TGFβ triggers miR-143/145 transfer from smooth muscle cells to endothelial

cells, thereby modulating vessel stabilization

h. 17.25 – 17.45

ORAL PRESENTATION Giuseppina Mastrototaro, PhD candidate Ablation of palladin in adult cardiac muscle causes dilated cardiomyopathy

Friday July 10, 2015

NEW FRONTIERS IN BIOTECHNOLOGY SESSION Moderators: Serena Bettoni and Federico Moro

h. 9.15 – 10.00

OPENING LECTURE Anna Caroli, PhD New frontiers in medical imaging in support of clinical research and diagnosis

h. 10.00 – 10.20

ORAL PRESENTATION Kanishka Sharma, PhD candidate Novel methods for total kidney volume computation in autosomal dominant

polycystic kidney disease

h. 10.20 – 10.40

ORAL PRESENTATION Marco Franzoni, PhD candidate In-vitro wall shear stress waveforms derived from arteriovenous fistula elicit

endothelial cell activation and neointimal hyperplasia related molecule

production


h. 11.00 – 11.20

ORAL PRESENTATION Stefano Finazzi, PhD candidate Kinetics of antibiotics in critically ill patients

h. 11.20 – 11.40

ORAL PRESENTATION Diego Baderna, PhD candidate Prevention starts from the environment: chemical and biological

approaches to study the potential adverse effects of pollutants

h. 11.40 – 12.30 POSTER SESSION III

IMMUNOLOGY SESSION Moderators: Andrea Tomirotti and Giovanni Castino

h. 12.30 – 13.15

OPENING LECTURE Paola Allavena, MD, PhD Immunity in health and disease

h. 13.15 – 13.35

ORAL PRESENTATION Rosita Rigoni, PhD candidate Microbial signals control inflammation and autoimmunity induced by


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hypomorphic RAG defects

h. 13.35 – 14.25 LUNCH

h. 14.25 – 14.45

ORAL PRESENTATION Naths Grazia Sukubo, PhD candidate microRNA-135b: the pivot of the macrophages polarization balance

h. 14.45 – 15.05

ORAL PRESENTATION Elena Pontarini, PhD candidate NK cells recruitment to the salivary glands regulates early viral control but is

dispensable for the formation of inducible tertiary lymphoid structures

h. 15.05 – 15.25

ORAL PRESENTATION Nadia Castioni, PhD candidate Role of SPARC and mast cells in non-Hodgkin B cell lymphomas

h. 15.25 – 16.15 POSTER SESSION IV


NEUROSCIENCE Moderators: Federico Moro and Andrea Tomirotti

h. 16.35 – 17.20

OPENING LECTURE Stefano Fumagalli, PhD Immune cell dynamics in the ischemic brain seen by in vivo two-photon

microscopy

h. 17.20 – 17.40

ORAL PRESENTATION Simonetta Papa, PhD candidate An early modulation of the pro-inflammatory response associated to the

activated microglia is pivotal to sustain healing processes in spinal cord injury

h. 17.40 – 18.00

ORAL PRESENTATION Valentina Iori, PhD candidate miR146a-based therapy against neuroinflammation has anti-ictogenic and

disease-modifying effects in murine models of seizures and epilepsy

h. 18.00 – 18.20

ORAL PRESENTATION Marco Rasile, PhD candidate Maternal immune activation provokes higher susceptibility to epilepsy in the

offspring

h. 18.20 – 18.40

ORAL PRESENTATION Elsa Ghirardini, PhD candidate The influence of mutant protein on synaptic structure and neurotransmission:

mechanisms of neuronal dysfunction in genetic prion diseases

h. 18.40 – 18.45 AWARD CEREMONY Enrico Garattini, PhD

h. 18.45 – 19.00 CONCLUDING REMARKS Domenico Delia, PhD


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ABSTRACTS


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Abstract #01 – Molecular Bases of Diseases

Effect of inhibition of the kynurenine pathway in a rat model of cardiac arrest and

cardiopulmonary resuscitation.

Roberta Affatato, Sabina Ceriani, Francesca Fumagalli, Jacopo Lucchetti, Claudia Fracasso, Ilaria Russo, Lidia Staszewsky, Serge Masson, Marco Gobbi, Roberto Latini, Giuseppe Ristagno. Mario Negri Institute for Pharmacological Research, Milan,Italy

Introduction. Kynurenine pathway (KP), involved in the pathogenesis of numerous cerebral disorders, is the major route of the tryptophane catabolism. We previously demonstrated that KP was activated early following cardiac arrest and correlated with neurological injury and survival both in animals and humans. We now hypothesized that modulation of KP through inhibition of the enzyme initiating the tryptophan catabolism into kynurenine, the IDO enzyme, could prevent KP activation and improve neurological recovery. Methods. Twenty-two rats received oral administration of 1-Methyl-DL-Tryptophan (1-MT, 400mg/Kg) (n=10) or corresponding vehicle (n=12), 24hr and 2hr before cardiac arrest. Ventricular fibrillation was induced and untreated for 8 min. Cardiopulmonary resuscitation was then initiated and continued for additional 8 min prior to defibrillation. Blood samples were obtained for high-sensitive cardiac troponin T (hs-cTnT) and neuron specific enolase (NSE) assays. Animals were observed up to 96hr for assesment of neurological recovery. Results. 90% of animals treated with 1-MT were successfully resuscitated compared to 75% of those treated with vehicle. However, only 5 animals in each group survived up to 96hr. Animals treated with 1-MT presented no activation of KP, as indicated by the significantly lower plasma levels of kynurenine and kynurenine/tryptophan ratio in comparison to vehicle ones. This KP inhibition was accompained by lower serum NSE and a significantly better neurological recovery compared. The magnitude of KP activation in plasma (r=0.94, p=0.0002) and in the hippocampus (r=0.73, p=0.019) was significantly related to the severity of the neurological deficit. Conclusions. Inhibition of KP improves neurological recovery after cardiac arrest.


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Abstract #02 – Cancer Biology

Fine tuning of platinum drug resistance by miR-483-3p in human ovarian carcinoma cells

Noemi Arrighetti, Giacomo Cossa, Loris De Cecco, Nives Carenini, Elisabetta Corna, Paolo Gandellini, Nadia Zaffaroni, Paola Perego, Laura Gatti Experimental Oncology and Molecular Medicine Department, Fondazione IRCCS Istituto

Nazionale dei Tumori, via Amadeo 42, 20133 Milan, Italy

In spite of the efficacy of platinum compounds, drug resistance represents a major limitation to the cure of ovarian carcinoma. Altered expression of microRNAs (miRNAs) may contribute to the drug-resistant phenotype of ovarian carcinoma by regulating different aspects of cell response to drugs. Thus, the aim of the present study was the analysis of the miRNAs pattern of expression in platinum-sensitive and –resistant cell lines using genome-wide and functional approaches, with specific reference to alterations conferring drug-resistance. The expression pattern of miRNAs in ovarian carcinoma cell lines resistant to platinum compounds (IGROV-1/Pt1 and IGROV-1/OHP) as compared to parental cells (IGROV-1) was examined. Six miRNAs were up-regulated in both resistant variants, among which we found miR-483-3p, which has been implicated in apoptosis and proliferation regulation. Functional studies were carried out by transfecting a synthetic precursor of miR-483-3p in parental cells. A consistent and marked upregulation of the miRNA levels at different times was observed. Over-expression of miR- 483-3p conferred mild levels of cisplatin resistance to IGROV-1 cells. A reduced proliferative rate upon miRNA over-expression in parental cells was observed in keeping with a down-regulation of CDK6 and PRKCA, which are predicted miR-483-3p targets. Moreover, cisplatin sensitivity of IGROV-1 cells decreased in the presence of the specific PKC-alpha inhibitor GO6983. Increased expression of miR-483-3p appears to confer low levels of cisplatin resistance by interfering with the proliferative potential of ovarian carcinoma cells. Because low levels of resistance have been proposed to be clinically relevant, our results may be of translational value.


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Abstract #03 – New Frontiers in Biotechnology

Prevention starts from the environment: chemical and biological approaches to study

the potential adverse effects of pollutants.

Diego Baderna, Filippo Cinà, Marco Lodi, Emilio Benfenati Laboratory of Environmental Chemistry and Toxicology, IRCCS Istituto di Ricerche

Farmacologiche “Mario Negri”

Environment and health are intimately connected: in fact it has been shown that exposure to pollutants in soil, water and air can cause the onset of diseases, exacerbate or make them more frequent. To assess the levels of pollutants in the environment around us, to determine the routes by which these compounds can reach and interact with human and ecological receptors and to define the toxicological profiles of individual pollutants and mixtures are important goals for the protection of quality of life starting from the environment. The potential risks to human health and the ecosystem approach can be hypothesized using a chemical-based risk analysis or by biological models of human interest or relevant for the ecosystem. Focusing on the biological approach, there are a lot of toxicological and ecotoxicological bioassays employed in monitoring campaigns as additional analytical tools and/or to support the processes of decision-making. The approach of the Laboratory of Environmental Toxicology and Chemistry and its application in some case studies from the field or from the lab will be described including the results from the Olona Valley and from the Lomellina and a preliminary study on phytotoxicity when soil is contaminated with single and multiple heavy metals. Finally, a fast overview of future improvements will be done to highlight possible collaborations with other researchers from different research fields.


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Functional human podocytes generated in organoids from amniotic fluid stem cells

Valentina Benedetti, Christodoulos Xinaris and Marina Morigi IRCCS – Istituto di Ricerche Farmacologiche ‘Mario Negri’, Centro Anna Maria Astori, Science

and Technology Park Kilometro Rosso, Bergamo, Italy.

The shortage of transplantable kidneys create an urgent need for tissue-engineered alternatives. However, to date no cell-based strategy has generated nephrons with intact 3D epithelial filtering barriers. First, we documented that organoids, made with E11.5 murine embryonic kidney cell suspensions, implanted under the renal capsules of athymic rats for 2 weeks, could recapitulate the complex 3D filtering structure of glomerular slits in vivo. The renal organoids accomplished selective glomerular filtration and tubular reabsorption of tracer macromolecules, consistent with their high degree of structural maturation. Then, by exploiting this technology, we mixed human amniotic fluid stem cells (AFSCs) with mouse kidney cells to establish 3D chimeric organoids that were cultured in vitro for up to 5 days. The majority of hAFSCs were distributed between Pax- 2-positive structures. To enhance integration of hAFSCs into chimeric organoids, human cells were genetically modified to express GDNF, a key factor in kidney organogenesis. GDNF-expressing hAFSCs were incorporated into Pax-2-positive structures surrounded by the basement membrane marker laminin. To achieve further maturation, chimeric organoids were implanted under the kidney capsule of athymic rats. After 1 week, organoids grew and formed vascularized glomeruli and tubules. Human cells contributed to the formation of glomerular structures, differentiated into podocytes with slit diaphragms, and internalized exogenously-infused bovine serum albumin, thereby attaining unprecedented degrees of specialization and function in vivo for donor stem cells. Human AFSC chimeric organoids may offer new ways of studying renal development and human podocyte disease, drug discovery, and translational research.


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INSIGHTS INTO THE EFFECTS OF COMPLEMENT FACTOR H ON THE

ASSEMBLY AND DECAY OF THE ALTERNATIVE PATHWAY C3

PROCONVERTASE AND C3 CONVERTASE

Serena Bettoni*, Stephanie Ngo†, Veronique Fremeaux-Bacchi†, Elena Bresin*, Marina Noris*, and Roberta Donadelli*

* IRCCS - Istituto di Ricerche Farmacologiche “Mario Negri”, Clinical Research Center for

Rare Diseases “Aldo e Cele Daccò”, Ranica, Bergamo, Italy

†Assistance Publique des Hopitaux de Paris, Hopital Europeen Georges Pompidou,

laboratoire d’Immunologie, Paris, France

In the central alternative complement pathway (AP) amplification step, C3b and factor B (FB) form the C3 proconvertase (C3bB), which is cleaved by factor D (FD) to an active C3 convertase (C3bBb). The role of factor H (FH) of dissociating C3bBb and/or inactivating C3b is well documented. At variance, FH ability to prevent C3bB assembly and its conversion to C3bBb has not been fully addressed. To investigate the effect of FH on the assembly and decay of the AP C3bB and C3bBb, we set up a new user-friendly assay based on combined microplate and western blot (WB) techniques,

exploiting the selective stabilization properties of Mn2+ and Mg2+ on C3bB and C3bBb, respectively.

C3bB(Mn2+), C3bBb(Mg2+) or C3bB/C3bBb(Ni2+) were obtained by incubation of FB (+/- FD) on C3b-coated wells with or without FH. Spontaneous or FH-mediated dissociation of the complexes was monitored by further incubation with buffer or FH. C3bB and C3bBb were selectively visualized on WB with anti-FB antibody as B (93 KDa) or Bb (60 KDa) bands, respectively.

FH, at physiological molar ratio with FB and FD, did not affect C3bB(Mn2+) and C3bB(Ni2+) formation indicating that FH does not efficiently compete with FB for binding to C3b. In

addition, FH did not dissociate C3bB(Mn2+ or Ni2+), suggesting that FH is not able to display

FB from C3b. On the other hand, FH had a strong C3bBb(Mg2+ or Ni2+) decay accelerating activity in a concentration-dependent manner, as previously documented and this effect was not

prevented by properdin. FH had also an apparent strong inhibitory effect on C3bBb(Mg2+ or

Ni2+) formation. By measuring Bb fragment levels in the supernatant of the reaction of FB, FD

on C3b-coated wells in the presence of Mn2+, higher Bb levels were found vs. the same reaction but without FD (200-500 ng/ml vs 75-98 ng/ml), while only C3bB was recovered from wells, indicating that in this condition C3bBb formed and rapidly dissociated. By adding FH in the mixture, low Bb levels (80-100 ng/ml) were found in the supernatant, while the formation of

C3bB(Mn2+) on wells was completely unaffected. Altogether, these results indicate that FH effect on C3bBb formation is likely the sum of inhibition of C3bB conversion to C3bBb and C3bBb decay acceleration. Our findings shed a new light on mechanisms underlying the regulation of C3 proconvertase and C3 convertase by FH.


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Abstract #06 - Neuroscience

EXPLORING MKK7 ROLE IN EXCITOTOXICITY AND CEREBRAL

ISCHEMIA: DESIGN NOVEL PHARMACOLOGICAL STRATEGY AGAINST

BRAIN INJURY.

Silvia Biggi1*, Alessandra Sclip1, Sara Cimini1, Daniele Di Marino3, Alessandro Vercelli2, Tiziana Borsello

1 IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri” Milan.

2 NICO-Neuroscience Institute Cavalieri Ottolenghi, University of Turin.

3 Università La Sapienza, Rome.

* Silvia Biggi: IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Via La Masa

19, 20156 Milano, Italy. e-mail: [email protected]; tel:+39 02 3901 4774

Excitotoxicity following cerebral ischemia elicits a molecular cascade, which leads to neuronal death. One key molecule underlying excitotoxic cell death is c-Jun-N-terminal kinase (JNK). We have previously shown that JNK blockade by specific cell-permeable inhibitor peptide significantly reduces infarct size and neuronal death in in vivo model of cerebral ischemia. However, systemic inhibition of JNK may have detrimental side effects due to blockade of its physiological function. Here, we designed a new inhibitor peptide (GADD45beta-I) targeting MKK7, an upstream activator of JNK, that mediates exclusively the pathological activation of JNK. GADD45beta-I was engineered by optimizing the domain of the growth arrest and DNA damage inducible 45beta (GADD45beta), able to bind to MKK7 and by linking it to the TAT peptide sequence, in order to allow penetration of cell membranes. GADD45betaI significantly reduced neuronal death in in vitro models of excitotoxicity, induced by NMDA exposure and by Oxygen Glucose Deprivation. Moreover, GADD45beta-I exerted neuroprotection in vivo, in two models of permanent ischemia, obtained by electrocoagulation and by thromboembolic occlusion of the Middle Cerebral Artery. By blocking MKK7 activation in vivo, GADD45beta-I reduced the infarct size when injected 30’ before the lesion in both models. Moreover the peptide was also effective when administrated 6h after lesion, as demonstrated in the electrocoagulation model. Targeting MKK7 could represent a new therapeutic strategy for the treatment of ischemia and other pathologies involving MKK7/JNK activation. Moreover, this new inhibitor can be useful to further dissect the physiological and pathological role of JNK pathway in the brain.


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Paclitaxel drives response to combination therapy with bevacizumab in ovarian cancer

preclinical models

Bizzaro F.1, D’Agostini E1, Decio A1, Falcetta F 2, Erba E 2, Ubezio P 2 and Giavazzi R1.

1 Laboratory of Biology and Treatment of Metastasis, Department of Oncology, IRCCS-Istituto di

Ricerche Farmacologiche Mario Negri, Milan, Italy.

2 Laboratory of Cancer Pharmacology, Department of Oncology, IRCCS-Istituto di Ricerche

Farmacologiche Mario Negri, Milan, Italy.

Introduction The choice of dose and schedule of chemotherapy combined with angiogenesis inhibitors is a relevant clinical issue. Carboplatin plus paclitaxel is the standard chemotherapy and the addition of bevacizumab is recommended for patients with ovarian cancer. Weekly schedule of paclitaxel (dose-dense) combined with carboplatin has shown clinical benefits (progression free survival and overall survival) compared with conventional tri-weekly paclitaxel, and it is well tolerated. However the advantage of adding bevacizumab (BEV) to chemotherapy with dose-dense paclitaxel (PTX) compared to conventional chemotherapy remain to be shown. The aim of our study was to compare the antitumor activity of different schedules of chemotherapy (i.e. conventional or dose- dense PTX) with or without bevacizumab in models of patient-derived epithelial ovarian cancer xenografts (EOC-PDX) with known sensitivity to cisplatin (DDP). Material and Methods Two high grade serous EOC-PDX, MNHOC18 and MNHOC84, were used. Mice bearing EOC- PDX were randomized to receive: i) vehicle; ii) chemotherapy (DDP/PTX conventional; DDP/PTX dose-dense); iii) chemotherapy plus bevacizumab (BEV+DDP/PTX conventional; BEV+DDP/PTX dose-dense). Dose-dense regimen was administered at two drug concentrations: equi-dose (total dose of PTX as for conventional) and high-dose (total dose of PTX 1.5 higher than conventional). Antitumor activity was evaluated as the best T/C%, growth delay and partial/complete responses; computer assisted analysis of tumor growth curves allowed to calculate scores of Log Cell Kill (LCK) and re-growth doubling time, supported by DNA flow cytometric analysis. The effect on tumor vasculature was evaluated as CD31 positive vessels. Results and discussion Dose-dense schedules of PTX were more effective in reducing tumor growth than conventional dose on both EOC-PDX models, with the high dose-dense being the most active. Bevacizumab added to conventional PTX was more effective than chemotherapy alone and when added to dose- dense PTX the outcome was improved in a dose-dependent manner, resulting in complete tumor responses. Analysis of the growth curves suggested that the triple treatment in the dose-dense regimen caused long term impairment of the re-growth kinetics of the tumors. Conclusion Dose-dense PTX was more efficacious than weekly PTX on EOC-PDX models and the response was improved by the addition of bevacizumab. The administration of dose-dense PTX combined with bevacizumab is likely offering the opportunity of better response than conventional treatment, with less toxicity for ovarian cancer. Supported by The Italian Association for Cancer Research (IG no.14532). A.D. is a fellow of

The Italian Foundation for Cancer Research (FIRC).


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Contribution of CCL2/CCR2 axis in motor neuronal pathology of Amyotrophic

Lateral Sclerosis (ALS).

B. Bosani1, G. Nardo1, MC. Trolese1, B. Savino 2,3, N.Caronni2,3, R. Bonecchi2,3, C. Bendotti1. 1) Dept. Neuroscience- IRCCS- Istituto di Ricerche Farmacologiche Mario Negri- Milano,Italy.

2) Humanitas Clinical and Research Center, Rozzano, Italy.

3) Department of Medical Biotechnologies and Translational Medicine, Università degli Studi di Milano, Rozzano, Italy.

A complex role of the innate and adaptive immune response has been suggested in the pathogenesis of ALS. Monocytes and lymphocyte infiltrates have been found in the spinal cord of patients and mouse models of ALS, although their effective role in the progression of the disease is controversial. CCL2/MCP-1 is one of the earliest chemokine detectable in the spinal cord of patients and ALS mouse models, and it is involved in the recruitment of immune cells through the interaction with the chemokine receptor CCR2. Previous evidence indicate an alteration in the peripheral level of monocytes and lymphocytes in the blood of ALS patients in respect to healthy controls. Thus, the aim of this study is to decipher the role of the CCL2/CCR2 axis in the induction and

maintenance of immune response in SOD1G93A mice, models of familiar ALS. As a result, we observed a sustained upregulation of CCL2 in motor neurons and peripheral

nerves of SOD1G93A mice during the disease course. CCR2 expression increased in

SOD1G93A glial cells during the symptomatic stage of the disease. However, we did not

find reduced levels of CCR2 in the monocytes of SOD1G93A mice. Accordingly, we did not find monocyte but pro-inflammatory T lymphocyte infiltration in the CNS while

immunomodulatory T-reg cells were reduced in spinal cord of SOD1G93A mice. Based on these results, the next step is to increase expression of CCR2 in T-reg cells to facilitate

their recruitment in the CNS of SOD1G93A mice and examine their effect on the disease progression.


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AXL sustains tumour-associated inflammation and is a prognostic indicator of poor

outcome in triple-negative breast cancers

G. Bottai1, C. Raschioni1, B. Szekely2, A. Losurdo3, L. Di Tommaso4, C. Tinterri5, J.

Kulka2, M. Roncalli4, L. Santarpia1 1Experimental Therapeutics Unit, IRCCS Clinical and Research Institute Humanitas,

Milan, Italy; 22nd Department of Pathology, Semmelweis University, Budapest, Hungary;

3Division of Oncology and Hematology, IRCCS Clinical and Research Institute Humanitas,

Milan, Italy; 4Department of Pathology, IRCCS Clinical and Research Institute Humanitas,

Milan, Italy; 5Breast Surgery Unit, IRCCS Clinical and Research Institute Humanitas, Milan,

Italy.

Background: Triple-negative breast cancers (TNBC) is a heterogeneous disease lacking effective therapeutic targets. This tumour subtype is characterized by an aggressive phenotype and usually associated with epithelial-to-mesenchymal transition (EMT) features and dense immune cell infiltration, which are both involved in tumour progression and response to chemotherapy. In this study we aimed to assess the potential correlation between tumour-associated macrophages (TAMs) and EMT-related kinases and their clinical relevance in TNBC. Methods: Real-time quantitative PCR and immunohistochemistry were used to assess the role of 30 EMT-related kinases and CD68, CD163 (as M2 macrophage polarized biomarker) in 203 TNBC. We validated our findings by in vitro experiments. Statistical analyses were performed using Spearman’s correlation, Fisher's exact tests, Kaplan-Meier and Cox regression analyses. Results: AXL was the most significant EMT kinase positively correlated with TAM, specifically to CD163-positive macrophages (rs = 0.503; p < 0.0001). TNBC patients who experienced tumour recurrence showed high expression of CD163 (p = 0.018) and AXL (p < 0.0001). AXL overexpression was particularly associated with metastasis to visceral organs (p = 0.036). Multivariate analysis confirmed that AXL was an independent prognostic markers in TNBC patients (RFS, p = 0.002; OS p = 0.001). AXL expressing mesenchymal-like TNBC cells polarized M2 human macrophages, which in turn induced EMT-driven tumourigenesis. Conclusions: AXL sustains TAM promoting tumour progression, and is a prognostic biomarker in TNBC patients. Selective AXL inhibition may represent an effective anti-cancer treatment for a subset of TNBC characterized by high expression of AXL associated with infiltration of M2 macrophages.


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GENERATION OF RENAL TUBULES BY USING MDCK CELL LINE AND

HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED URETERIC BUD

PROGENITOR-LIKE CELLS

Valerio Brizi IRCCS – Istituto di Ricerche Farmacologiche “Mario Negri”, Centro Anna Maria

Astori, Science and Technology Park Kilometro Rosso, Bergamo, Italy.

Creating renal organoids from human induced pluripotent stem cells (hiPSCs) would be important for understanding kidney development and disease, and establishing new therapeutic approaches. However, at present no cell-based strategies can effectively support the generation of human tissues with normal anatomies and functions. One major problem is the inability of existing methods to allow for the proper branching of the ureteric bud (UB), the precursor tissue of the adult renal collecting system. Here, to solve this problem we developed a three-dimensional (3D) culture system in which hiPSC-derived kidney cells are directed to form human tubular structures. First, we optimized culture conditions using MDCK cells - a distal tubule/collecting duct-derived cell line. Specifically, cells were embedded in a collagen I-based gel, seeded into 3D-printed PDMS moulds and cultured in the presence of HGF a potent inducer of UB elongation. After 48h, engineered tubes were transferred to a 3D extracellular matrix-mimicking gel and induced to branch by adding GDNF (the primary signal for UB survival and branching), and further cultured for 48-72h. Immunofluorescence analysis showed that the MDCK-derived structures were homogeneous E-cadherin-positive tubules, endowed with a single and continuous lumen and surrounded by the basement membrane marker Laminin. Also, upon GDNF induction tubules showed early signs of branching. To examine whether this technology can be applied to generate human tubular structures, hiPSCs were stimulated to adopt a UB fate by using a combination of BMPs, retinoic acid and activin A, and then cultured under the conditions described above. Immunofluorescence analysis showed that differentiated hiPSCs could form well-defined and elongated tubular-like structures presenting a linear lining of E-cadherin-positive cells along both sides of the structure. These studies could be the basis for engineering human kidney tissues in vitro to then use for developing novel investigative and therapeutic approaches.


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Base Excision Repair modulation of cisplatin activity in KRAS mutated NSCLC cells

Elisa Caiola2 ,Roberta Frapolli2, Monica Lupi2, Rossana Valerio1, Marina Chiara Garassino3,

Massimo Broggini1 and Mirko Marabese1

1Laboratory of Molecular Pharmacology, Department of Oncology, IRCCS - Istituto di Ricerche

Farmacologiche “Mario Negri”, Milan, Italy.

2Laboratory of Cancer Pharmacology Department of Oncology, IRCCS - Istituto di Ricerche

Farmacologiche “Mario Negri”, Milan, Italy.

3Department of Medical Oncology, National Cancer Institute, Milan, Italy

Introduction. KRAS is one of the best characterized genes in cancer but still remains practically undruggable. KRAS mutations in NSCLC are supposed to be associated to a poor prognosis and poor response to chemotherapy but this feature lacks a mechanistic evidence so far. In tumors, KRAS is mutated mostly at codons 12, but this process has as result a pool of mutations differing in the base replacement and the amino acid substitution. We hypothesized that different KRAS mutations in NSCLC patients may differently impact on drug sensitivity. We generated isogenic NSCLC clones expressing different KRAS mutations to determine the response to cisplatin, the election drug for the first line treatment of NSCLC in the clinic. The G12C clone was less sensitive in vitro and in vivo to cisplatin, but MAPK and PI3K pathways were similarly modulated by cisplatin in all the clones. Material and method. DNA damage response was determined by western blotting, immunofluorescence and Real-Time PCR. Platinum adducts on DNA were revealed by DRC-ICP- MS. Results and discussion. Cisplatin was able to enter the cells and reach its major target, the DNA, in all the KRAS mutant clones and, even if a slight difference was observed in cisplatin intracellular uptake and processing, this did not explain the different behaviour of the clones after cisplatin treatment. At early time points after cisplatin exposure, a similar amount of DNA adducts was observed in all the clones; however, in the cells carrying the G12C mutation a much faster decrease was observed 24 hours after treatment start. In addition, the G12C clone did not show H2AX activation and G2/M cell cycle phase arrest compared to all the others. Systematic analyses of DNA damage repair systems involved in cisplatin adducts removal revealed that Base Excision Repair (BER) could be responsible of KRAS G12C clone resistance to cisplatin: in fact, concomitant treatment of cells with different BER inhibitors at non toxic concentrations was able to almost completely restore the sensitivity of KRAS G12C mutant clone to cisplatin. Conclusion. The results seem to indicate that KRAS G12C mutation could stimulate BER, promoting platinum removal from DNA. These preclinical findings could represent the starting point to design more tailored therapies and new trials in the clinical setting. Supported by CARIPLO (2010-0794) and AIRC (IG 12915).


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High proliferative potential of endothelial progenitors isolated from Kaposi’s Sarcoma

patients

Francesca Calcaterra1,2, Antonello Ferrazzano1,2, Lucia Brambilla3, Domenico Mavilio1,2,

Silvia Della Bella1,2 1Department of Medical Biotechnologies and Translational Medicine (BioMeTra), University of

Milan, Milan, Italy; 2Laboratory of Clinical and Experimental Immunology, Humanitas Clinical and Research

Center, Rozzano (MI), Italy; 3Department of Dermatology, IRCCS Ospedale Maggiore, Milan, Italy

Kaposi’s sarcoma (KS) is a lymphoangioproliferative tumor of endothelial origin whose causative agent is human herpesvirus-8 (HHV8) which is present in the majority of KS tumor cells, the characteristic spindle cells.Endothelial progenitor cells (EPCs) are rare bone-marrow derived cells highly involved in vasculogenesis and angiogenesis. We previously demonstrated that EPCs obtained from KS patients, isolated and cultured as endothelial colony-forming cells (ECFCs), are HHV8-infected suggesting that EPCs may be involved in KS pathogenesis. In this study we investigated whether ECFCs isolated from KS patients have features similar to the spindle cells. ECFCs were obtained from 82 KS patients and 41 controls, using a culture protocol optimized in our lab. Cultures were analyzed for efficiency of ECFC colony isolation, cell growth, immunophenotype, cytokine production and in-vitro vasculogenesis. HHV8-infection was assessed as expression of latency-associated nuclear antigen (LANA) detected by immunofluorescence. During the isolation phase, ECFC colonies isolated from KS patients appeared earlier than those isolated from controls (p<0,05) and a significantly higher number of ECFC colonies was obtained from KS patients (p<0,001). During the expansion phase, ECFCs obtained from both KS and controls were endowed with the typical characteristics of ECFCs, but only ECFCs isolated from KS patients contained HHV8-infected cells. Moreover, ECFCs isolated from KS patients had higher proliferative and angiogenic ability and produced higher levels of IL-6 than control ECFCs. These results demonstrate that ECFCs obtained from KS patients have features that may be particularly relevant to KS pathogenesis, suggesting that ECFCs may contribute to KS lesions development.


18


d16HER2 splice variant regulates the activity of HER2-positive breast cancer- initiating

cells

Lorenzo Castagnoli1, Ada Koschorke1, Gaia Cristina Ghedini1, Lorenzo Galvani1, Valentina

Ciravolo1, Cristina Ghirelli1, Arianna Palladino2, Alessia Lamolinara3, Manuela Iezzi3,

PierLuigi Lollini2, Tiziana Triulzi1, Patrizia Nanni2, Elda Tagliabue1, Serenella M. Pupa1.

1 Molecular Targeting Unit, Fondazione IRCCS, Istituto Nazionale dei Tumori, Milan, Italy,

2Department of Experimental Pathology, University of Bologna, Bologna, Italy

3Aging Research

Centre, G. D’Annunzio University, Chieti; Italy.

The transmembrane tyrosine kinase receptor HER2, overexpressed in ~20% of human breast cancers (BCs), identifies an aggressive tumor subtype and is an important regulator of breast cancer-initiating cell activity (BCIC). We found that ~90% of HER2+ BC patients express a splice isoform of wild-type HER2 (WTHER2) gene characterized by the lack of exon 16 (d16HER2), a deletion that promotes the formation of stable and constitutively activated d16HER2 homodimers. Our comparison of the tumorigenic potential of the human d16HER2 and WTHER2 genes in the corresponding transgenic mouse models revealed a significantly shorter tumor latency period (p< 0.001) and a higher tumor incidence in the d16HER2 mice (p<0.001), suggesting a role for this variant in HER2-positive BCICs. In this context, our analyses of HER2-positive primary mammary tumor cell lines MI6 and MI7 derived from transgenic d16HER2 mice showed a significantly higher mammosphere-forming efficiency and higher levels of stem cell marker transcripts, compared to transgenic mouse WTHER2 tumor cells (WTHER2_1 and WTHER2_2). Mammospheres generated from human HER2-overexpressing BC cell lines BT474 , which also express the d16HER2 variant, exhibited an increase in the relative abundance of d16HER2 mRNA compared with that in the same parental cells cultured in adhesion conditions,. Experiments in mice injected into the mammary fat pad with the d16HER2- and WTHER2-positive cell lines at different serial dilutions indicate a higher “stemness potential” of MI6 and MI7 cells compared to WTHER2_1 and WTHER2_2 cells, strongly suggesting that the d16HER2 variant plays a relevant role in regulating BCICs of HER2-driven mammary tumors.

Supported by Minister of Health and AIRC


19

Abstract #14 - Immunology

Occurrence of Tertiary Lymphoid Tissue in Pancreatic Adenocarcinoma

Giovanni F Castino1, Francesca Bergomas1, Giuseppe Di Caro2, Fabio Grizzi2, Cristina

Ridolfi4, Francesca Gavazzi4, Alessandro Zerbi2,4, Luigi Laghi2,3, Alberto Mantovani1,5, Paola

Allavena1, Federica Marchesi 1,5.

1 Department of Immunology and Inflammation,

2 Laboratory of Molecular

Gastroenterology, 3Department of Gastroenterology,

4 Section of Pancreatic Surgery,

Department of Surgery, Humanitas Clinical and Research Center, Via Manzoni 56, 20089

Rozzano, Italy, 5 Department of Biotechnology and Translational Medicine, University of Milan,

Milan, Italy.

Objective. We are currently investigating the occurrence of Tertiary Lymphoid Tissue in human Pancreatic Ductal Adenocarcinoma (PDAC) and its relationship to other leukocyte populations. Moreover we are setting up a protocol of immunotherapy to stimulate the formation of such intratumor lymphoid tissue, and to boost the immune response against tumor cells, in a PDAC preclinical model. The immunotherapy approach might be a promising alternative to conventional treatments, that have been so far ineffective in contrasting PDAC progression. Methods. Occurrence of TLT has been evaluated by immunohistochemistry in PDAC tissue specimens from consecutive patients who underwent surgical resection at the Humanitas Clinical and Research Centre. A dendritic-cell based vaccine has been used to immunize mice injected with Panc02 murine cells. Results. In human PDAC tissue specimens, we identified organized lymphoid tissue, including compartmentalized T and B cell areas, dendritic cells and high endothelial venules (HEV). TLT occurred preferentially in the stromal compartment, rich in mesenchymal cells. The density of TLT correlated to the density of intra tumor CD8 T cells. However, despite CD8+ T lymphocytes in TLT displayed an activated phenotype (CD45RO+), they were devoid of Granzyme B granules, thus indicating a defective activation status. In a preclinical model of PDAC the immunotherapy regimen sporadically induced formation of aggregates of B and T cells, resembling the ones occurring in human PDAC. Further studies are ongoing to evaluate whether adjuvant immunostimulating agents improve the immune response in intratumor TLT in preclinical models.


20


Role of SPARC and mast cells in non-Hodgkin B cell lymohomas

Nadia Castioni, Sabina Sangaletti, Claudio Tripodo, Mario Paolo Colombo Molecular immunology unit, Department of Experimental Oncology and Molecular

Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori

Non‐Hodgkin B‐cell lymphomas (B-NHL) are a heterogeneous group of hematologic cancers arising from the neoplastic transformation of B‐lymphocytes. Although most of the processes implicated in cancer progression are inherent to the neoplastic clone, the crosstalk with a faulty microenvironment is also involved. Indeed, the defective extracellular matrix (ECM) deposition due to deficiency of the secreted protein acidic and rich in cysteine

(SPARC) induced, in the autoimmunity‐prone Faslpr/lpr mice, the development of lymphomas mimicking human chronic lymphocytic leukemias, that are actually characterized by low SPARC expression and low ECM deposition. Conversely, other subtypes of B-NHLs, such as the diffuse large B‐cell lymphoma (DLBCL), express high levels of SPARC and deposit more abundant ECM. Noticeably, in a set of B‐NHL patients characterized by high SPARC expression, programs related to mast cell (MC) function were also over-activated. Indeed, MCs, other than in allergic diseases, are involved in tumorigenesis and cancer progression. Impressively, MCs have been shown to promote the proliferation of the lymphomatous clone in the bone marrow (BM) of splenic marginal zone lymphoma patients. Whether MCs have a role in DLBCL development as well as their possible functional regulation by ECM molecules is unknown. By using a murine transplantable model of DLBCL, the A20 cell line, we observed degranulated MCs infiltrating lymphoma-bearing spleen and BM of mice; also, treatment of inoculated mice with cromolyn, a MC stabilizer, seemed to inhibit A20 cell infiltration of spleen and BM, highlighting a possible role of MCs in favoring lymphoma establishment in those organs.


21


SIRT3-dependent deacetylation of mitochondrial FoxO3A regulates colorectal cancer cell

fate

Valentina Celestini1, Tugsan Tezil2, Alessia Peserico1,2, Cristiano Simone1,2,3

1 Division of Medical Genetics, DIMO, University of Bari “Aldo Moro”, Policlinico, P.zza Giulio Cesare 11, 70124 Bari, Italy 2 National Cancer Institute, IRCCS Oncologico “Giovanni Paolo II”, Bari, 70124, Italy 3 Cancer Genetics Laboratory, IRCCS “S.De Bellis”, Castellana Grotte (BA), 70013, Italy

Cancer cells metabolize glucose in a different way compared to healthy cells: they show increased glycolytic activity even in the presence of high oxygen. This metabolic difference results in an enhanced oxidative stress, which may link to mitochondrial dysfunction. Previously, we have reported that glucose restriction triggers FoxO3A accumulation in non-cancerous cells in an AMPK-dependent manner. This event promotes the assembly of a protein complex containing FoxO3A, SIRT3 and mtRNAPol on mtDNA in cultured cells and in vivo. Our results show that SIRT3 activity is not required for FoxO3A mitochondrial localization, nor it is necessary for its interaction with mtRNAPol or SIRT3 itself; however, it is necessary for mtDNA binding and transcription of mitochondrial-encoded core or catalytic subunits of the OXPHOS machinery. Additionally, pharmacological inhibition or genetic ablation of SIRT3 increased acetylated FoxO3A in mitochondrial fractions and prevented its mtDNA binding. We further evaluated the functional role of these proteins in colorectal cancer cells. Our results indicate that SIRT3-dependent deacetylation of FoxO3A in mitochondria is required for survival of cancer cell in low nutrient condition.


22


Protein and RNA-immunoprecipitation for the identification of new biomarkers in

autoimmune diseases

Angela Ceribelli1,2, Natasa Isailovic 1, Maria De Santis 1, Elena Generali 1, Marco

Massarotti 1, Minoru Satoh 3, Carlo Selmi 1,2

1 Rheumatology Unit, Humanitas Research Hospital, Rozzano, Milan, Italy;

2BIOMETRA

department, University of Milan, Italy; 3 University of Occupational and Environmental

Health, Kitakyushu, Fukuoka, Japan.

Background and aims. Serum autoantibodies are the cornerstone of diagnosis, follow-up and management of rheumatic diseases. Their lack is responsible for delayed diagnosis, as for psoriatic arthritis (PsA) in subjects with skin psoriasis, and Systemic Sclerosis (SSc) in early phases of disease onset. We aim to apply protein- and RNA-immunoprecipitation (IP) for the identification of new serologic markers in these rheumatic diseases. Materials and Methods. Sera from 29 adult PsA and 19 SSc patients were collected in the Rheumatology Unit- Humanitas Research Hospital, and 42 sera were tested as controls. Sera were analyzed by indirect immunofluorescence (IIF) for anti-nuclear antibodies (ANA), by protein- immunoprecipitation

(IP) using K562 cell extract radiolabeled with 35S-methionin, and by RNA-immunoprecipitation using K562 RNA extracted with phenol/chloroform/isoamyl alcohol analyzed by silver staining. Results. In SSc cases, 13/19 (68%) express anti-centromere and 4/19 (21%) anti-topo I antibodies. One SSc case tested positive for anti-RPA (replication protein A) antibodies, while RNA- IP did not show autoantibody specificities. In PsA patients, IP identified specific bands corresponding to anti-Ago2/Su in 2 cases, anti-Ki/Sl in 2 cases and one weak anti- PM/Scl. In 6/29 (20%) PsA cases, bands of variable molecular weight (48-180kD) were identified by IP, but these do not correspond to known autoantibodies and no common band was detected. RNA-IP did not show any specific autoantibody in PsA patients. Conclusions. We identified serum autoantibodies in PsA and support the autoimmune pathogenesis of this condition. We also detected anti-RPA antibodies in one SSc patient, a specificity previously reported in other connective tissue diseases.


23


Identification and characterization of potential novel targets in thyroid carcinoma:

evidence of “non-oncogene addiction” unveiling tumor cell vulnerabilities

Cetti Elena1, Anania Maria Chiara1, Gasparri Fabio2, Fraietta Ivan2, Miranda Claudia1,

Mazzoni Mara1, Colombo Riccardo2, Galvani Arturo2, Pierotti Marco A.3 and Greco Angela1 1Molecular Mechanisms Unit, 3Scientific Directorate, Fondazione IRCCS Istituto Nazionale dei

Tumori, Milan, Italy; 2 Nerviano Medical Sciences Srl, Oncology, Nerviano (MI), Italy.

The “non-oncogene addiction” paradigm states that the tumorigenic state relies on the activity of genes and pathways which are essential to support the phenotype of cancer cells but not required to the same degree for normal cell viability. We faced this concept in order to identify nodal points for potential therapeutic intervention in thyroid carcinoma, the most frequent endocrine tumor with an incidence rapidly increasing. A fraction of thyroid tumors develops resistance to therapy and progresses into the most aggressive forms with poor prognosis. Target therapies are often unsuccessful, therefore there is still the need of better understanding the mechanisms of thyroid carcinogenesis and of identifying novel therapeutic opportunities. To identify “non-oncogenes” essential for the tumorigenic phenotype, we screened a commercial siRNA library, targeting the human druggable genome, in the thyroid cancer cell line BCPAP

(carrying BRAFV600E oncogene) in comparison with immortalized normal human thyrocytes Nthy-ori3-1. Data obtained in the two cell models revealed a panel of 386 putative BCPAP-specific survival genes, and 15 out of 28 genes were technically confirmed. We performed validation studies of some hits with independent siRNAs and confirmed their inhibitory effect on BCPAP cell growth. Moreover, their silencing impaired the growth of various thyroid tumor cell lines. Our work identified putative “non-oncogenes” essential for sustaining the oncogenic phenotype of thyroid tumor but not normal cells. We found that these genes play a role in the biology of thyroid tumors and may represent promising targets for new therapeutic strategies.


24


From whole-exome sequencing to functional characterization of a novel candidate gene

for inherited nonsyndromic hearing loss.

C. Chiereghin2,3, M. Robusto2,3, V. Massa1, S. Duga2,3, G. Soldà2,3, R. Asselta2,3

1Department of Health Sciences, Università degli Studi di Milano, Milan,

Italy 2Biomedical Sciences Department, Humanitas University, Rozzano (Milan), Italy 3Humanitas Clinical and Research Center, Rozzano (Milan), Italy Inherited nonsyndromic hearing loss (NSHL) is characterized by a very high level of genetic heterogeneity, making extremely challenging to obtain a molecular diagnosis with traditional screening methods. In recent years, whole exome sequencing (WES) has been introduced as an alternative approach to search for alleles underlying Mendelian disorders and has been successfully applied to disease gene discovery. Three siblings (two affected and one normal-hearing) from an Italian family segregating with recessive NSHL were previously subjected to WES. Data analysis and variant prioritization, followed by segregation analyses, pointed out a promising NSHL-causing candidate gene (here referred to as DFNX ). In this frame, the present project is aimed at functionally validating the mutation identified in DFNX, as well as at characterizing the role of this candidate gene in auditory function. We confirmed, by both RT-PCR and immunohistochemical studies on wild-type mice at different developmental stages, that DFNX ortholog protein is expressed in the mouse inner ear from 15.5 days post coitum. In particular, co-localization studies with the cell-specific markers MyoVIIA and P75 allowed the definition of the specific expression of the protein in cochlear sensory and supporting cells, suggesting a possible role of DFNX in hearing function. In parallel, immunofluorescence assays are being set up in human cell lines to evaluate the effect of the mutation on DFNX localization and function.


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Abstract #20 - Molecular Bases of Diseases

Molecular characterization of new Wilms tumor genes

Sara Ciceri1, Beatrice Gamba1, Patrizia Mondini1, Paola Corbetta1, Paola Collini2, Filippo

Spreafico3, Paolo Radice1, Daniela Perotti1. 1Molecular Bases of Genetic Risk and Genetic Testing Unit, Department of Preventive and

Predictive Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

2Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei

Tumori, Milan, Italy. 3Pediatric Oncology Unit, Department of Hematology and Pediatric

Hemato-Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

Wilms tumor (WT), the most frequent malignant childhood renal tumor, shows a high degree of genetic heterogeneity. In a consistent fraction of cases the underlying genetic defect is unknown. To disclose further genes possibly involved in WT development, a genome-wide single nucleotide polymorphism (SNP) array analysis was performed on 96 WT samples. After excluding chromosomal regions polymorphic for copy number (CN) variations, this analysis highlighted eight different regions affected with CN/allelic ratio anomalies, in which genes with a putative role in tumorigenesis and/or organogenesis are mapped. These genes are: MYCN (2p24.3) DIS3L2 and miR-562 (2q37.1), HACE1 (6q21), GLI3 (7p14.1), CDKN2A and CDKN2B (9p21.3), PALB2 (16p12.1), BTG3 (21q21.1), and CHEK2 (22q12.1). Through genomic sequencing analyses, we identified 18 previously reported and 4 novel missense variations (3 in DIS3L2, 2 in HACE1, 5 in GLI3, 1 in CDKN2B, 6 in PALB2, 5 in CHEK2). These variations have been investigated for their impact on protein product through bioinformatic tools, and, for previously reported variations, the allele frequency difference between cases and controls was studied. One CHEK2 mutation was demonstrated to cause exon skipping, resulting in an altered ratio among two physiological isoforms, and also in the production of new out of frame mRNAs. Promoter methylation analysis of HACE1, CDKN2A and CDKN2B disclosed partial methylation affecting HACE1 in 79.2% of cases. Gene expression array analysis revealed high levels for MYCN, and low levels for CDKN2A, CDKN2B and HACE1. Our data suggest that, with the exception of miR-562 and BTG3, investigated genes may have a role in WT development and/or predisposition. ACKNOWLEDGEMENTS: This work was supported by grants from the Associazione Bianca Garavaglia, Busto Arsizio, Varese, Italy


26

Abstract #21 - Molecular Bases of Diseases

TGFb Triggers miR-143/145 Trnasfer from Smooth Muscle Cells to Endothelial Cells,

Thereby modulating vessel Stabilization

Montserrat Climent1, Manuela Quintavalle2, Michele Miragoli2-5, Ju Chen6, Gianluigi

Condorelli2-3-4, and Leonardo Elia2-3 1Multimedica Research Hospital, Via Fantoli 16/15, 20138, Milan, Italy; 2Humanitas

Clinical and Research Center, Via Manzoni 113, 20089 Rozzano (MI), Italy; 3Milan Unit,

Institute of Genetic and Biomedical Research, Via Manzoni 113, 20089 Rozzano (MI), Italy;

4University of Milan, Via Manzoni 113, 20089 Rozzano (MI), Italy; 5Center of Excellence

for Toxicological Research (CERT), Department of Clinical and Experimental Medicine,

University of Parma, Parma, Italy; 6University of California, San Diego, 9500 Gilman Drive La Jolla, California 92093-0613C, USA.

Aim: The endothelium plays a critical role modulating vessel biology and its alteration may induce vessel pathologies such as atherosclerosis. Neo-arteriogenesis involves the strict association between endothelial cells (ECs) and mural cells (pericytes and smooth muscle cells (SMC)) leading to vessel maturation and stabilization. Although miR-143/145 cluster regulates SMCs phenotypic switch and vascular homeostasis, its role in the neighboring ECs is not well known. Our aim was to study whether SMCs control EC functions through the transfer of miR-143/145. Methods: We assessed the transfer of miR-143/145 using SMC/EC co-cultures and intact vessels. Using high-resolution microscopy and live imaging we were able to identify the way of miR transfer between the two cell types. We also studied the molecular mechanism by which miR-143/145 transfer is regulated and defined its biological effects in ECs. Results: We proved that miR-143/145 cluster is transferred from SMCs to ECs and that TGFβ is involved and plays a crucial role in this communication. Moreover, miR-143/145 and its targets modulate EC angiogenesis. We confirmed our findings in vivo using a new genetic modified animal model in which the miR-143/145 cluster is specifically deleted in the SMC layer. Conclusion: We demonstrate that miR-143/145 acts as communication molecule between SMCs and ECs affecting EC maturation and vessel stabilization, and that this process is modulated by TGFβ. This finding could have relevant implications for pathological and therapeutic angiogenesis.


27


Dual prognostic significance of Tumor Associated Macrophages in human pancreatic

adenocarcinoma treated or not with chemotherapy

Nina Cortese1,3, Giuseppe Di Caro1,3, Giovanni Francesco Castino1, Fabio Grizzi1, Francesca

Gavazzi2, Cristina Ridolfi2, Giovanni Capretti2, Alessandro Zerbi2, Paola Allavena1, Alberto

Mantovani1, Federica Marchesi1

1 Department of Immunology and Inflammation, Humanitas Clinical and Research Center,

Rozzano, Italy. 2 Section of pancreatic Surgery, Department of Surgery, Humanitas Clinical

and Research Center, Rozzano, Italy 3These authors contributed equally to the work.

Tumor-associated macrophages (TAMs) play key roles in tumor progression. Recent evidence suggests that TAMs critically modulate the efficacy of anticancer therapies, raising the prospect of their targeting in human cancer. In a large retrospective cohort study involving 110 pancreatic ductal adenocarcinoma (PDAC) patients, we assessed the density of CD68-TAM immune reactive area (IRA%) at the tumor-stroma interface and addressed their prognostic relevance in relation to post-surgical adjuvant chemotherapy treatment (CTX). In vitro, we dissected the synergism of chemotherapy and TAMs. In human PDAC, TAMs predominantly exhibited an immunosuppressive M2-like profile, characterized by expression of scavenger receptors (CD206, CD163) and production of IL-10. Surprisingly, while the density of TAMs associated to worse prognosis and distant metastasis, CTX restrained their protumor prognostic significance. High density of TAMs at the tumor margin positively dictated prognostic responsiveness to CTX independently of T cell density. Accordingly, in vitro, Gemcitabine-treated macrophages became tumoricidal, activating a cytotoxic gene expression program and switching to an antitumor phenotype. In human PDAC patients, neoadjuvant CTX was associated to a decreased density of M2-like CD206-TAMs at the tumor margin. Overall, our data highlight TAMs as critical determinants of prognostic responsiveness to CTX and provide clinical and in vitro evidence that CTX directly reeducates TAMs to an anti-tumor phenotype. These results suggest that the quantification of TAMs could be exploited to select patients more likely to respond to CTX and provide basis for novel strategies aimed at reeducating macrophages in the context of chemotherapy treatment.


28


Expression quantitative trait analysis reveals fine germline transcript regulation in mouse

lung tumors

Alice Dassano 1,§, Chiara E. Cotroneo 1,§, Francesca Colombo 1, Angela Pettinicchio 1, Matteo

Dugo 2, Loris De Cecco 2, Tommaso A. Dragani 1, Giacomo Manenti 1 1 Department of Predictive and Preventive Medicine, 2 Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS, Istituto Nazionale dei Tumori, Milan, Italy. § These authors contributed equally to the work. Alterations in gene expression are involved in the development and progression of tumors. Genetic variations affecting gene expression could be detected by expression quantitative trait loci (eQTL) analysis, that combines genotype and gene expression data. Herein, we carried out a whole-genome genetic linkage analysis on the transcriptome of induced-lung tumors in an (A/J x C57BL/6)F4 mice population, whose individual lung tumor multiplicity (Nlung, LOD = 48.2) is linked to the genotype at the Pulmonary adenoma susceptibility 1 (Pas1) locus. We found that expression levels of 1,179 and 1,579 genes are modulated by cis- and trans-eQTLs, respectively (LOD score > 5). In particular, we observed that Pas1 plays a major role in the regulation of gene expression in already developed lung tumors; indeed, its genomic area was linked to 857 and 14 genes located in trans and in cis, respectively. Moreover, we detected a linkage between Pas1 and the mean lung tumor volume (Vlung, LOD = 4.82), meaning that this locus is involved in regulating tumor progression as well as susceptibility. We then identified 1,124 and 180 transcripts associated with Nlung and Vlung, respectively. The overlaps among these two sets of genes and the ones controlled by the Pas1 surrounding area indicated that 67 transcripts were common to all three sets, and that the majority of the Vlung-associated transcripts were also associated with Nlung. Overall, this work gives an overview of the germline transcriptional regulation existing in mouse lung cancer, and points to a novel role of the Pas1 locus in already developed tumors.


29


Role of inflammation in the developmental switch of GABA signaling.

Genni Desiato1,3, Filippo Mirabella1, Michela Matteoli1,2 and Davide Pozzi1 1 Laboratory of Pharmacology and Brain Pathology, Humanitas Clinical and Research Center,

Milan; 2 CNR, Institute of Neuroscience, Milan; 3 Department of Translational Surgery and

Medicine, University of Milano-Bicocca, Monza

GABAergic transmission plays a very important role in brain functioning at different developmental stages. In mature brain, GABA-mediated inhibitory transmission is involved in regulating excitatory activity, thus allowing a fine control of neuronal circuits. Conversely, during early postnatal development, GABAergic synaptic transmission is endowed with excitatory activity, playing a trophic role in neuronal growth and development. The transition of GABA signalling from excitatory to inhibitory represents a critical process for neuronal development, and its alteration has been found to be involved in the pathogenesis of several neurodevelopmental disorders, such as autism and epilepsy. Moreover, recent studies reported the occurrence of elevated levels of proinflammatory cytokines in patients affected by these diseases, suggesting that an inflammatory state can contribute to the pathogenesis of such disorders. Hence, our project aims at investigating whether an inflammatory-like state, characterized by high levels of specific proinflammatory cytokines, might alter the GABA switch. To address this issue, we are taking advantage of primary neuronal cultures and of a multidisciplinary approach based on functional and morphological techniques. Our preliminary data confirmed that GABA signalling itself is involved in the GABA switch. We are currently investigating whether pro- or anti-inflammatory cytokines have different roles in the GABA switch. Results from these studies will allow us to clarify the role of immune molecules in this fundamental process and, possibly, in the pathogenesis of neurodevelopmental diseases.


30


Opposite effect of miR-9 and miR-200c on PDGFRbeta-mediated vasculogenic mimicry in

triple negative breast cancer

Elvira D’Ippolito1*, Ilaria Plantamura 1*, Bongiovanni Lucia2, Patrizia Casalini3, Sara Baroni1 ,

Claudia Piovan1, Rosaria Orlandi3, Filippo De Braud4, Elda Tagliabue3, Tripodo Claudio2 and

Marilena V. Iorio1 1Start Up Unit, Department of Experimental Oncology, Fondazione IRCCS Istituto Nazionale

Tumori; 2Tumor Immunology Unit, Department of Health Sciences, Human Pathology Section,

University of Palermo; 3Molecular Targeting Unit, Department of Experimental Oncology,

Fondazione IRCCS Istituto Nazionale Tumori; 4 Medical Oncology Department , Fondazione

IRCCS Istituto Nazionale Tumori.

*these authors equally contributed to this work Vasculogenic mimicry is the ability of tumor cells to contribute to the tumor vasculature generating perfusable vascular-like channels, and in triple negative breast cancer (TNBC) PDGFR-β is crucial in mediating this phenomenon. Epithelial to mesenchymal transition (EMT) can induce vasculogenic mimicry and activate autocrine PDGF networks essential for the maintenance of the post-EMT phenotype. We aimed at investigating microRNAs able to regulate the vasculogenic mimicry mediated by PDGFR-β in TNBC, particularly focusing on miR-9 and miR-200c because of their known involvement in EMT. In MDA-MB-231 and MDA-MB-157 TNBC cell lines, transfection of miR-9 or miR-200 family (miR-200b and miR-200c) promoted or inhibited vasculogenic mimicry, respectively. We found that miR-9 was induced upon stimulation of PDGFR-β-CREB axis, and its action on vasculogenic mimicry relied in part on the negative regulation of STARD13, direct target validated by luciferase reporter assay. MiR-200 family, instead, can indirectly suppress PDGFR-β at protein level through ZEB1 targeting. We then evaluated in a cohort of TNBC the expression of miR-9 and miR-200c, by qRT- PCR, and PDGFR-β, by immunohistochemistry; interestingly, the expression of miR-200c negatively associated with both the presence of vascular lacunae and tumor nests positive for PDGFR-β. Finally, in situ hybridization analysis on human TNBC revealed that miR-200c was mainly expressed by tumor cells. Our results suggested that miR-9 and miR-200c can mediate or suppress the vasculogenic mimicry regulated by PDGFR-β, playing an opposite role. EMT, through the activation of the PDGFR- β-miR-9 axis and the suppression of miR-200c, represents the trigger event of this phenomenon.


31

Abstract #26 - Cancer Biology

Dissecting melanoma heterogeneity by integrative genomic analysis for tailored anti-cancer

therapy

Matteo Dugo1, Marialuisa Sensi1, Silvana Canevari1 1Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto

Nazionale dei Tumori, Milan, Italy

Selective BRAF inhibition (BRAFi) has become approved standard treatment in BRAFV600

mutated advanced-stage melanoma. Most patients treated with BRAFi develop, however, resistance and some of them are intrinsically unresponsive to treatment. We recently reported that melanoma cell lines belonging to the Cancer Cell Line Encyclopedia (CCLE) pharmacogenomic study could be classified into three different subtypes by integrating basal receptor tyrosine kinases (RTKs) gene expression profiles with a previously reported signature related to the two known transcriptional states representing the invasive and proliferative melanoma phenotypes. In particular, the subtype displaying the highest and lowest expression of EGFR and ERBB3

(EGFRHIGH/ERBB3LOW–invasive subtype) respectively, included BRAF-mutant tumors, all intrinsically resistant to BRAFi, as assessed by analysis of the CCLE pharmacological data and by in vitro growth inhibition assays. To identify molecular aberrations causative of resistance we reproduced our classification in metastatic melanoma samples (n = 367) from The Cancer Genome Atlas (TCGA) and we identified features unique of each subtype at the copy number, expression and proteomic level. Results from copy number data pointed out that resistant tumors exhibit on average a more stable genome compared to the other two subtypes. In addition gene expression analysis highlighted the activation and inhibition of several biological processes, reflected also at the proteomic level. These results will have significant implications for the understanding of the biology underlying tumors with intrinsic resistance to BRAFi and for the development of alternative treatments in predefined subsets of unresponsive patients.


32

Abstract #27 - Cancer Biology

miR-875-5p enhances prostate cancer cell sensitivity to ionizing radiation

Rihan El Bezawy, Denis Cominetti, Valentina Profumo, Paolo Gandellini, Nadia Zaffaroni Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale per lo Studio e la Cura

dei Tumori, Milan, Via Venezian 1/Via Amadeo 42, 20133 Milan, Italy

Radiotherapy is a standard treatment for organ-confined prostate cancer (PCa). Although technical optimization has greatly improved local tumor control, a significant percentage of patients still undergo recurrence. The onset of radioresistance remains poorly understood. Mounting evidence supports a role for microRNAs (miRNAs) in determining cell responsiveness to ionizing radiation. However, the few studies carried out so far in PCa cells produced controversial results. The present study is aimed at identifying specific miRNAs that regulate key cellular pathways mediating radioresponsiveness, with special reference to epithelial-mesenchymal transition (EMT), a phenotypic switch known to be involved in radioresistance. We focused on miR-875-5p, a miRNA we found to be downregulated in PCa samples compared to matched normal tissues and never reported before in the literature. The relevance of miR-875-5p in PCa radiation response was based on the observation that miRNA reconstitution in DU145 and PC3 cell lines induced changes in cell morphology and cytoskeleton architecture, reduced migration ability and modulated mRNA and protein levels of EMT markers. Most importantly, miR-875- 5p was able to increase cell radiosensitivity as demonstrated by clonogenic survival reduction and comet tail moment increase. In addition, among genes predicted as putative targets of miR-875-5p and known to be involved in pathways relevant to radiation response, EGFR was identified and validated as a direct target by luciferase assay. Given that EGFR is known to counteract radiation-induced EMT and that miR-875-5p was demonstrated to impair this process, our data suggest the miR-875-5p-EGFR axis as a possible relevant determinant in PCa radiation response.


33


Biomarkers as tools to evaluate the toxicity of water bodies in coal mining area: the case of

Das Pedras stream (Brazil)

Luciana Fernandes de Oliveira, Millena Cabral, Claudia Martinez Department of Animal and Plant Biology, Londrina State University, Rodovia Celso Garcia Cid

Km 380, 86.057-970 Londrina, Brazil

Coal mining is one of the highest potentially impactful industry activities and represents an example of human input source of metals in the environment. Therefore, the aim of this study was to evaluate the sub-lethal effects of Das Pedras stream (Brazil), located near coal mining area, on freshwater bivalve Anodontites trapesialis, using a biomarker approach. Bivalves were maintained into cages for 96 h at four points along a stream, two located upstream and two downstream from the mining area. At each experimental location, physical and chemical variables were measured as the collection of water and sediment to determinate several metal concentrations. To evaluate the toxicity effects of Das Pedras water, we applied the following biomarkers on abovementioned bivalve: total antioxidant capacity, lipid peroxidation, protein carbonylation, glutathione S-transferase activity and metallothionein content. The results showed the influence of coal mining area on Das Pedras water by alterations in its physical/chemical variables (high metal concentrations, conductivity, total dissolved solids) at the two points located downstream. However, biomarker alterations occurred mainly at downstream point farther from the mining area and didn’t follow the same pattern; A. trapesialis presented higher levels of lipid peroxidation and protein carbonylation indicating oxidative stress and elevated metallothionein content. In this context, it is important to take into account that other contaminant sources could be responsible for observed alterations. This research encourages further studies regarding the potential adverse effects in aquatic organisms by polluted rivers on coal mining areas.


34


The role of the sarcomeric protein myopalladin in skeletal muscle growth

Filomena MCa,b, Yamamoto DLc,e, Mastrototaro Gb,f, Caremani Mg, Lieber Rh, Giacca Mi,l,

Nigro Vm,n, Linari Mg, Chen Jh, Bang MLb,d

aDepartment of Medical Biotechnologies and Translational Medicine (BIOMETRA),

University of Milan, bHumanitas Research Hospital, Rozzano, Milan, Italy .cInstitute of Biomedical

Technologies (ITB) and dInstitute of Genetic and Biomedical Research (IRGB), Milan unit, National

Research Council, Milan, Italy.eMultimedica Hospital, Scientific and Technology Pole, Milan,

Italy.fDoctoral School in Molecular and Translational Medicine (DIMET), University of Milano-

Bicocca, Milan, Italy.gDepartment of Biological Evolution, University of Florence, Italy. hDepartment

of Medicine, University of California San Diego, USA. iUniversity of Trieste and lICGEB, Trieste,

Italy.mDepartment of General Pathology, Second University of Naples, and nTelethon Institute of

Genetics and Medicine (TIGEM), Naples, Italy. Corresponding author: Marie-Louise Bang, Humanitas Research Hospital, Via Manzoni 113, 20089 Rozzano, Milan, Italy, Tel: (+39) 0282245210; Fax: (+39) 0282245290; E-mail: [email protected] Myopalladin (MYPN) is a sarcomeric protein belonging to the palladin/myopalladin/myotilin family of actin-associated immunoglobulin-containing

proteins1. Within the sarcomeric Z-line, myopalladin binds to a-actinin, nebulin, and PDZ-

LIM proteins1,2, while in the I-band it binds to the stress-inducible transcriptional cofactor CARP, which, in turn, binds to the I-band region of titin, suggesting a potential role of

MYPN in mechanosensing3. We further demonstrated that MYPN can bind to and bundle filamentous actin and thereby promote actin polymerization. The important role of MYPN in striated muscle is illustrated by the identification of MYPN mutations in human

cardiomyopathy4,5,6 and limb-girdle muscular dystrophy patients. MYPN knockout (MKO) mice show no signs of myopathy but are significantly smaller compared to wildtype (WT) littermates and have an about 30% reduction in skeletal muscle fiber cross-sectional area at all ages. Consistently, reduced differentiation rate and myotube width was observed in primary skeletal muscle cultures derived from MKO mice. Studies of muscle performance in 2-month-old MKO mice showed reduced isometric force and power during isotonic shortening at any loads as a result of the reduced cross sectional area. By up- and downhill treadmill running, MKO and WT mice performed similarly. However, while the performance of WT mice was unaffected following 4 consecutive days of downhill running (eccentric contractions), the performance of MKO mice decreased progressively and signs of muscle regeneration following muscle damage were observed. Ongoing investigations are focused on understanding the molecular basis for the reduced muscle fiber size and consequent reduced skeletal muscle performance. 1. Bang et al., J Cell Biol 153, 413-27, 2001. 2. von Nandelstadh et al., Mol Cell Biol 29, 822-34, 2009. 3. Miller et al., J Mol Biol 333, 951-64, 2003 4. Duboscq-Bidot et al., Cardiovasc Res 77, 118-25, 2008. 5. Purevjav et al., Hum Mol Genet 21, 2039-53, 2012. 6. Meyer et al., Eur J Hum Genet. 3, 294-300, 2013.


35


Biological and clinical significance of circulating tumor cells in breast cancer

Emanuela Fina1, Loredana Cleris1, Carolina Reduzzi1, Rosita Motta1, Gloria Morandi1,

Francesca D’Aiuto1, Maurizio Callari1, Serena Di Cosimo2, Giulia Bianchi2, Antonia

Martinetti2, Janine Wechsler3, Vera Cappelletti1 and Maria Grazia Daidone1

Biomarkers Unit, Department of Experimental Oncology and Molecular Medicine1, Medical

Oncology Unit2, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy

ScreenCell, Paris, France3

Circulating tumor cells (CTC) represent a unique biological source as they offer the opportunity to monitor disease evolution and assess tumor heterogeneity using minimally-invasive approaches. In this study the presence of CTCs and metastases was investigated in several breast cancer (BC) xenograft models and their transcriptome compared to those of solid lesions. In parallel, CTC fluctuations and molecular features have been monitored during therapy in patients with early (T2N1M0) or metastatic BC. CTCs can be found in MDA-MB-231, MDA-MB-453, BT-474 and MDA-MB-468 xenograft models. MDA-MB-231-derived CTCs were proved to have peculiar molecular features as a signature of 65 genes exclusively up-regulated in CTCs compared to primary tumor and metastases (log2FC≥2, Padj.<0.01) was identified. Among such genes, TFF3 (trefoil factor 3 secreted peptide) transient silencing was proved to affect MDA-MB-231 cell migratory properties and anoikis-resistance (P=0.0001 and P=0.0010, respectively), but not cell proliferation, compared to control cells. Interestingly, distinct CTC subpopulations can be observed according to the clinical setting as a higher frequency of EPCAM positive CTCs and a lower frequency of CTC clusters was observed in M+ compared to M0 cases (45 vs 0%, P=0.0075, and 27 vs 78%, P=0.0002, respectively). These results suggest that integrating basic and clinical research could help to gain a deeper knowledge of molecular mechanisms that regulate the metastatic process and identify novel prognostic and predictive biomarkers. In this perspective, the biological role of TFF3 gene will be further investigated in murine models and its clinical relevance validated in BC case series.


36


Kinetics of antibiotics in critically ill patients

Stefano Finazzi, Elena Garbero, and Guido Bertolini Laboratory of Clinical Epidemiology, IRFMN–IRCCS

Infections are one of the most common problems in the care of critically ill patients. They often yield high-mortality conditions as severe sepsis or septic shocks. In addition, the effectiveness of antibiotic therapies is undermined by several factors, associated either with critical patient conditions or with the interaction with other treatments, as the simultaneous administration of several drugs or renal replacement therapies (RRT). We are currently running a project to create a decision support system to perform real-time

pharmacokinetic simulations, with the aim of assisting clinicians in the design of effective personalized antibiotic therapies. This tool will be integrated in Margherita 3 (M3), an electronic clinical record developed for both clinical and research purposes by the Laboratory of Clinical Epidemiology, IRFMN. As a first study, we have developed a pharmacokinetic model to describe the temporal

evolution of plasma concentration of a common antibiotic molecule, vancomycin, in critically ill patients. The dependence of drug clearance and distribution volume as a function of patient clinical conditions has been determined using a multilevel model, built on data collected through M3 on a very heterogeneous sample. Variables have been chosen on the basis of clinical relevance and introduced according to statistical significance. We have found that variability of drug distribution volume is explained by albumin concentration in serum. Variability of drug clearance is explained by creatinin clearance, use of RRT, and sex. Interestingly, total clearance is comparable in patients undergoing RRT and in patient with normal renal function.


37

Abstract #32 – New Frontiers in Biotechnology IN-VITRO WALL SHEAR STRESS WAVEFORMS DERIVED FROM

ARTERIOVENOUS FISTULA ELICIT ENDOTHELIAL CELL ACTIVATION AND

NEOINTIMAL HYPERPLASIA RELATED MOLECULE PRODUCTION

M. Franzoni1, I. Cattaneo1, B. Ene-Iordache1 and A.Remuzzi2 1IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, Bergamo, Italy 2Department of Industrial Engineering, University of Bergamo, Dalmine (Bergamo), Italy.

Native arteriovenous fistula (AVF) is recommended as first choice to create a vascular access

(VA) in dialysis patients1. AVF outcome however is affected by ] 40% failure rate within 2

years2. First cause of AVF failure is vessel stenosis caused by intimal hyperplasia (IH) not

adequately compensated by outward remodeling3. IH development has a complex pathway, which involves endothelium, smooth muscle cells and fibroblasts. Disease location is strongly

related to local hemodynamics4. Both in-vivo and in-vitro studies show that reciprocating and disturbed wall shear stress (WSS) stimuli induce an activated phenotype and a pro- inflammatory

signalling in endothelial cells (EC)5-6. High risk stenosis areas of AVF, as the venous side near

the anastomosis, are exposed to reciprocating and disturbed WSS waveforms7-8. The aim of the present study was to investigate the in-vitro effect of AVF- derived WSS wave forms on morphological adaptations, gene and protein expression of EC, related to development of IH.

RT-PCR and ELISA. According to manufacturer instructions, we performed RT-PCR to quantify Krüppel-like factor 2 (KLF2) gene expression and ELISA to quantify interleukin 8 (Il-8) and monocyte chemo-attractant protein 1 (MCP1) release.

Statistical analysis. We performed statistical analysis using one-way ANOVA and the differences among groups were established using the post-hoc Bonferroni multiple comparisons test. Data are presented as mean value ± SD. Statistical significance level was defined for P<0.05.


38


Identifying key regulators of the immune/inflammatory response for developing novel

anticonvulsive and antiepileptogenic approaches

Federica Frigerio1, Anna Torello1, Michel Neveux2, Catherine Vandenplas2, Patrick Foerch2,

Rafal Kaminski2 and Annamaria Vezzani1 1IRCSS-“Mario Negri” Institute for Pharmacological Research, Milano, Italy; 2UCB Biopharma SPRL, Braine l’Alleud, Belgium Rationale. Long-lasting neuroinflammation is induced after epileptogenic injuries in animal models of epilepsy. This process contributes to experimental seizures generation likely because of lack of activation of efficient resolution mechanisms in the brain. Resolution of inflammation is a highly coordinated process chiefly controlled by endogenous pro-resolving lipids or peptides. Our hypothesis is that brain inflammation after epileptogenic injuries is inefficiently controlled by pro- resolving mediators, i.e Resolvin D1 (RvD1) and LipoxinA4 (LXA4) and their cognate receptors ChemR23 and LXA4 (ALXR), thus potentially contributing to epileptogenesis. We studied resolution during epileptogenesis to explore the therapeutic potential of boosting this endogenous response with pharmacological interventions to arrest epilepsy development. Methods. Adult male mice were exposed to Status Epilepticus (SE) induced by intra-amygdala kainate, then killed 2, 24, 72 h and 1 week (i.e., time of epilepsy onset) later. The pro-inflammatory cytokines IL-1β and TNF-α, pro-resolving receptors ChemR23 and ALXR, endogenous mediators LXA4 and RvD1 and their biosynthetic enzymes Lipoxygenase (LOX)-5 and -15 were analyzed by RT-PCR, western blot (WB) and immunohistochemistry (IHC) in the hippocampus. IL-1β and TNF-α expression was analyzed by IHC, 48 h post-SE in mice treated with BML111, a LXA4 stable analogue. Results. Pro-resolving receptors were induced during epileptogenesis and mRNA levels of LOX-5 and -15 were up-regulated in the hippocampus. Treatment with BML-111 strongly reduced IL-1β and TNF-α expression 48 h post-SE. Conclusions. Neuroinflammation following SE precedes the up-regulation of pro-resolving mechanisms, thus suggesting delayed activation of resolution during epileptogenesis. Treatment with drug mimicking LXA4 strongly reduced neuroinflammation following SE. This set of evidence supports the hypothesis that pharmacological intervention with pro-resolving drugs may reduce neuroinflammation and inhibit epileptogenesis.


39


Amplitude spectrum area to guide defibrillation: a validation on 1617 patients with

ventricular fibrillation.

Fumagalli F1, Mauri T2, Cesana G3, Li Y4, Finzi A1, , Rossi G2, Grieco N5, Migliori M5,

Andreassi A5, Latini R1, Fornari C3, Pesenti A2,5, Ristagno G1; Azienda Regionale Emergenza Urgenza Research Group. 1IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy 2Department of Anesthesia and Intensive Care, San Gerardo Hospital, Monza, and University of

Milano- Bicocca, Milan, Italy 3Research Center on Public Health, Department of Statistics and Quantitative Methods,

University of Milano-Bicocca, Milan, Italy 4School of Biomedical Engineering, Third Military Medical University and Chongqing

University, Chongqing, China 5 Azienda Regionale Emergenza Urgenza (AREU), Milan, Italy

Background. This study sought to validate the ability of amplitude spectrum area (AMSA) to predict defibrillation success and long-term survival after of out-of-hospital cardiac arrests. Methods. ECGs recorded by automated external defibrillators were obtained from patients with cardiac arrests occurring in 8 city areas. A database, including 2447 defibrillations, was used as the derivation group, and an additional database, including 1381 defibrillations, served as validation. A 2-second ECG window before defibrillation was analyzed, and AMSA was calculated. Univariable and multivariable regression analyses and area under ROC curve were used for associations between AMSA and study end points: defibrillation success, sustained return of spontaneous circulation, and long- term survival. Results. Among the 2447 defibrillations of the derivation database, 26.2% were successful. AMSA was higher before a successful defibrillation than a failing one (13 ± 5 versus 6.8 ± 3.5 mV-Hz) and was an independent predictor of defibrillation success (odds ratio, 1.33; 95% confidence interval, 1.20-1.37) and sustained return of spontaneous circulation (odds ratio, 1.22; 95% confidence interval, 1.17-1.26). Area under the ROC curve for defibrillation success prediction was 0.86 (95% confidence interval, 0.85-0.88). AMSA was also associated with long-term survival. The following AMSA thresholds were identified: 15.5 mV-Hz for defibrillation success and 6.5 mV-Hz for defibrillation failure. In the validation database, AMSA ≥ 15.5 mV-Hz had a positive predictive value of 84%, whereas AMSA ≤ 6.5 mV-Hz had a negative predictive value of 98%. Conclusions. AMSA was validated as an accurate predictor of defibrillation success and as a predictor of long-term survival.


40


Cardiovascular comorbidities in rheumatoid arthritis: analysis of a real life cohort of

patients treated with anti CTLA-4 Ig blocking T cell co- stimulation

Elena Generali1, Greta Carrara2, Carlo Selmi1,3, Carlo A. Scirè2 1) Humanitas Research Hospital; 2) Epidemiology Unit, Italian Society for Rheumatology; 3)

BIOMETRA Department, University of Milan

Background and objectives. Rheumatoid arthritis (RA) is a chronic disease associated with high cardiovascular (CV) risk. RA treatment with biologic agents counteracts chronic inflammation and may lower CV risk, however the magnitude of this latter effect is poorly defined, especially in real life studies. We aim to ascertain in a real life population the effect of anti-TNFα (etanercept) and anti-CTLA-4 (abatacept) as first-line biologic agents on the development of heart failure (HF). Materials and methods. Among the 10 million residents of Lombardy region, we identified RA cases and HF incidence using disease-specific payment exemptions, hospital discharge forms, diagnosis-related group codes and anatomical-therapeutic- chemical codes. Confounding factors, such as disease duration, methotrexate (MTX), NSAIDs and oral steroids use, previous CV risk factors as hypertension, dyslipidemia, diabetes were analysed. Results. A total of 1.545 RA patients treated with etanercept (n=1.254) or abatacept (n=291) as first-line biologic agent were identified. The competing crude and adjusted risk analysis showed and increased risk of HF in the abatacept cohort, respectively 2.95 (95%CI 0.98-8.87; p=0.054) and 1.54 (95%CI 0.40-5.91; p=0.530). HF incidence rate was higher in the abatacept (550 person year, 9.09, 95% CI 3.78-21.85) than in the etanercept (3.398 person year, 2.94, 95% CI 1.58-5.47) cohort. Conclusions. We show that HF is a frequent morbidity condition in the RA population. HF risk is not reduced in the cohort treated with anti-CTLA-4, however this biologic agent may be prescribed to a more severe RA population in consideration of its safety profile.


41


The influence of mutant prion protein on synaptic structure and neurotransmission:

mechanisms of neuronal dysfunction in genetic prion diseases

E. Ghirardini1,2, R. Morini1, E. Restelli3, D. Ortolan3, R. Chiesa3 and M. Matteoli1 1IRCCS Humanitas, Rozzano, Milano; 2Dip di Biotecnologie Mediche e Medicina

Traslazionale, Università di Milano; 3 IRCCS Istituto di Ricerche Farmacologiche Mario

Negri, Milano

Genetic prion diseases are fatal neurodegenerative disorders linked to mutations in the gene

encoding the cellular prion protein (PrPC). PrPC mutations favor the conformational conversion of the protein into a pathogenic isoform that kills neurons through an unknown mechanism. Different mutations are associated with distinct clinical and neuropathological phenotypes, including familial Creutzfeldt-Jakob disease (fCJD) and fatal familial insomnia (FFI).

There is evidence that PrPC engages in functional interaction with proteins that are important for synaptic function, such as glutamate receptors and other proteins involved in dendritic spine regulation. We hypothesize that aberrant interactions of mutant PrPs with these proteins affect glutamatergic transmission and synaptic plasticity, ultimately leading to neuronal death. We started to address this possibility by carrying out biochemical and morphological analyses, electrophysiological recordings and functional imaging in neurons from transgenic mice expressing mutant CJD and FFI PrPs. Our preliminary data indicate that mutant PrP alters the cellular localization of AMPA and NMDA receptors and induces a reduction in dendritic spine density and in pre- postsynaptic coupling. In CJD neurons this is accompanied by a decrease in basal glutamatergic transmission and impaired homeostatic plasticity, a form of synaptic plasticity that maintains stability in neuronal outputs in response to changes in network activity. Intertestingly, none of these functional alterations is present in FFI neurons, suggesting isoform-specific effects. Our data indicate that mutant PrP induces both morphological and functional alterations at the synapse which may represent a major cause of neuronal dysfunction and degeneration in genetic prion diseases.


42


LONG-TERM PROGNOSIS OF EPILEPSY AND DRUG RESISTANCE IN A

POPULATION BASED COHORT FROM NORTHERN ITALY

Giussani G, Canelli V, Bianchi E, Erba G, Beghi E.

Laboratory of Neurological Disorders, Department of Neuroscience, IRCCS –

Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy

Aim of the study was to understand the timing and outcome of prolonged periods of seizure freedom in patients with epilepsy and investigate the long-term outcome of patients with drug-resistant epilepsy in a population-based cohort. The medical records of 123 general practitioners were reviewed to identify local residents fulfilling the diagnosis of epilepsy and drug-resistant epilepsy, confirmed by a neurologist, during the period 2000-2008. Prognostic patterns were classified as follows: 1. Early remission: 2-year seizure freedom within two years from treatment start and still in remission at last follow- up; 2. Late remission: 2-year remission after more than two years after treatment start and in remission at last follow-up; 3. Remission followed by relapse: early or late remission but not in remission at last follow-up; 4. Remission never: never entering remission during the entire follow-up. The study population included 527 patients. Prognostic patterns were the following: early remission: 101 (19.2%); late remission: 175 (33.2%); remission followed by relapse: 85 (16.1%); remission never: 166 (31.5%). Lower chance of remission (whether early, late with or without relapse) was predicted by drug-resistant epilepsies, shorter follow-up, generalized symptomatic/cryptogenic epilepsy or cryptogenic/symptomatic partial epilepsy. The long-term prognosis of epilepsy is favorable in most cases. However, early seizure remission is not invariably followed by terminal remission. Prolonged seizure remission and prognostic patterns can be both mostly predicted by the classification of a patient in broad syndromic categories and drug-resistance. Drug-resistant epilepsy does not preclude the possibility to enter seizure remission.


43


The Epigenetic cofactor UHRF1 controls the metabolic re-programming of

hypertrophic cardiomyocytes

CM Greco1, P Carullo1, P Kunderfranco1, Papait1 and G Condorelli1 1Humanitas Clinical and Research Center (ICH)

Cardiac hypertrophy and failure is characterized by major alterations in the bioenergetics of cardiomyocytes. However, the mechanisms underlying myocardial energy metabolism alterations in the hypertrophic heart are not completely understood.

DNA methylation is a key element of transcriptional repression; genome-wide studies have indicated that this epigenetic modification could be involved in the pathogenesis of heart failure.

By performing massive DNA sequencing on cardiomyocytes isolated from adult mice subjected to transverse aortic constriction (TAC) for one week, we found genome-wide alterations in the DNA methylation profile, and, more specifically, increased DNA methylation at promoters of genes involved in the metabolic remodeling of the hypertrophic heart.

Expression screening of genes involved in DNA modifications revealed that the Uhrf1 gene was highly re-expressed in hypertrophic cardiomyocytes. UHRF1 epigenetically silenced genes involved in the TCA cycle, OXPHOS/ETC, and ATP synthesis pathways. In addition, hyper-methylated promoters were specifically enriched in binding sites for nuclear respiratory factor-1 (NRF1), a transcription factor co-activated by PGC1]and responsible for transcriptional activation of many genes encoding mitochondrial proteins. Importantly, methylation prevented the binding of NRF1 to the promoters of several metabolic genes.

Finally, in vivo knock down of Uhrf1 with serotype 9 adeno-associated vectors (AAV9) expressing anti-Uhrf1 shRNAs, prevented the development of cardiac and mitochondrial dysfunction in TAC mice.

This is the first study showing a causal role of DNA methylation in the metabolic remodelling of the hypertrophic heart and identifying a key player and its underlying mechanism of action during this process.


44


COMPLEMENT GENE VARIANTS DETERMINE THE RISK OF IMMUNE-

COMPLEX- MEDIATED MPGN AND C3 GLOMERULOPATHY AND PREDICT

LONG-TERM RENAL OUTCOME

Paraskevas Iatropoulos1*, Marina Noris1*, Caterina Mele1, Rossella Piras1, Elisabetta Valoti1,

Elena Bresin1, Manuela Curreri1, Elena Mondo2, Anna Zito1, Sara Gamba1, Serena Bettoni1,

Luisa Murer3, Veronique Fremeaux-Bacchi4, Marina Vivarelli5, Francesco Emma5, Erica

Daina1*, and Giuseppe Remuzzi1,2* 1IRCCS - Istituto di Ricerche Farmacologiche “Mario Negri”, Clinical Research Center for Rare Diseases “Aldo e Cele Daccò”, Ranica, Bergamo, Italy 2 Unit of Nephrology and Dialysis, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy

3Unit of Pediatric Nephrology, Dialysis and Transplantation, Azienda Ospedaliera of Padova,

Italy 4 Department of Immunology, Assistance Publique-Hopitaux de Paris, Hopital Europeen George- Pompidou and INSERM UMRS 1138, Cordelier Research Center, Team “Complement and Diseases”, Paris, France 5 Division of Nephrology and Dialysis. “Bambin Gesù” Pediatric Hospital. Roma, Italy

*Equally contributed Background: Membranoproliferative glomerulonephritis (MPGN) is an uncommon cause of chronic nephropathy recently reclassified into immune-complex-mediated MPGN (IC-MPGN) and C3 glomerulopathy (C3G). In this study we aimed: 1. to unravel whether complement gene variants correlate with bioptic findings and disease manifestations; 2. to establish whether complement gene variants predict long-term evolution. Methods: In a large cohort of 142 patients with IC-MPGN or C3G we performed gene screening and correlated genetic, biochemical and histologic data with clinical features to search for outcome predictors. Results: We found variants in genes encoding alternative pathway complement proteins in both IC- MPGN and C3G, and mutation prevalence was similar among histologic subgroups. C3 was the most frequently affected gene and CFB, encoding for the other component of C3 convertase, emerged as a new MPGN-associated gene. The common variants CFH 62I and THBD 473V, encoding the complement regulators factor H and thrombomodulin respectively, reduced the IC- MPGN/C3G risk in subjects carrying complement gene mutations, underscoring the complex inheritance of these diseases. Using multivariate analysis we found that a higher age at onset, the presence of nephrotic syndrome at onset, and the complement gene profile, but not histologic type, efficiently predict long-term renal outcome. Conclusions: We provide new insights into the pathogenesis of IC-MPGN/C3G. Our findings may also be useful to clinicians for predicting individual renal survival probabilities, directing therapy and designing or interpreting clinical trials.


45


miR146a-based therapy against neuroinflammation has anti-ictogenic and disease-

modifying effects in murine models of seizures and epilepsy

Valentina Iori1, Anand M. Iyer2, Luca Beltrame3, Lara Paracchini3, Sergio Marchini3, Milica

Cerovic1, Teresa Ravizza1, Riccardo Brambilla4, Maurizio d’Incalci3, Eleonora Aronica2,3 and

Annamaria Vezzani1 1Department of Neuroscience and Department of Oncology3, IRCCS-Istituto di Ricerche

Farmacologiche “Mario Negri”, Milano, Italy; 2Department of (Neuro)Pathology,

Academisch Medisch Centrum, Amsterdam and 3Epilepsy Institute in The Netherlands

Foundation (Stichting Epilepsie Instellingen Nederland, SEIN), Heemstede, The Netherlands;

4Institute of Experimental Neurology (INSPE), Division of Neuroscience, IRCCS San Raffaele

Scientific Institute

Neuroinflammation is a major pathogenic factor in epilepsy, as supported by experimental and clinical evidence. A mechanism of efficient upstream control of pathologic neuroinflammation in epilepsy is represented by micro(mi)RNAs, key posttranscriptional regulators of the innate immune response. Using models of acute and chronic seizures induced by kainic acid in C57BL/6 mice, we demonstrate the anti-ictogenic, anti-epileptogenic and disease-modifying properties of a synthetic mimic of miR146a which regulates the activation of the pro-inflammatory IL-1 receptor/Toll-like receptor signaling, a major ictogenic pathway. Intracerebroventricular injection of mimic, inducing a transient 4-fold increase in miR146a hippocampal levels and a concomitant reduction in IL-1 receptor/Toll-like receptor signal associated proteins, decreased by >50% acute symptomatic seizures evoked by a low kainate dose. In mice developing epilepsy following status epilepticus (SE) evoked by a high kainate dose, miR146a mimic administration for 2 weeks starting at the time of spontaneous seizure onset, precluded the 3-fold progression in seizure frequency over the following 2 months, as compared to control mice. The mimic injection after SE up to before the onset of spontaneous seizures, afforded neuroprotection in forebrain and rescued the cognitive deficit observed in the novel object recognition test. Transcriptomic analysis of differentially expressed genes (DEGs) done in the hippocampus of SE-exposed mice at the end of miR146a mimic treatment, showed 1281 DEGs specific to the mimic-treated group, mostly related to the immune/inflammatory response with a notable down-regulation of cytokine-related signal transduction proteins. Thus, specific anti-inflammatory miRNAs might be targeted for preventing the pathologic consequences of neuroinflammation in epilepsy, thereby providing a new class of treatments that favourably modify the disease course.


46

Abstract #41– Cancer Biology

Evaluation of novel biomarkers associated to Pancreatic Carcinogenesis

Mohammad Azhar Kamal1,2, Imran Siddiqui2, Silvia Schiarea3, Daniela Pistillo2, Stefania

Mitola1, Marco Presta1, Chiara Chiabrando3 and Paola Allavena2

1Department of Molecular and Translational Medicine, University of Brescia, Italy.

2Department of Immunology and Inflammation, Humanitas Clinical and Research Centre,

Rozzano-Milan, Italy. 3Department of Environmental Health Sciences, IRCCS - Istituto di

Ricerche Farmacologiche "Mario Negri", Milano, Italy.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive forms of cancer and estimated to become the second leading cause of cancer death in United States by the year 2020. Early detection of this cancer is the key-determining step to improve the survival rate and prognosis of PDAC patients. In the last decade, several approaches have been employed to identify biomarkers to detect PDAC in the early stage of its progression, but none has been reached full validation. We report here the evaluation of some potential PDAC biomarkers chosen among the proteins that we found overexpressed in an in-vitro model of early PDAC tumorigenesis, i.e; Human Pancreatic Ductal Epithelial (HPDE) cells made tumorigenic by transduction with mutated KRas, which often occurs in early-stage PDAC. A quantitative proteomic approach was used for global identification and quantification of secretome proteins (Stable Isotope Labeling with Amino acids in Cell culture (SILAC) followed by analysis of conditioned media by 1DE-LC-mass spectrometry analysis with LTQ Orbitrap XL). Among the

proteins overexpressed by KRasG12V we identified proteins possibly linked to early metastasis and inducers of epithelial-to-mesenchymal transition (EMT). A panel of matrix-associated proteins, metabolic enzymes and proteins related to the KRas-signaling was selected for overexpression validation in clinical samples. Tumor tissue from PDAC patients, analyzed by quantitative real-time PCR, confirmed high mRNA expression for these proteins. Furthermore, plasma levels of the matrix proteins were significantly higher (p<0.0001) in PDAC patients (n=87) than healthy donor (n=72), indicating their potential value as candidate biomarkers for the diagnostic of PDAC.


47


Investigating inflammatory pathways as targets for anti-epileptogenesis and biomarkers of

disease progression using functional imaging of IL-1beta expression

Kostoula C, Kulbida R, van Loo KM, Schoch S, Becker AJ, Vezzani A. Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Milan, Italy

Department of Neuropathology, University of Bonn Medical Center, Bonn, Germany

Experimental and clinical studies demonstrated a prominent activation of the IL-1beta signaling in epileptogenic foci from experimental models and drug-resistant human epilepsies. Importantly, this inflammatory pathway significantly contributes to the mechanisms of seizure generation, and may also play a role in drug-resistance. In order to interfere with the IL-1beta signaling activation in epilepsy for developing preventative, or disease-modifying treatments, it is crucial to gather detailed information on the extent and timing of IL-1beta induction in brain after a primary epileptogenic injury. We are developing, therefore, a method for detecting the IL-1beta induction in longitudinal studies in vivo, using clinically relevant animal models of epilepsy, by bioluminescence functional imaging of IL-1beta promoter activation. We focused on the generation of a recombinant viral vector expressing the luciferase gene under the control of the IL-1beta promoter. These constructs, upon introduction into RAW264.7 cells, allowed testing the activity of the IL-1beta promoter in response to an inflammatory stimulus (LPS), as analyzed by luciferase assays. After evaluating the construct with the most strongly LPS-induced IL-1β promoter sequence we will use it in vivo for targeting astroglia after stereotaxic transfer into the hippocampal region in order to investigate the brain expression of IL-1beta during epileptogenesis in clinically relevant animal models of epilepsy. The expected outcome is to determine the extent and timing of IL-1beta induction during epileptogenesis for designing a pharmacologic intervention to interfere effectively with the inflammatory cascade and to assess if IL-1beta induction in astrocytes is a biomarker of epilepsy development and/or its progression.


48


DBC1 is required for Chk2-dependent KAP1 phosphorylation and repair of heterochromatic

DNA lesions

Martina Magni1, Vincenzo Ruscica1, Giacomo Buscemi2, Enrico Fontanella1, Domenico Delia1

and Laura Zannini1.

1Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto

Nazionale dei Tumori, Via Amadeo 42, 20133 Milan, Italy. 2 Department of Biosciences, University of Milan, via Celoria 26, 20133 Milan, Italy.

Deleted in breast cancer 1 (DBC1, also known as CCAR2 or KIAA1967) is a nuclear protein largely involved in DNA damage response, apoptosis, metabolism, chromatin structure and transcription regulation. In response to DNA lesions, the apical kinases ATM/ATR phosphorylate DBC1, enhancing DBC1 binding to SIRT1 and leading to SIRT1 inhibition, p53 acetylation and p53-dependent apoptosis. Recently, we reported that also the checkpoint kinase Chk2 and the proteasome activator REGγ are required for efficient DBC1-mediated induction of p53-dependent apoptosis. Here we show DBC1 requirement for the repair of heterochromatic DNA lesions. Using the CRISPR/Cas9 system for genome engineering, we generated human osteosarcoma U2OS cells knock-out for DBC1. With this system model, we found that cells lacking DBC1 retain, at late time-points after genotoxic treatment, abnormal levels of DNA damage-associated 53BP1 nuclear foci. The timely resolution of these foci is reinstated by HP1β depletion, thus demonstrating the heterochromatic localization of these breaks. We also found that this impairment is caused by defective activation and activity of Chk2 towards its substrate KAP1, whose phosphorylation is required for mobilization of HP1 family proteins and induction of chromatin relaxation and DNA repair. These studies further extend the role of DBC1 in the DNA damage response and DNA repair and illustrate a new mechanism of Chk2 activity regulation. The involvement of DBC1 in the repair of heterochromatic DNA breaks also suggests a new role for this protein in the maintenance of chromosomal stability, which is necessary to prevent cancer formation.


49


Comparison of toxicity induced by some illicit drugs to the zebra mussel

Stefano Magni, Camilla Della Torre, Marco Parolini, Andrea Binelli Department of Biosciences, University of Milan, Via Celoria 26, 20133 Milan, Italy

Illicit drugs are considered emerging aquatic contaminants because of their high use worldwide and the inability of traditional wastewater treatment plants to efficiently remove them from wastewater. Although their occurrence in the aquatic ecosystems is well-known, few research regarding their potential toxicity on aquatic communities have been performed. Therefore, the aim of this study was to evaluate the sub-lethal effects caused by some common illicit drugs found in the aquatic ecosystems, namely ∆-9- tetrahydrocannabinol (∆-9-THC), morphine, 3,4-methylenedioxymethamphetamine (MDMA), amphetamine, benzoylecgonine and ecgonine methyl ester, on the freshwater bivalve, the zebra mussel (Dreissena polymorpha), by a biomarker battery. For each tested compound, we exposed the bivalves at the same concentration (0.5 µg/L) for 14 days. To assess the oxidative stress, we monitored the activity of antioxidant enzymes, as well as the levels of lipid peroxidation and protein carbonylation. In addition, genotoxicity was evaluated on bivalve haemocytes by Comet Test, Micronucleus Test and DNA Diffusion Assay. The obtained results were integrated into a synthetic index, called Biomarker Response Index (BRI), to draw a toxicity scale on the basis of the effects observed with each individual biomarker. Our data showed that the ∆-9-THC is the most toxic drug to the zebra mussel, since it induced both oxidative and genetic damage. The two cocaine metabolites (benzoylecgonine and ecgonine methyl ester) appeared to have a lower environmental toxicity compared to ∆-9-THC, while morphine, MDMA and amphetamine exhibited the lowest toxicity, at least for the considered end-points.


50


Analysis of in vitro effects of metformin alone or in combined treatments in colorectal

cancer

Maria Valeria Maiorana1,2, Angela Mogavero1,2, Filippo Pietrantonio3, Giacomo Manenti1,

Marco A. Pierotti2, Manuela Gariboldi1,2 1 Department of Experimental Oncology and Molecular Medicine Fondazione IRCCS Istituto

Nazionale dei Tumori, Milano, Italy 2 Molecular Genetics of Cancer, Fondazione Istituto FIRC Oncologia Molecolare (IFOM),

Milano, Italy 3 Department of Medical Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori,

Milano, Italy

Several retrospective cohort studies suggest that metformin treatment is associated with reduced cancer risk and/or mortality, and distinct in vitro and in vivo studies on different cancer types seem to confirm these results. However the mechanism for metformin anti-cancer activity in colorectal cancer (CRC) needs to be clarified. The aim of this study is to investigate metformin effects on three CRC cell lines (HT29, HCT116 and HCT116P53-/-). A clonogenic assay showed that metformin strongly reduced both the number and size of colonies formed. Metformin treatment induced a moderate cell cycle arrest in G0/G1 phase, confirmed by a decrease of cyclin D1 and c-Myc expression. The antiproliferative activity of the drug resulted mediated by suppression of mTOR and of its downstream targets S6 and 4EBP1, both in an AMPK dependent and independent way and also downregulation of the activity of IGF1R. However metformin does not induce irreversible phenomena such as apoptosis, senescence or autophagy. To assess the effects of metformin in combination with the standard chemotherapy used for CRC (5- Fluorouracil (5FU), Oxaliplatin (OXA) or Irinotecan (IRI)) viability assays were performed. Drugs have been tested in different combined treatments: alone, administered contemporary and in different sequences. Contemporary administration 5FU-metformin and OXA-metformin resulted the best treatment. Positive results for combined treatment with IRI were obtained only at longer time exposition to IRI before metformin. Results were confirmed when metformin was added to standard regimens (OXA-5FU and IRI-5FU) used in clinics.


51


Investigating the role of the inhibitor of apoptosis proteins (IAPs) in metastasis formation

Maria Teresa Majorini1, Daniele Lecis1, Annalisa Conti1, Enrico Fontanella1, Giacomo

Manenti2 and Domenico Delia1 1Molecular Mechanisms of Cell Cycle Control Unit, Department of Experimental Oncology and

Molecular Medicine, 2Molecular Basis of Genetic Risk, Polygenic Models Unit, Department of

Predictive and Preventive Medicine - Fondazione IRCCS Istituto Nazionale dei Tumori

Inhibitor of apoptosis proteins (IAPs) are known to prevent apoptosis and are frequently over-expressed in tumors where they promote chemoresistance and disease progression. Therefore, IAPs represent potential pharmacological targets and, accordingly, a new class of antagonizing compounds, called smac mimetics (SMs), has been developed. SMs target and induce the degradation of cellular IAP1 (cIAP1) and cIAP2, which are regulators of several pathways and interestingly are involved in cancer cell dissemination. To test whether the inhibition of the IAPs prevents the metastatic process, we treated NOD-SCID mice engrafted with the invasive breast cancer cell line MDA-MB231 by means of daily injections of SM83, a novel SM developed in our lab. Our findings show that SM83 treatment results in the reduction of spontaneous lung metastasis, supporting the hypothesis that IAPs are promoters of the metastatic process. Moreover, gene expression profiling of primary tumors collected from SM83-treated mice showed a down-regulation of Snai2 which is a mediator of the epithelial-mesenchymal transition (EMT), a process that favors cancer cell detachment and dissemination. We then investigated the mechanisms responsible for Snai2 regulation and demonstrated that cIAP1, and not other IAPs, favors Snai2 expression. Moreover, we determined that the NF-kB pathway mediates at least in part the cIAP1-mediated regulation of Snai2, even though MAPK and PI3K/AKT signaling cascades could also play a role. In conclusion, our findings support the notion that cIAP1 favors metastasis by promoting the expression of pro-metastasis genes. Therefore, SMs, by antagonizing IAPs, could result in an anti- metastatic effect exploitable in cancer therapy.


QSAR model for cell viability of silica

cells

S. Manganelli, C. Leone, E. Benfanti

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

A predictive model for cell viability (%) of 20 and 50 nm silica nanoparticles has

been built using so-called optimal descriptors as mathematical functions of size, concentration and exposure time. These parameters have been encoded into 31 combinations‘size– concentration-exposure’.CORAL software (http://www.insilico.eu/coral/)systems into training and test sets. The statistical quality of the best model for the cell viability(%) of cultured human embryonic kid

concentrations of silica nanoparticles measured by MTT assay is the following: n =22, R

ranging from 0.7096 to up to 0.8778 (training set) and n = 9, R

0.8996, and > 0.5 (test set).

external validation, providing intrinsic property, as well as experimentalmodeling scheme, after being

References: (a) Toropova AP, Toropov AA,SiO2 nanoparticles on human lung fibroblasts; J Nanopart Res (2014)(b) Wang F, Gao F, Lan M, Yuannanoparticle-induced cytotoxicity in human embryonic kidney cells, Toxicology in Vitro808–815 (c) A. P. Toropova, A. A. Toropov,into the prediction of membrane(2013) 93:2650–2655

[PhD Students Meeting

New Frontiers in Biotechnology

QSAR model for cell viability of silica nanoparticles on human embryonic kidney

, C. Leone, E. Benfanti

Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy

A predictive model for cell viability (%) of 20 and 50 nm silica nanoparticles hascalled optimal descriptors as mathematical functions of size,

exposure time. These parameters have been encoded into 31 combinationsexposure’. The calculation has been carried outhttp://www.insilico.eu/coral/) using three random splits of the obtained

training and test sets. The statistical quality of the best model for the cell cultured human embryonic kidney cells (HEK293) containing different

nanoparticles measured by MTT assay is the following: n =22, R

to 0.8778 (training set) and n = 9, R2 ranging from 0.7734 up to

set). One more set of 9 combined system has been used for the

> 0.5. The advantage in the use of thisexperimental condition, can be easily introduced

being encoded and combined into a ‘quasi-SMILES’.

AA, Benfenati E, Korenstein R. QSAR modelnanoparticles on human lung fibroblasts; J Nanopart Res (2014) 16:2282

Yuan H, Huang Y, Liu J. Oxidative stress induced cytotoxicity in human embryonic kidney cells, Toxicology in Vitro

Toropov, Optimal descriptor as a translator of eclecticmembrane damage by means of various TiO2 nanoparticles.

PhD Students Meeting] [2015]

52

human embryonic kidney

A predictive model for cell viability (%) of 20 and 50 nm silica nanoparticles has called optimal descriptors as mathematical functions of size,

exposure time. These parameters have been encoded into 31 combinations out by means of the

using three random splits of the obtained training and test sets. The statistical quality of the best model for the cell

ney cells (HEK293) containing different

nanoparticles measured by MTT assay is the following: n =22, R2

ranging from 0.7734 up to

One more set of 9 combined system has been used for the

this method is that any introduced within the

SMILES’.

model for cytotoxicity of

contributes to silica induced cytotoxicity in human embryonic kidney cells, Toxicology in Vitro (2009) 23:

eclectic information nanoparticles. Chemosphere


53


Effects of inflammation on functionality and development of inhibitory circuits in the

central nervous system

Cristina Mantovani1, Alice Canzi1, Romana Tomasoni1, Raffaella Morini1, Elisabetta

Menna1,2, Michela Matteoli 1,2 1 Humanitas Clinical and Research Center, via Manzoni 56, 20089 Rozzano, Italy; 2 National Research Council, Institute of Neuroscience, Milan, Italy. Several key evidence indicate that inflammation and inflammatory mediators contribute to neurodegenerative or psychiatric disorders. Aim of the project is to investigate whether and at which extent inflammation originates synaptic dysfunctions which may eventually lead to cognitive impairments. To this aim we will take advantage of a genetic mice model defective in a well characterized immune pathway, the TIR8/SIGIRR-deficient mice. TIR8/SIGIRR is a key regulator of inflammation and TIR8/SIGIRR-deficient mice show inability to dampen signaling from IL-Rs and TLRs family members (Qin et al., 2005; Huang et al., 2006; Gulen et al., 2010). For this reason, TIR8/SIGIRR-deficient mice represent a perfect genetic model of inflammation and, indeed, an excellent tool to investigate the impact of inflammation on brain development and functionality. Our preliminary results indicated that TIR8/SIGIRR-deficient neurons display defects in excitatory synapse structure and function (Tomasoni et al., in preparation). We will now investigate whether the inhibitory neuronal network is also affected by deregulation of inflammatory pathways. By using a combination of whole cell patch-clamp recordings and confocal analysis in different in vitro models from TIR8/SIGIRR- deficient mice (i.e., primary cell cultures, acute hippocampal slices, organotypic slice cultures), we are currently investigating the possible occurrence of morphological and functional defects of inhibitory synapses originated by inflammation. To further investigate the synaptic consequences of inflammation, we will perform electrophysiological experiments and confocal analysis in wild type hippocampal slices or cultures treated with IL-1β.


54


CX3CR1+ intestinal macrophages at the crossroad of tissue repair and tumor development

Giulia Marelli1,2, Marco Erreni1, Cristina Belgiovine1, Alberto Mantovani1,2 and Paola

Allavena1

1Clinical and Research Institute Humanitas, Milano, Italy; 2Department of Medical

Biotechnology and Translational Medicine, University of Milano, Italy.

Colorectal cancer (CRC) is the third cause of death worldwide and is considered a paradigm of cancer related inflammation. Macrophages infiltrating CRC are associated with better prognosis, but it is not clear which type of macrophages performs this role. In this study we focused on a particular population of resident macrophages that we and other

groups have characterized as CX3CR1+, CD64+, F4/80+, CD11b+. These cells are able to maintain homeostasis and they prevent the aberrant immune response to harmless antigens. Moreover they are able to regulate inflammation during colitis to avoid the establishment of chronic disease. However, the role of these cells in colon carcinogenesis is unknown. We analyzed the behavior of these macrophages in mice lacking the CX3CR1 receptor and in wild type mice. Using the AOM/DSS model of colitis-induced carcinogenesis, we found a significantly higher number of polyps, higher score of tissue damage and higher number of immune cells in the intestine of KO mice. Furthermore, KO mice are not able to switch off inflammation in the adenomatous colonic tissue, as demonstrated by a deregulation of both pro and anti- inflammatory cytokines. This inability to regulate inflammation results in delayed tissue repair compared to WT mice. Taken together our results show that CX3CR1+ macrophages are important in the maintenance of homeostasis but they are also indispensable for the resolution of inflammation. When CX3CR1 receptor is lacking, macrophages tissue repair response is inefficient and it results in a prolonged inflammatory response that leads to a greater tumor development.


55


Dot1L and miR29 epigenetic-mediated modulation of atherosclerosis neointimal formation

Marzo Stefano, Manuela Quintavalle Istituto Clinico Humanitas

Atherosclerosis is one of the major cause of death in the western world. Hallmark of this pathology is the formation of blood vessels lesions that are characterized by the thickening of the intimal layer due to the smooth muscle cells hyperproliferation. The limits of the symptomatic pharmacological treatment impose a research on alternative strategies to revert the induced vascular damage. Epigenetic mechanisms are recently recognized as a suitable therapeutic target to normalize the altered gene expression profile of several diseases, including cardiac hypertrophy and heart failure. On these premises a previous gene expression screening performed in our lab, has revealed that the epigenetic enzyme DOT1-like histone H3K79 methyltransferase (Dot1l) is up-regulated in the aorta of atherosclerosis developing mice models, suggesting that this enzyme could be involved in the intima hyperplasia. Further in vitro experiments confirmed that Dot1l is a key player to promote smooth muscle cells proliferation after exposure to known de- differentiating stimuli and that is subjected to regulation by the microRNA miR29. Aim of this PhD project is to characterize the role of Dot1l on a mouse conditional KO gene model and to asses whether its downregulation by miR29 could influence the atherosclerotic phenotype. On the same KO cells isolated from this mice we will perform in vitro chip-seq analysis to identify Dot1l genomic targets among genes and miRNA. In parallel a screening for other target of miR29 will be performed to obtain a better view of the interplay between these two potential actors.


56


Ablation of palladin in adult cardiac muscle causes dilated cardiomyopaty

Mastrototaro G1,2 , Carullo P2,3 , Zhang J4, Chen J4, Bang ML2,3 1 University of Milan-Bicocca, Milan, Italy 2 Humanitas Research Hospital, Rozzano, Milan, Italy 3Institute of Genetic and Biomedical Research, Milan Unit, National Research council,Milan, Italy 4University of California, San Diego School of Medicine, La Jolla, CA, USA Palladin (PALLD), myopalladin (MYPN), and myotilin (MYOT) make up a small protein family of immunoglobulin-containing proteins in the Z-line of the sarcomere. All three proteins interact with α-actinin and are essential for the organization of the actin cytoskeleton (1,2). While MYPN and MYOT are expressed in striated and skeletal muscle, respectively, PALLD is ubiquitously expressed in several isoforms and associated with actin-associated structures in various cell types. The longest 200 kDa PALLD isoform is expressed predominantly in striated muscle and is highly homologous in structure to MYPN, suggesting that the two proteins may play similar roles in heart and skeletal muscle (3). However, while several mutations in MYPN gene are been identified in patients with dilated, hypertrophic and restrictive cardiomyopathies (4-6), the role of PALLD in the heart remains unknown. PALLD knockout mice are embryonic lethal due to neural tube closure defects (7) thus, to investigate the role of PALLD in heart, we generated constitutive (cPKO) and inducible (cPKOi) cardiac specific PALLD knockout mice. Detailed analyses of cPKO mice revealed no cardiac phenotype either at basal conditions or following mechanical pressure overload by transaortic constriction (TAC), suggesting that MYPN may compensate for the absence of PALLD. On the other hand, cPKOi mice at adult stage resulted in progressive systolic dysfunction, leading to dilated cardiomyopathy. Our results show that PALLD might be involved in the modulation of actin cytoskeleton through the serum response factor-MRTFA pathway and MAPK signaling. Further experiments are being developed in order to better understand these molecular mechanisms. 1. Otey et al., Int Rev Cytol 246, 31-58, 2005. 2. Otey et al., Cell Motil Cytoskeleton 66, 618-634, 2009. 3. Wang and Moser. Dev Dyn 237, 3342-3351, 2008. 4. Duboscq-Bidot et al., Cardiovasc Res 77, 118-125, 2008 5. Purevjav et al., Hum Mol Genet 21: 2039-2053, 2012. 6. Meyer et al., Eur J Hum Genet 21, 294-300, 2013. 7. Luo et al., Mol Cell Neurosci 29: 507-515, 2005.


57


Interleukin‐1 receptor 8 (IL‐1R8) plays a crucial role in Natural Killer (NK) cell

differentiation and function

Martina Molgora1, Eduardo Bonavita1, Andrea Ponzetta1, Marialuisa Barbagallo1, Giorgia Benigni2, Giovanni Bernardini2, Angela Santoni2, Alberto Mantovani1, Cecilia Garlanda1

1Humanitas Clinical Research Center, via Manzoni 56, 20089 Rozzano (Milano), Italy 2Dipartimento di Medicina Molecolare Istituto Pasteur-Fondazione Cenci Bolognetti, Università di

Roma "La Sapienza," 00161 Rome, Italy

IL-1R8 is an Interleukin-1R like receptor (ILR) family member and acts as a negative regulator of ILRs and Toll-like receptors (TLRs) signaling. Our previous data show that both murine and human Natural Killer (NK) cells express high levels of IL-1R8 and the aim of my PhD is to analyze its role in modulating ILR and TLR signaling in NK cells. IL-1R8 is differentially expressed during NK cell maturation in the mouse. IL-1R8 deficiency is associated with higher frequency and absolute number of mature NK cells in blood, spleen, bone-marrow and liver. IL-1R8 deficient NK cells display an increased Interferon-γ (IFN-γ) production and an higher cytotoxic activity. IL-1R8 deficiency is also associated with an higher level of NK cell activating receptors, a more differentiated phenotype of NK cells and an increased activation of the mTOR pathway. IL-1R8 expression was then evaluated in the three distinct subsets of human NK cells, associated with different stages of NK cell maturation. IL-1R8 was up-regulated during NK cell maturation, both in terms of mRNA and protein expression. Although the molecular mechanisms of IL-1R8-dependent regulation of NK cells are still under investigation, the results presented here suggest that IL-1R8 is a novel regulator of NK cell development, IFN-γ production and cytotoxic activity. IL-1R8 plays therefore a nonredundant role in the regulation of NK cell biology and could be a crucial regulator of NK cell antiviral and antitumoral activity.


58

Abstract #53

Chronic n-acetylcysteine treatment promotes extinction of conditioned cue-induced nicotine-

seeking behavior in the rat

F. Moro1, G. Giannotti2, F. Fumagalli2 and L. Cervo1 1IRCCS - Mario Negri Institute for Pharmacological Research, Experimental Psychopharmacology,

Department of Neuroscience, Via La Masa 19, Milan, Italy 2University of Milan, Dept. of Pharmacological and Biomolecular Sciences, Via Balzaretti 9,

Milan, Italy

Although some pharmacological and psychosocial support can help smokers to quit, the high

relapse rates indicate a need for more efficacious treatments. The cysteine pro-drug N-acetylcysteine (N-AC) may have beneficial therapeutic effects in the treatment of drug addiction. Pre-clinically N-AC reduced conditioned cues-induced cocaine- and heroin-seeking by restoring cystine-glutamate exchange system Xc- and the glial glutamate transporter GLT1, normalizing extracellular glutamate, thus blunting the activation of glutamatergic neurons associated with drug cues-induced reinstatement.

Although nicotine-cues reinstate drug-seeking is still unclear whether N-AC would inhibit cue-induced reinstatement in abstinent rats after nicotine self-administration. Moreover, it is unknown whether restoring Glu homeostasis by chronic N-AC treatment would enhance the outcome of cue-exposure therapy for smoking cessation. To gain such information, we used an animal model of cue-induced robust and lasting nicotine-seeking in abstinent rats. We found that a single dose of N-AC (100 mg/kg) induced a short-term reduction of cues-induced nicotine-seeking. When N-AC (100 mg/kg) was given chronically during abstinence or in combination with nicotine-associated cues, it was found that only in the latter condition N-AC not only showed efficacy when biologically available during testing, but also produced a long-lasting anti-relapse activity that was still present 50 days later. To provide a mechanism for behavioral results, separate groups of animals that received either N-AC (100 mg/kg) or saline treatments in combination with nicotine-associated cues were killed for proteins analysis. Interestingly, western blot analysis of punches containing Nacc-core and Nacc-shell revealed that chronic N-AC reverted nicotine-induced changes in the levels of the catholic subunit of system Xc- and GLT1. These results support and extend previous preclinical findings suggesting that N-AC might offer a therapeutic opportunity for nicotine addiction


59


ChrXq27.3 miRNAs cluster in epithelial ovarian cancer: decipher their role to provide new

avenues for treatment.

Nicoletti R1, Cacciamali A1, Granata A1, Alberti P1, De Cecco L1, Zhang W2, Raspagliesi

F1, Canevari S1, Pignata S3, Mezzanzanica D1, Bagnoli M1.

1 Fondazione IRCCS Istituto Nazionale dei Tumori, Milan – Italy

2 The University of Texas MD Anderson Cancer Center, Huston, USA.

3 Fondazione G. Pascale Istituto Nazionale Tumori, Naples – Italy

Epithelial ovarian cancer (EOC) is a heterogeneous and aggressive disease with poor prognosis, mainly due to late diagnosis. EOC is characterized by tumor cells’ spreading into peritoneal cavity with the acquisition of mesenchymal phenotype, and development of resistance to chemotherapy. A better understanding of cellular and molecular biology of this disease could improve the outcome for EOC patients. Due to their master regulatory role, microRNAs (miRNAs) are considered powerful tools to obtain representative molecular portraits of specific tumor characteristics and behaviors. MiR-506, belonging to the ChrXq27.3 miRNAs cluster that we identified as down-modulated in early relapsing advanced stage EOC patients, seems to play a pivotal role in different biological processes such as drug resistance and epithelial-to-mesenchymal transition (EMT). We showed that miR-506, besides targeting the E-cadherin repressor SNAI2, simultaneously suppress vimentin and N- cadherin thus representing a new class of miRNAs which regulate master players of EMT and metastatization process. On the other hand, among miR-506 target genes involved in drug sensitivity/resistance, we validated RAD51 and RAD17, both involved in DNA damage signaling and repair pathways by guiding BRCA1/2 to DNA strand breaks stabilizing active signaling. In EOC, germline and somatic mutational inactivation of DNA repair genes (BRCA1/2) is associated with better prognosis, due to sensitization to DNA damaging drugs. Interestingly, we observed that miR-506 expression increased EOC cell lines Platinum-sensitivity and is strongly associated to patients’ response to Platinum treatment thus mimicking BRCA1/2 inactivation. Overall our data support the role of miR-506 in regulating both cell plasticity and drug sensitivity/resistance.


60


B7-1 is not induced in podocytes of human and experimental diabetic nephropathy

*Rubina Novelli, *Elena Gagliardini, *Daniela Corna, *Carla Zoja, *Barbara Ruggiero, *Ariela

Benigni and *°Giuseppe Remuzzi *IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, Centro Anna Maria Astori, Science

and Technology Park Kilometro Rosso, Via Stezzano 87 - 24126 Bergamo °Unit of Nephrology and Dialysis, Azienda Ospedaliera Papa Giovanni XXIII, Piazza OMS 1

– 24127 Bergamo, Italy

The incidence of progressive kidney disease associated with diabetes continues to rise worldwide. Only partial renoprotection is achieved by current standard therapy with angiotensin-converting enzyme inhibitors and/or angiotensin receptor blockers, increasing the need for novel therapeutic approaches. The induction of B7-1, an immune-related protein found on antigen-presenting cells, was recently described in podocytes of proteinuric patients including those with recurrent focal and segmental glomerulosclerosis (FSGS) and type 2 diabetic nephropathy (DN). These findings sparked great excitement among the renal community implying that abatacept, a costimulatory inhibitor that targets B7-1, could be used as a novel therapy for recurrent FSGS and DN. However, doubts about the validity of the immunohistochemical assays used to detect B7-1 on renal specimens, as well as the erratic clinical responses to abatacept, raised several concerns. Here, we wanted to investigate B7-1 expression in human and experimental DN before embarking in clinical studies on the use of B7-1 targeting strategies. Immunohistochemical analysis on kidney specimens using different antibodies and protocols revealed that B7-1 was not induced in podocytes of 31 patients with DN independently on the stage of the disease nor in BTBR ob/ob mice, a model of type 2 diabetes, and in type 1 diabetic C57BL/6 mice injected with streptozotocin. These results provide an important cautionary note about the relevance of B7-1 expression on podocytes as a biomarker and do not support the use of abatacept as a potential novel therapeutic strategy for the prevention or treatment of DN, at least to target podocyte B7-1.


61


Exome analysis to understand the genetic basis of Acrofrontofacionasal Dysostosis 1

Eleonora Palagano1,2, Dario Strina1,2, Ciro Menale1,2, Stefano Mantero1,2, Paolo

Prontera3, Maria Leine Guion-Almeida4, Paolo Uva5, Andrea Angius5,6, Anna Villa1,2,

Cristina Sobacchi1,2 1CNR-IRGB, Milan Unit, Milan, Italy; 2Humanitas Clinical and Research Center,

Rozzano, Italy; 3CRR Genetica Medica, Università ed Azienda Ospedaliera di Perugia,

Perugia, Italy; 4Department of Clinical Genetics, Hospital of Rehabilitation of

Craniofacial Anomalies, University of Sao Paulo, Sao Paulo, Brasil; 5CRS4, Science and

Technology Park Polaris, Piscina Manna, Pula, Italy; 6CNR-IRGB, Monserrato, Italy

Acrofrontofacionasal Dysostosis 1 (AFFND1) is a rare human syndrome (1:1,000,000), characterized by bone abnormalities, multiple congenital anomalies and mental retardation. Only four AFFND1 families have been described so far. The patients are severely affected, displaying mental retardation, short stature, facial and skeletal abnormalities characterized by cleft lip/palate, campto-brachy-poly-syndactyly and marked anomalies of foot structures. Overall, they have a poor quality of life which seriously impacts also on their families. An autosomal recessive pattern of inheritance is hypothesized, but the causative genetic defect is not known, yet. In order to define the molecular basis of AFFND1, we exploited exome sequencing as a powerful sequencing technique able to detect nearly all the coding variations present in an individual genome. We collected DNA samples from 1 Indian and two Brazilian families, exome sequencing was performed on patients and their parents. After sequencing, appropriate filtering was performed, also considering the pattern of inheritance of the disease, frequency and parental consanguinity (in 2 out of 3 families). We identified a short list of variants in the 3 families investigates, with no shared candidates, suggesting the possibility of genetic heterogeneity. Thus far, we addressed more in detail the analysis of the results obtained in the Indian family. Among the 17 variants identified, based on data in literature, we selected 5 variants and confirmed them by Sanger sequencing. We focused on NBAS gene, which is known to play a role in embryogenesis from studies in Zebrafish. Further in silico analyses as well as functional evaluations are ongoing.


62


AN EARLY MODULATION OF THE PRO-INFLAMMATORY RESPONSE

ASSOCIATED TO THE ACTIVATED MICROGLIA IS PIVOTAL TO SUSTAIN

HEALING PROCESSES IN SPINAL CORD INJURY

S. Papa1, R. Ferrari2, E. Erba1, N. Panini1, M. De Paola1, A. Mariani1, I. Caron1, F. Rossi2,

D. Pozzer1, D. Moscatelli2, P. Veglianese1.

1 IRCCS Istituto di Ricerche Farmacologiche Mario Negri, Milano

2 Politecnico di Milano - Department of Chemistry, Materials and Chemical Engineering

“Giulio Natta”

Spinal cord injury (SCI) is the result of a mechanical damage (primary injury) followed by a post-traumatic degeneration of the cord, called “secondary injury”. Many efforts have been performed to demonstrate the role of the peripheral macrophages during the progression of SCI, revealing a pleiotropic effect associated to different phenotypes (M1 and M2), that are spatially and temporally recruited in the injured site. Differently, the role of activated microglia during the secondary injury was poorly investigated, because of the difficult to target and modulate selectively activated microglia in the injury site. To overcome these limitations and clarify the involvement of the pro-inflammatory response associated to activated microglia, we exploited a delivery nanovector tool able to treat selectively this cell population. We found that a selective treatment with minocycline, a well-known anti-inflammatory drug, only administered promptly in an early phase of SCI, is able to modulate efficiently resident microglial cells, reducing the pro- inflammatory response, maintaining a pro-regenerative milieu and ameliorating the behavioral outcome up to 63 days post injury. Furthermore, we demonstrate that resident microglia have a key role in the recruitment of M1 peripheral macrophages via CCL2 chemokine during the progression of SCI confirming a detrimental role of the pro- inflammatory microglia in the first phase of the injury.


63


Crosstalk between the long pentraxin PTX3 and the Complement system in the immune

response to Aspergillus fumigatus

Raffaella Parente1, Francesca Petroni1, Marina Sironi1, Sonia Valentino1, Marco Gobbi2,

Barbara Bottazzi1, Alberto Mantovani1,3, Antonio Inforzato1,4

1Humanitas Clinical and Research Center, Rozzano, Italy 2Mario Negri Institute for Pharmacological Research, Milan, Italy 3Humanitas University, Rozzano, Italy 4Department of Translational Medicine, University of Milan, Rozzano, Italy

The opportunistic fungus Aspergillus fumigatus (AF) is the etiologic agent of invasive

aspergillosis (IA), a life-threatening infection amongst immunocompromised individuals. The host response to AF is mainly sustained by both cellular and humoral components of innate immunity, including polymorphonuclear neutrophils (PMNs) and Complement. The long pentraxin PTX3 is as a soluble pattern recognition molecule that cooperates with Complement and PMNs in the opsono-phagocytosis and killing of AF [1]. Here, we studied the molecular crosstalk between PTX3 and Complement in the immune

response to AF, using FACS, ELISA, Electrophoresis, Chromatography and Surface Plasmon Resonance techniques. We found that PTX3 inhibits the interaction of AF conidia with factor H (FH, the major soluble inhibitor of Complement), which is a critical immune evasion strategy of AF. This is due to competition for common binding sites on the conidial wall and is recapitulated by the N-terminal domain of PTX3. Consistent with this, the cofactor activity of FH on AF conidia (factor I-mediated cleavage of C3b to iC3b) is inhibited by PTX3 and its N-terminus. Furthermore, we observed that PTX3 forms a stable complex with C3b in an FH-dependent fashion, and recruits C3b onto AF conidia. Given that C3b is the prototypic opsonin of Complement (that is recognized by the

phagocytic receptor CR1), we propose that PTX3 enhances C3b deposition onto AF via a novel FH-dependent mechanism, thus counteracting the Complement escape strategies of this fungus and enhancing its phagocytosis and killing by PMNs. 1. Cunha et al (2014) N Engl J Med 370(5):421-32


64


THE TS3R DOMAIN OF THROMBOSPONDIN-1 AFFECTS TUMOR

VASCULARIZATION AND IMPROVES RESPONSE TO CHEMOTHERAPY

Denise Pinessia, Andrea Resovia, Lavinia Morosib, Patrizia Borsottia, Raffaella Giavazzia,

Massimo Zucchettib, and Giulia Tarabolettia

aTumor Angiogenesis Unit, Laboratory of Biology and Therapy of Metastasis, IRCCS- Istituto

di Ricerche Farmacologiche Mario Negri, Bergamo; bClinical Cancer Pharmacology Unit,

Laboratory of Cancer Pharmacology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri,

Milano

Antiangiogenic agents can lead to a transient reorganization of the tumor vasculature, improving the delivery and efficacy of chemotherapy. Thrombospondin-1 (TSP-1) is a major endogenous inhibitor of angiogenesis. Our previous studies identified a new site in the type III repeats domain (TS3R) of TSP-1, that binds FGF-2 and inhibits angiogenesis. Aim of this study was to investigate the effect of TS3R on tumor growth, vascularization and response to chemotherapy. We used a human ovarian carcinoma cell line A2780- 1A9, transfected to express and secrete the TS3R domain or a truncated form lacking the FGF-2 binding site. In vivo, tumor cells expressing the TS3R domain had a decreased tumorigenicity compared to control and tumor cells lacking the FGF-2 binding site. Moreover, TS3R-expressing tumors showed a greater sensitivity to paclitaxel (PTX 10mg/kg, i.v.) and cisplatin (DDP 4 mg/kg, i.v.), the standard-of-care drugs for ovarian carcinoma. Histological analysis showed that TS3R-expressing tumors presented evidence of vascular reorganization with an increased number of CD31 positive vessels and decreased vessel area and diameter. Pharmacokinetic analysis and MALDI imaging mass spectrometry analysis on tumors collected 4h after a single administration of paclitaxel (60 mg/kg, i.v.) showed a more homogeneous distribution and higher concentration of paclitaxel in tumors expressing TS3R. The TS3R domain of TSP-1 has the potential to affect the tumor microenvironment and improve tumor drug delivery, setting the basis for new therapeutic strategies.


65

Abstract #60

Drug prescription and perinatal determinants during the first year of life

Daniele Piovani1, Antonio Clavenna1, Chiara Pavoni1, Maurizio Bonati1

1 IRCCS – Istituto di Ricerche Farmacologiche “Mario Negri”, Laboratory for Mother and Child Health, Department of Public Health

The aim of the study was to investigate the drug prescription profile during the first year of life in a cohort of newborns, and the influence of some perinatal and socio- demographic factors on drug prescription. A total of 61,479 neonates born in 2011 were included. The data source was the database of reimbursed prescriptions of the Lombardy region, Italy. Drug prescriptions were classified according to the Anatomical Therapeutic Chemical (ATC) classification system. Drug prevalence was calculated as the percentage of neonates receiving at least one drug prescription in one year. Chi-squared and t-test were used to compare categorical and continuous variables. In 2011, 42,204 newborns (68,7%) received at least one drug prescription. The prevalence of drug

prescription was higher (χ2= 178 p<0.01) in males (71.1%) than females (66.1%). The most used drug classes in the first year of life were antibiotics (39.5% of children), anti-asthmatics (32.6%), and corticosteroids for systemic use (9.8%). The first prescription was filled, on average, at 20.6 (IQR 9.4-33.3) weeks of life. Males received the first prescription (20.1; IQR 9.3-32.6) about one week before females (21.3; IQR 9.7-34.0). At the first prescription anti-asthmatics (40.5%), and antibiotics (37.9%) were most used drug classes, and amoxicillin (16.3%), beclomethasone (16.0%), and amoxicillin clavulanate (15.0%) were the most used active substances. In conclusion, in the first year of life, males were more exposed to drugs than females. The data is in agreement with the epidemiology of diseases in neonates. The analysis concerning the influence of perinatal factors is on-going.


66


Multiple abnormalities in the CFHR gene cluster in a patient with DDD.

Rossella Piras1, Bertha Martin2, Elisabetta Valoti1, Nicolò Borsa2, Marta Alberti1, Carla

Nishimura2, Elena Bresin1, Gregorini Gina3, Giuseppe Remuzzi1,4, Richard Smith2, Marina

Noris1.

1 IRCCS ‐ Istituto di Ricerche Farmacologiche “Mario Negri”, Clinical Research Center for Rare Diseases “Aldo e Cele Daccò”, Ranica, Bergamo, Italy.

2 Molecular Otolaryngology and Renal Research Laboratories, Carver College of Medicine, University of Iowa, Iowa City, United States.

3 Department of Nephrology and Dialysis, Azienda Ospedaliera Spedali Civili,

Brescia, Italy. 4 Unit of Nephrology and Dialysis, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy C3 glomerulopathy (C3G) is a rare kidney disease characterized by dominant complement C3 deposition in glomeruli. Uncontrolled complement alternative pathway (AP) activation due to genetic and/or acquired abnormalities plays a main pathogenetic role. By Electron Microscopy findings, C3G is distinguished into Dense Deposit Disease (DDD) and C3 Glomerulonephritis (C3GN). CFH and C3 mutations and genomic rearrangements in CFHRs genes resulting in AP dysregulation have been reported in DDD. We studied a patient who presented at 26 years of age with proteinuria (2 g/day) and low C3 levels (C3=45 mg/dl). Kidney biopsy findings were consistent with DDD. She received 3 kidney grafts (the first 2 lost for relapse). The patient was evaluated, while on dialysis, before the third transplant: she had slight C3 reduction (72.5 mg/dl), normal C4 (23 mg/dl) and sC5b9 (269 ng/ml); C3NeF was negative. Genetic screening by using a NGS diagnostic 6 gene‐panel (CFH, C3, MCP, CFI, CFB and THBD) did not show any mutation. NGS Sequencing by a wider complement and coagulation panel (CasCADE) disclosed two heterozygous stop codons in CFHR2 causing interruption in SCR4 of the protein (p.Q211X and p.R254X). We performed Copy Number Analysis in CFH‐CFHR gene cluster using Multiplex Ligation Dependent Probe Amplification (MLPA), a quantitative multiplex PCR (to target CFHR4 intron 1 and exon 2), and read alignment and quantification on the Integrative Genomics Viewer (IGV). DNAs from healthy individuals with normal Copy Numbers in CFH‐CFHR cluster were analyzed in parallel as controls. Combined data from the above approaches revealed: 1) CFHR1: zero copy; 2) CFHR3: only one copy which however lacks exons 5 and 6; 3) CFHR4: one wild‐type copy and one copy lacking exons 1 to 8. Results would suggest that the patient has an aberrant genomic segment including exons 1,2,3,4 of CFHR3 fused to exons 9,10 of CFHR4. Further studies are necessary to localize the breakpoint, to characterize the novel FHR31,2,3 ‐ FHR48,9 hybrid protein, and to investigate the pathogenetic role of CFHR2 stop codons. Since FHRs are circulating proteins, the abnormalities in this patient were not corrected by kidney transplant, which is consistent with relapsing disease in the grafts. These results confirm the pathogenetic role of CFHRs genomic abnormalities in DDD.


67


NK Cells Recruitment to the Salivary Glands Regulates Early Viral Control but is

Dispensable for the formation of Inducible Tertiary Lymphoid Structures.

E. Pontarini1*, D. Lucchesi2*, L. Fossati-Jimack2, C. Croia2, M. Bombardieri2 and D.

Mavilio1. 1 Clininal and Experimental Immunology, Humanitas Clinical and Research Centre, Milan 2 Experimental Medicine and Rheumatology, Queen Mary University of London, London. * E.P and D.L contributed equally to this work. Natural Killer (NK) cells are a central component of the innate immune system and are crucial in containing and eliminating viral infections. The salivary glands (SG) represent a permissive site for the persistence of several viruses that show specific sialotropism. We investigated the relevance of NK cells in the early phases of the immune response upon Adenovirus (AdV) infection within the SG. In our murine model, intra-SG AdV delivery triggers the formation of inflammatory infiltrates that progressively acquire features of tertiary lymphoid structures (TLS). Herein, we characterized the NK cell response to AdV at glandular mucosal sites in order to investigate whether NK cell-mediated viral control might affect the virus-induced TLS formation. We found that in response to AdV delivery, NK cells were enriched within the SG, displaying an activated phenotype and a functional cytotoxic potential. Peripheral NK cells are recruited within AdV infected SG, undergo proliferation and contribute to the clearance of AdV-infected cells in the early phases of the immune response. Nonetheless, selective depletion of NK cells did not affect the number, organization and functionality of TLS in SG. We conclude that, in the present model NK cells are able to exert an early control of viral infection but are dispensable for the formation of inflammatory aggregates. Moreover, our AdV-induced SG inflammation model is an ideal platform to study the immune response to viral infections within the SG and might lead to a better understanding of the mechanisms underlying virus- induced formation of TLS in this organ.


68


MATERNAL IMMUNE ACTIVATION PROVOKES HIGHER SUSCEPTIBILITY TO

EPILEPSY IN THE OFFSPRING

Marco Rasile2,3, Elisa Focchi1,2, Flavia Antonucci1,2, Elisabetta Menna1,3, Maria Luisa

Malosio1,3, Irene Corradini1,2, Michela Matteoli1,3

1CNR Institute of Neuroscience, Milan, Italy;

2Dept Medical Biotechnology, University of

Milan, Italy; 3Humanitas Clinical and Research Center, Rozzano, Italy;

Synapses are fundamental brain structures that mediate information transfer between neurons and control cognition, attention and learning. Recently, evidence accumulated indicating that immune deregulation or inflammation are linked to several types of psychiatric diseases (Patterson, 2009). Accordingly, perinatal infections have been associated with increased risk for neurodevelopmental disorders (Meyer et al., 2006; Harvey and Boksa, 2012), including schizophrenia (Miller et al., 2013) and depression/anxiety (Enayati et al., 2012). Interestingly, recent evidence was provided demonstrating that prolonged inflammation during pregnancy is associated with increased hippocampal excitability in the offspring in an animal model (Pineda et al., 2013). By using the Poly I:C (polyinosinic-polycytidylic acid) model of inflammation, we addressed the possibility that a single inflammatory event during pregnancy may alter neurodevelopment, leading to neurologic disorders in the offspring. Our results demonstrate that a single injection of Poly I:C at gestation day 9 causes higher susceptibility to kainate-induced seizures in the offspring. This is associated with increased permeability of the Blood-Brain Barrier (BBB), due to alterations in tight junctions, as demonstrated by a reduction of Claudin-5 expression levels.

No changes in the expression levels of synaptic proteins was observed in the offspring of Poly I:C- treated mothers. Electrophysiological recordings of neuronal cultures obtained from mouse embryos from Poly I:C-injected mothers and relative controls showed no defects in the excitation- inhibition balance.

Our results thus open to the possibility that inflammation during early pregnancy may increase susceptibility to epilepsy by interfering with normal brain vascularization and BBB formation.


69


Microbial signals control inflammation and autoimmunity induced by hypomorphic

RAG defects

Rosita Rigoni1,2, Elena Fontana3, Virginia Maina2, Stefano Mantero4, Veronica

Marrella4,2, Graziano Pesole5, J. Rodrigo Mora6, Simone Guglielmetti7, Luigi Poliani3,

Fabio Grassi1,8, Anna Villa4,2 and Barbara Cassani4,2 (1) Department of Medical Biotechnology and Translational Medicine, University of Milan,

Italy

(2) Humanitas Clinical and Research Center, Rozzano, Italy

(3) Department of Molecular and Translational Medicine, Pathology Unit,

University of Brescia

(4) UOS/IRGB, Milan Unit, CNR, Milan, Italy

(5) Department of Bioscienze, Biotecnologie e Biofarmaceutica, University of Bari, Italy

(6) Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School,

Boston, Massachusetts, USA.

(7) Department of Food, Environmental and Nutritional Sciences, University of Milan, Italy

(8) Institute for Research in Biomedicine (IRB), Bellinzona, Switzerland

Hypomorphic mutations in the RAG genes result in profound lymphopenia associated with multisystem autoimmune manifestations in humans and mice. The role of gut homeostasis and microbial signals in the immune dysfunctions and disease pathogenesis is still debated.

The Rag2R229Q/R229Q mutant mice developed an inflammatory bowel disease involving the small intestine, characterized by marked infiltration of CD4+T cells and, intriguingly, also of Treg cells, in the lamina propria compartment. Increased expression of the gut homing receptors CCR9 and α4β7 on peripheral CD4+ T cells confirmed the abnormal lymphocyte trafficking to this environmental interface. A pro-inflammatory profile, characterized by a Th1/Th17 skewing, distinguished the intestinal immune

responses in the Rag2R229Q/R229Q mice. Remarkably, similar pattern was also evident in the periphery. On the contrary, B cells were poorly present in the gut of mutant mice and the faecal level of IgA was obviously reduced. Interestingly, these findings correlated with augmented intestinal permeability and enhanced epithelial innate responses. Metagenomic analysis revealed substantial changes in the composition of the gut microbial communities in the mutant mice, with an overall reduced bacterial biodiversity. Importantly, decreasing microbial load with antibiotic treatment significantly limited the gut lymphocytic infiltration, decreased the frequency of peripheral gut-tropic CCR9+ T cells, and ameliorated both the intestinal and systemic inflammation by dampening pro-inflammatory Th1/Th17 immune responses. Overall, these results suggest that microbial factors play a substantial role in the pathogenesis of human autoimmune disease associated with hypomorphic RAG defects.


70


The role of metal ions in promoting the cardiotoxicity of immunoglobulin light chains

unveils novel therapeutic openings

Margherita Romeo1, Paola Rognoni 2, Claudia Foray3, Giovanni Palladini 2,4, Robert

A. Cherny 5, Vittorio Perfetti6, Fabio Fiordaliso3, Giampaolo Merlini2,4, Mario Salmona 1 Luisa Diomede 1. 1Department of Molecular Biochemistry and Pharmacology, IRCCS-Istituto di Ricerche

Farmacologiche “Mario Negri”, Milan, Italy; 2Amyloid Research and Treatment Center,

Foundation IRCCS Policlinico San Matteo, Pavia, Italy; 3Bio-imaging Unit, Department of

Cardiovascular Research, IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Milan,

Italy; 4Department of Molecular Medicine, University of Pavia, Italy; 5The Florey Institute of

Neuroscience and Mental Health, Kenneth Myer Bldg, The University of Melbourne, Royal Pde,

Parkville, VIC, Australia Prana Biotechnology Ltd., 369 Royal Pde, Parkville, VIC, Australia;

6Medical Oncology Unit, Foundation IRCCS Policlinico San Matteo, Pavia, Italy.

Immunoglobulin light chain amyloidosis (AL), the most common form of systemic amyloidosis, are caused by the proteotoxic effects of aberrant, misfolded immunoglobulin light chains (LC) produced by a bone marrow plasma cell clone. An heterogeneous picture related to the organs most clinically affected by amyloid deposition characterizes this disease. In most cases, patients experience rapid worsening of cardiac failure because are too fragile to tolerate aggressive chemotherapy. A better understanding of the molecular and biochemical mechanisms underlying the disease process, particularly those guiding the cardiotoxicity of LC, is crucial to design innovative therapeutic strategies and improve patients’ outcome. . Efficacious and well- tolerated therapies for patients suffering from cardiac AL are needed and a deep knowledge of the mechanism of heart damage may indicate innovative treatment modalities. Using Caenorhabditis elegans as valuable animal model we investigated the pathogenic effects of monoclonal LC in vivo. We show that cardiac amyloid LC, but not to myeloma LC, enhanced reactive oxygen species (ROS) production and activated the FOXO transcription factor DAF-16, heat shock protein, superoxide dismutase as well as pharyngeal mitochondrial damage. Metal chelating compounds 8-hydroxyquinoline and PBT2 interrupted the vicious cycle of oxidative stress blocking the mechanism of toxicity. These observations indicate that ROS are actively involved in LC toxicity and that metal ions represent an innovative, disease-relevant, druggable target in AL.


71


A SMYD3 Small-Molecule Inhibitor Impairing Cancer Cel Growth

Paola Sanese1, Alessia Peserico1,2, Armenio Jorge Barbosa4, Raffaella Fittipaldi5, Edoardo

Fabini6, Carlo Bertucci6, Greta Varchi7, Giuseppina Caretti5, Alberto Del Rio4,7, and Cristiano

Simone1,2,3

1Division of Medical Genetics, DIMO, University of Bari “Aldo Moro”, Policlinico, Piazza

Giulio Cesare 11, 70124 Bari, Italy 2National Cancer Institute, IRCCS Oncologico “Giovanni Paolo II”, 70124 Bari, Italy 3Cancer Genetics Laboratory, IRCCS “S. De Bellis”, Castellana Grotte (BA) 70013,

Italy 4Department of Experimental, Diagnostic and Specialty Medicine (DIMES), Alma

Mater Studiorum University of Bologna, Via S. Giacomo 14, 40126 Bologna, Italy 5Department of Biosciences, University of Milan, 20133 Milan, Italy 6Dipartimento di Farmacia e Biotecnologie, University of Bologna, Via Belmeloro 6,

40126 Bologna, Italy 7Institute of Organic Synthesis and Photoreactivity (ISOF), National Research Council

(CNR), Via P. Gobetti 101, 40129 Bologna, Italy

SMYD3 is a histone lysine methyltransferase that plays an important role in transcriptional activation as a member of an RNA polymerase complex, and its oncogenic role has been described in different cancer types. We studied the expression and activity of SMYD3 in a preclinical model of colorectal cancer (CRC) and found that it is strongly upregulated throughout tumorigenesis both at the mRNA and protein level. Our results also showed that RNAi-mediated SMYD3 ablation impairs CRC cell proliferation indicating that SMYD3 is required for proper cancer cell growth. These data, together with the importance of lysine methyltransferases as a target for drug discovery, prompted us to carry out a virtual screening to identify new SMYD3 inhibitors by testing several candidate small molecules. Here we report that one of these compounds (BCI-121) induces a significant reduction in SMYD3 activity both in vitro and in CRC cells, as suggested by the analysis of global H4K5me and H3K4me2/3 levels. Of note, the extent of cell growth inhibition by BCI-121 was similar to that observed upon SMYD3 genetic ablation. Most of the results described above were obtained in CRC; however, when we extended our observations to tumor cell lines of different origin, we found that SMYD3 inhibitors are also effective in other cancer types, such as lung, pancreatic, prostate, and ovarian. These results represent the proof of principle that SMYD3 is a druggable target and suggest that new compounds capable of inhibiting its activity may prove useful as novel therapeutic agents in cancer treatment.


72


Novel Methods for Total Kidney Volume Computation in Autosomal Dominant Polycystic

Kidney Disease

Kanishka Sharma1,2, Anna Caroli1, Loic Peter2, Christian Rupprecht2, Lichao Wang2,

Nassir Navab2, Maximilian Baust2, Andrea Remuzzi1

1 IRCCS - Istituto di Ricerche Farmacologiche “Mario Negri”, Bergamo, Italy

2 Computer Aided Medical Procedures, Technische Universität München, Germany

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by evolution and expansion of non-uniform fluid filled cysts leading to gradual enlargement of both kidneys, and eventually renal failure. Quantifying total kidney volumes (TKV) is important to study effect of drugs potentially slowing ADPKD progression. To fulfill this aim, efficient image based segmentation methods are necessary. However, irregular cyst formation, adjacent liver cysts exhibiting similar intensities and presence of haemorrhagic cysts makes the segmentation process complex. Current methods such as manual outlining and stereology tend to be strenuous and/or time consuming. We investigated different semi-automatic TKV computation methods potentially improving existing methods. First proposed method, known as livewire, is based on lowest cost path Dijkstra algorithm entailing user interaction with faster kidney segmentation. Results show that this method improves time required for segmentation w.r.t manual outlining (time required: 24 vs 35 min), while maintaining the same segmentation accuracy (Intra- and Inter rater concordance correlation coefficient (CCC) =1; CCC with manual tracing =1). Further, we investigated 3D segmentation method requiring user input only on a single slice. This method is based on geodesic distance volumes introduced to a random forest classifier. The mean dice coefficients for both left and right kidneys are 0.7, indicating good segmentation similarity between proposed methodology w.r.t manual outlines. The above methods can potentially reduce time and effort for volumetric measurements in ADPKD.


73


Characterization of mediators of Aβ oligomers toxicity by genome wide analysis in C.

elegans

Matteo Stravalaci1, Margherita Romeo1, Silvana Pileggi1, Alun Williams2, Luisa Diomede1,

Marco Gobbi1

1IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy

2 Department of Veterinary Medicine, University of Cambridge, Cambridge, UK

Soluble oligomers of the amyloid-β (Aβ) protein are though to play a key role in the pathogenesis of Alzheimer’s disease (AD), the most common neurodegenerative disorder. It is, however, poorly understood how these aggregates are formed and how they can exert their toxicity. In order to search for mediators of Aβ oligomers formation and toxicity we employed an inducible transgenic C. elegans model of AD in which the expression of Aβ oligomers causes complete paralysis of the worm after 48 hours from the induction. Upon chemical mutagenesis we identified different clones with different paralysis kinetics. One of them, called prp-1 (paralysis-rescued-phenotype-1), showed a total lack of paralysis, even at longer times of induction. Notably, this strain did not bear mutations in the Aβ coding region, and accumulates Aβ transcript levels comparable to that of the non-mutated strain. In this study, we described the first part of the project in which we investigated the induction of genes involved in the age-related protein homeostasis of the worm, in particular the insulin/IGF-1-like signalling (IIS) pathway. qRT-PCR analysis showed specific down-regulation of some genes in prp-1 strain, but not in the non mutated strain. Among them, we found two genes belonging to the hsp-16 family that encode for a class of chaperons, which are homologous to αB-Crystallin (CRYAB) in humans. These results suggested that Aβ oligomer toxicity could be mediated through previously unexplored pathways, which could open up new insights for research on age-related, neurodegenerative diseases.


74


MicroRNA-135b: the pivot of the macrophage polarization balance.

N. G. Sukubo 1, M. Pesant 2, M. Locati1,2

1 Department of Medical Biotechnologies and Translational Medicine, University of Milan,

Rozzano (MI), Italy.

2Humanitas Clinical and Research Center, Rozzano (MI), Italy.

Introduction: MiRNAs are non-coding RNAs acting as gene expression regulators at posttranscriptional level. Recently has been show that they are involved in the inflammatory response, but their role in the activation of macrophages due to different stimuli is not well defined. The aim of this work was to typify the miRNome of human polarized macrophages and to define its relevance for the polarization process. Methods and results: Monocyte-derived macrophages were polarized to classic (M1; LPS + INFγ) or alternative (M2; IL-4) cells and 733 miRNAs were profiled using TaqMan MicroRNA Arrays. Out of these miRNAs, 69 resulted differentially expressed among the subset. In particular, MiR-135b was strongly associated with M1 phenotype. Furthermore, its expression profile were intimately linked to macrophage polarity as its expression levels were decreased in M1 re-polarized to M2 and enhanced in M2 re-polarized to M1. In silico analysis identified miR-135b as a potential modulator of the alternative activation pathway through direct targeting of the key transcriptional factors c- MYC, STAT6 and KLF4. These targets were validated by luciferase assay and Western blot analysis. Consistent with these findings, miR-135b overexpression led to a significant reduction in the production of M2-associated chemokines (CCL17, CCL18 and CCL22), promoting polarization to the M1 phenotype. Conclusions: We show that macrophage phenotypes are characterized by differential expression of miRNA profiles and we identified miR-135b as an M1-associated miRNA, targeting the M2- associated transcription factors c-MYC, STAT6 and KLF4, thus suppressing M2 polarization. Altogether these data indicate miR-135b enforces macrophage classical activation.


75


Inhibition of the VEGFR3 pathway as a novel therapy for Colorectal Cancer

Carlotta Tacconi1, Carmen Correale1, Philippe Fonteyne1, Luciana Petti1, Andrea Piontini1,

Federica Ungaro1,2, Marco Genua1, Stefania Vetrano1,3, Silvia D’Alessio1,2 & Silvio

Danese1

1Division of Gastroenterology, Humanitas Clinical and Research Center, Rozzano, Italy

2Department of Medical Biotechnologies and Translational Medicine, University of Milan, Milan,

Italy 3Humanitas University, Rozzano, Milan, Italy

Colorectal Cancer (CRC) is one of the major causes of cancer related mortality. Neo- lymphangiogenesis promotes metastasis to regional lymph nodes, but no mechanistic data is available on the molecules involved in modulating lymphangiogenesis in early CRC stages. VEGFC is a key molecule in lymphangiogenesis that signals through its receptor VEGFR3 whose blockade has been successful in several models of cancer metastasis. Besides lymphangiogenesis, VEGFR3 signaling can modulate macrophage polarization in experimental colitis. We hypothesized that blocking VEGFR3 signaling may be therapeutically effective in CRC. Methods: We addressed these issues in vivo by systemic inhibition of VEGFR3 or delivery of VEGFC in the dextran sulfate sodium (DSS)/azoxymethane (AOM) model of CRC. Tumor density and size were measured at the end of the protocol. Area density of the lymphatic vessels and macrophages were measured within tumoral and peritumoral regions. Moreover, in vitro co-culture experiments of macrophages and T cells were performed. Results: Inhibition of VEGFR3 prevented lymphangiogenesis in tumoral and peritumoral regions and reduced tumor density and size, in comparison with untreated mice. In contrast, tumor density and growth was enhanced by systemic delivery of VEGFC. Interestingly, VEGFC converted macrophages into immunosuppressive cells able to suppress CD8 T cell proliferation in a VEGFR3 dependent manner. Conclusion: Our findings demonstrate that VEGFC/VEGFR3 pathway plays a role not only in metastasis dissemination, but also in early stages of tumor growth by modulating lymphangiogenesis and macrophage polarization. Therefore, inhibition of VEGFR3 may represent a promising target for the prevention and the treatment of CRC.


76


GlucoCEST, In vitro and vivo MRI Imaging of glucose

Daniele TOLOMEO1, Edoardo MICOTTI1, Michael CHAPPELL2, Gianluigi FORLONI1 1 Department of Neuroscience, IRCCS Istituto di Ricerche Farmacologiche “Mario

Negri”, Milano, Italy 2 Institute of Biomedical Engineering, Department of Engineering

Science, University of Oxford, UK

Amyloid-β amyloidosis and alteration of metabolism are the earliest events in the Alzheimer’s disease (AD) pathological cascade. It is well established that brain glucose uptake/metabolism is impaired in AD. Understanding the metabolic changes induced by AD is than a task of major relevance allowing the identification of clinical biomarkers suitable for an early diagnosis. Chemical exchange saturation transfer (CEST) is a new contrast enhancement technique that makes MRI sensitive to the concentrations of endogenous metabolites, like glucose. Although glucose is hardly visible in the physiological proton spectrum, it can be detected indirectly through the water signal using selective radio-frequency saturation of the hydroxyl (OH) protons. The resulting saturated spins are rapidly transferred to water protons leading to partial saturation of the water signal. This method provides a technique to image glucose uptake and metabolism in vivo without the need for isotopic labeling of the molecules.. Experiments will be performed on a 7T small bore animal Scanner (Bruker Biospec, Ettlingen, Germany) equipped with two actively decoupled radio frequency coils: a volume coil of 7.2 cm diameter used as transmitter and a surface coil as receiver. The objective of my phD thesis will be to develop a similar glucoCEST method in order to investigate changes in the metabolism of AD transgenic mice.


77

Abstract #72

Bone marrow as sensor of cancerous transformation

Andrea Tomirotti1, Claudia Chiodoni1, Matteo Dugo2, Claudio Tripodo3, Mario Paolo Colombo1

1. Molecular Immunology Unit, Department of Experimental Oncology and Molecular

Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy

2. Functional Genomics and Bioinformatics, Fondazione IRCCS Istituto Nazionale dei

Tumori, Milan, Italy

3. Tumor Immunology Unit, Department of Health Science, Human Pathology Section,

University of Palermo

Emergency granulopoiesis represents a fast bone marrow (BM) response to peripheral perturbation and, on this line, cancer progression is associated with expansion of myeloid derived suppressor cells (MDSC). We hypothesize that stroma modification in peripheral tissues, where neoplastic transformation is occurring, may be sensed by the BM, which modulates its own stroma and hematopoiesis. This study aims at identifying stromal modifications/rearrangements in the BM of tumor bearing mice by cytofluorimetry and gene expression profile (GEP). FACS analysis on BM of BALB/NeuT transgenic mice (developing spontaneous mammary carcinomas) at 24 weeks of age, when invasive tumors are presents, showed an expansion of myeloid cells (CD11b+) and a contraction of B cells (B220+) in comparison to wild type (wt) mice. The analysis of progenitor cells, showing accumulation of granulocyte-monocyte progenitors (GMP) in NeuT mice, confirmed a boost in myelopoiesis. FACS data are supported by immunohistochemistry analysis in which an increase in neutrophils and in morphologically immature myeloid progenitors is observed in NeuT BM. On the same BM samples we performed GEP analysis: 208 genes (61 up-regulated and 147 down-regulated genes) were found to be significantly modulated. Gene Ontology analysis showed a down-modulation of genes related to lymphocyte differentiation and activation, hemopoiesis, hematopoietic organ development, and more in general, immune responses, and an up-regulation of genes related to inflammatory response. Taking together the results demonstrate profound alterations in the BM environment of tumor-bearing mice that may be further investigated for the identification of new potential biomarkers.


78


The influence of tumour microenvironment in osteosarcoma

Ilaria Torselli1, Lucia Bongiovanni2, Claudio Tripodo2, Mariella Parenza1, Katia Scotlandi3,

Mario P. Colombo1 and Claudia Chiodoni1

1. Molecular Immunology Unit, Department of Experimental Oncology and Molecular

Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy 2. Tumor Immunology Unit, Department of Health Science, Human Pathology Section,

University of Palermo School of Medicine, Palermo, Italy. 3. CRS Development of Molecular Therapies, Laboratory of Experimental Oncology,

Istituto Ortopedico Rizzoli, Bologna, Italy.

Osteosarcoma (OS) is the most common malignant bone tumour with a peak of incidence in children and adolescence, mostly associated with poor prognosis due to high rate of pulmonary metastases. OS is a high-grade stromal tumour thought to derive from osteoblast precursors that do not proceed to terminal differentiation and produce defective osteoid, newly synthesized bone matrix that does not undergo mineralization. SPARC is a matricellular protein known to have a role in bone formation and in neoplastic progression of several tumour histotypes. We have developed transplantable murine OS models, with various differentiation stages and histotypes, highly resembling the human disease. These models have been used to investigate the role of SPARC in OS progression and metastasis when it is produced either by tumour or host cells, using gene silencing of tumour cells and SPARC-deficient mice, respectively. SPARC down-modulation in a well-differentiated OS line enhanced tumour growth, suggesting an inhibitory role for SPARC on cell proliferation; additionally it severely reduces cell differentiation and bone matrix deposition, whereas leukocytes infiltration is increased. Conversely, in a poorly differentiated OS line SPARC down-modulation does not affect either cell proliferation or differentiation. On the other hand, when OS cells are injected in sparc-/- mice, tumour growth is severely compromised. Bone marrow transplantation and in vivo T cell depletion suggest the involvement of an adaptive immune response. Our data indicate that SPARC modulation in tumour and in host cells results in opposite phenotypes caused by the alteration of different functions of the same multifaceted protein.


79


Tumor dependence on HER2 signaling as a player in immune infiltration required for

trastuzumab activity

1Tiziana Triulzi, 1Viola Regondi, 1Luca Forte, 1Gaia Ghedini, 2Maria Luisa Carcangiu, 1Elda Tagliabue. 1Molecular Targeting Unit, Dept. of Experimental Oncology and Molecular Medicine,

2Anatomic Pathology A Unit, Department of Pathology, Fondazione IRCCS Istituto Nazionale

dei Tumori, Milan, Italy

Recent clinical studies have shown that either tumor dependence on HER2 signaling or the presence of high levels of tumor-infiltrating lymphocytes is predictive of trastuzumab sensitivity. Preclinical data suggest that only a fraction of the HER2+ breast carcinoma (BC) cells are truly addicted to the oncogene and that innate and adaptive immune cells contribute substantially to the therapeutic effect of trastuzumab. Based on these data, we aimed to determine whether tumor cells addicted to HER2 guide the development of tumor immune cell infiltration required for trastuzumab cytotoxic activity. Gene expression profile analysis of 53 HER2+ BC patients treated with adjuvant trastuzumab and chemotherapy identified a 41-gene classifier able to predict tumor relapse risk in both adjuvant and neoadjuvant settings. Tumors classified as low-risk presented high ERBB2 mRNA levels, were mainly HER2-enriched based on PAM50 signature and showed significantly enhanced immune cell pathways and T cell infiltration as compared with high-risk samples. Gene-set enrichment analysis identified chemokine and cytokine ligands and receptor pathways enriched in low-risk patients. The high CXCL9 expression in low-risk tumors was validated by immunohistochemistry on FFPE tumor slides. Mean expression levels of relevant chemokines in human and murine HER2+ BC cell lines as analyzed by qRT-PCR were higher in HER2- addicted than non-addicted lines. Analysis of PBMC migration induced by tumor cells showed a higher recruitment of immune cells by HER2-addicted than non-addicted cells in vitro. Overall, our results support the notion that the activated HER2 oncogene induces production of chemokines/cytokines that recruit the immune infiltrate favorable to trastuzumab activity.


80


MECHANISMS OF CROSS TALK BETWEEN THE MOTOR NEURONS AND THE

T-LYMPHOCYTES IN AMYOTROPHIC LATERAL SCLEROSIS

M.C. Trolese1*, G. Nardo1, B. Bosani1,C. Bendotti1

1Molecular Neurobiology Unit; Dept. Neuroscience; IRCCS - Istituto di Ricerche

Farmacologiche Mario Negri, Via La Masa 20156, Milan, Italy. Tel. +39(0)239014206

Mail: [email protected]

Growing evidence suggest a prominent role of the immune system in the pathoprogression of Amyotrophic Lateral Sclerosis (ALS), the most common and fatal adult-onset motor neuron disease.

In the spinal motor neuron (MN) of SOD1G93A mouse model of familial ALS, we found a significant upregulation of the immunoproteasome subunit LMP7, which starts at the presymptomatic stage and increase during disease course. Immunoproteasome recognizes and degrades polyubiquitinated protein substrates to generate fragments that can be loaded by MHC

class-I, and the associated β2-microglobulin (β2m), and exposed for CD8+ T cells recognition.

In this study we examined the MHCI expression at the CNS and PNS of SOD1G93A mice during disease progression. We report that MN and surrounding cells (microglia, olygodendrocytes, but not astrocytes) exhibit the activation of LMP7, MHCI and β2m at very early disease stage. Notably, while LMP7 and β2m were highly expressed in the perikaria of MNs and motor axons, the MHCI immunoreactivity was increased exclusively in the motor axons and neuromuscular junction of

SOD1G93A mice during the disease course. Consistently, we found CD8+ T lymphocytes

infiltrates in the spinal cord, sciatic nerve and muscle of SOD1G93A as demonstrated by CD3 positive immunoreactivity and qRT-PCR, suggesting the interaction of MNs with cytotoxic T cells through MHCI. These data point out that the activation of the adaptive immune system, both at central and peripheral level, may take part in the pathoprogression of ALS. Studies are ongoing to

investigate the beneficial or detrimental effect of this immune response in SOD1G93A mice.


81


The fate of melanoma tumor initiating cells could be determined by immunomodulatory

factors via autocrine/paracrine signaling

Alessandra Tuccitto1,Valeria Beretta1, Marcella Tazzari1, Francesca Rini1, Claudia Miranda1,

Angela Greco1, Mario Santinami2, Licia Rivoltini1, Chiara Castelli1* and Michela Perego1*. *Authors with equal contributions

1Department of Experimental Oncology, Unit of Immunotherapy of Human Tumors, Fondazione

IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

2Melanoma and Sarcoma Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan,

Italy.

Melanoma is a highly heterogeneous tumor, in which the cells could switch from a status of tumor initiating cells (TIC) to a more differentiated one. Recent evidences support a model of dynamic stemness in which stem properties are in part orchestrated by the tumor microenvironment (TME). We focused on the role of cyto/chemokines in shaping TIC isolated directly from tumor specimens of two melanoma patients. We found that TIC grown in vitro as melanospheres had a complex secretory profile as compared to their differentiated counterparts. We tested the ability of cyto/chemokines to influence TIC self-renewal and differentiation. We shown that IL-6, released by differentiated cells, reduced TIC self-renewal and induced TIC differentiation. Conversely, IL-10 strongly promoted TIC self-renewal through paracrine/autocrine action. Neutralization of IL-10 obtained by gene silencing combined with antibody-mediated blocking of the IL-10Rα was required to sensitize melanoma cells to IL-6- induced differentiation. These results provide the first evidence that homeostasis and functional heterogeneity of melanoma could be directly influenced by TME soluble factors, with IL-6 favoring TIC differentiation, and IL-10 supporting TIC self-renewal. Understanding the TME role in modulating melanoma TIC phenotype will be instrumental to identify novel therapeutic targets to obtain long-lasting regression of metastatic melanoma.


82

Abstract #77

How to improve trabectedin efficacy: the identification of “intelligent” drug combinations.

Uboldi Sarah1, Laura Carrassa1, Eugenio Erba1 and Maurizio D’Incalci1

1 IRCCS – Istituto di Ricerche Farmacologiche Mario Negri, Department of Oncolgy, Milan, Italy

The ability of trabectedin to displace chimeric proteins hallmark of translocated soft tissue sarcomas from target genes results in a significant antitumor activity of the drug in these neoplasms. These chimeric proteins act as deregulated oncogenic transcription factors. The observation that in Ewing sarcoma cells trabectedin transiently suppresses several genes regulated by the chimera, could be exploited to make these cells more vulnerable to other drugs. This hypothesis is supported by the finding that in Ewing sarcoma trabectedin causes a transient downregulation of Werner syndrome, RecQ helicase-like (WRN) gene - that is regulated by EWS-FLI1 chimera - with a consequent sensitization of cells to topoisomerase-I inhibitors, an observation confirmed in vivo too. In order to identify possible other unknown synthetic lethal combinations we have set up experiments in Ewing sarcoma cells treated with trabectedin (4h) and then transfected with a siRNA kinase library. The initial results suggested that the downregulation of BLK, CSK, FGFR1 and PDK3 genes induced an enhancement of trabectedin efficacy. Studies are ongoing in order to validate these findings using specific inhibitors both in in vitro and in vivo models. We envision that this approach will lead to novel more effective targeted sequential combinations of trabectedin - used as modulator of gene transcription - with drugs selected based on preclinical demonstration of synergism.


83


Effects of cannabinoid receptor type 2 (CB2) agonist in a mouse model of type 2 diabetic

nephropathy

Sebastian Villa, Carlamaria Zoja IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Centro Anna Maria Astori,

Science and Technology Park Kilometro Rosso, Via Stezzano, 87, Bergamo

Type 2 diabetes is the major cause of progressive chronic kidney disease in the world. Inhibitors of RAS can slow the progression of diabetic nephropathy (DN). However, the effectiveness of RAS blocking agents depends on when treatment is started, and imperfect renoprotection occurs if therapy begins at late disease stage. Novel strategies targeting pathogenetic pathways other than angiotensin II need to be explored for diabetic patients who remain at risk of poor renal outcome. Studies demonstrated the critical involvement of the endocannabinoid/cannabinoid receptor system in diabetes. Activation of the cannabinoid receptor type 2 (CB2) had a protective role in early experimental type 1 diabetes. We investigated the effects of a CB2 agonist given at a phase of overt disease in BTBR ob/ob mice, a model of type 2 DN. BTBR ob/ob mice received from 10 to 21 weeks of age vehicle, CB2 agonist or ACE inhibitor, as standard therapy for comparison. BTBR wild-type mice served as controls. Results are shown in the table:

Groups

Albuminuria (µg/24h) 21wks

Mesangial matrix

expansion (score)

Mesangial sclerosis

%

Podocyte nephrin expression

%

Inflammatory cells in glomeruli (no.)

Glomerular MCP-1 (score)

D+vehicle 245±41** 1.33±0.10** 9.34±2.03** 1.44±0.17** 5.34±1.39** 1.30±0.13**

D+CB2 150±23° 0.91±0.05**° 3.54±0.90° 1.80±0.13°° 2.47±0.36° 0.86±0.16*°

D+ACEi 111±24° 0.90±0.11**°° 3.10±0.73° 1.74±0.20*° 1.96±0.50° 0.90±0.23*

Control 36±8 0.01±0.01 0.02±0.02 2.06±0.27 0.34±0.06 0.14±0.04

Data are mean±SE; *p<0.05, **p<0.01 vs control; °p<0.05,°°p<0.01 vs Diabetes(D)+vehicle Treatment with CB2 agonist reduced progressive albuminuria of BTBR ob/ob mice. The antiproteinuric effect was associated with amelioration of the defective nephrin expression in podocytes of diabetic mice. CB2 agonist limited mesangial matrix expansion and sclerosis. Glomerular infiltration of Mac-2-positive monocytes/machrophages was attenuated by CB2 agonist, at least in part due to the drug’s ability to reduce MCP-1 chemotactic signals. Renoprotective effects of CB2 were similar to those achieved by ACE inhibitor. Treatments did not affect hyperglycemia and dyslipidemia of diabetic mice. These results suggest that CB2 agonism is a potential option to be added to the available therapeutic armamentarium for type 2 DN.


84


Tph2 gene deletion enhances amphetamine-induced hypermotility: effect of 5-HT

restoration and role of striatal noradrenaline release

Villani C, Carli M, Kostoula C, Sacchetti G, Mainolfi P, Anastasia A, Invernizzi RW IRCCS - Istituto di Ricerche Farmacologiche “Mario Negri” Dept. Neuroscience Lab.

Neurochemistry and Behavior;Via G. La Masa 19, 20156 Milano, Italy

Variants of tryptophan hydroxylase-2 (Tph2), the gene encoding the enzyme responsible for the synthesis of brain serotonin (5-HT) has been associated to neuropsychiatric disorders, substance abuse and addiction. This study assessed the effect of Tph2 gene deletion on motor behavior and found that motor activity induced by 2.5 and 5 mg/kg amphetamine was enhanced in

Tph2-/- mice. Using the in vivo microdialysis technique we found that the ability of amphetamine to stimulate noradrenaline (NA) release in the striatum was reduced by about 50%

in Tph2-/- mice while the release of dopamine (DA) was not affected. Tph2 deletion did not affect the release of NA and DA in the prefrontal cortex. The role of endogenous 5-HT in enhancing the effect of amphetamine was confirmed showing that treatment with the 5-HT precursor 5-hydroxytryptophan (10 mg/kg) restored tissue and extracellular levels of brain 5-HT

and the effects of amphetamine on striatal NA release and motor activity in Tph2-/- mice. Treatment with the NA precursor dihydroxyphenylserine (400 mg/kg) was sufficient to restore

the effect of amphetamine on striatal NA release and motor activity in Tph2-/- mice. These findings indicate that amphetamine-induced hyperactivity is attenuated by endogenous 5-HT

through the inhibition of striatal NA release. Tph2-/- mice may be a useful preclinical model to assess the role of 5-HT-dependent mechanisms in psychostimulants action.


85

PARTICIPANTS INDEX: Affatato R., p. 6 Arrighetti N., p. 7 Baderna D., p. 8 Benedetti V., p. 9 Bettoni S., p. 10 Biggi S., p. 11 Bizzaro F., p. 12 Bosani B., p. 13 Bottai G., p. 14 Brizi V., p. 15 Caiola E., p. 16 Calcaterra F., p. 17 Castagnoli L., p. 18 Castino G., p. 19 Castioni N., p. 20 Celestini V., p. 21 Ceribelli A., p. 22 Cetti E., p. 23 Chiereghin C., p. 24 Ciceri S., p. 25 Climent M., p. 26 Cortese N., p. 27 Dassano A., p. 28 Desiato G., p. 29 D'Ippolito E., p. 30 Dugo M., p. 31 El Bezawy R., p. 32 Fernandes de Oliveira L., p. 33 Filomena M.C., p. 34 Fina E., p. 35 Finazzi S., p. 36 Franzoni M., p. 37 Frigerio F., p. 38 Fumagalli F., p. 39 Generali E., p. 40 Ghirardini E., p. 41 Giussani G., p. 42 Greco C., p. 43 Iatropoulos P., p. 44 Iori V., p. 45 Kamal M.A., p. 46 Kostoula C., p. 47 Magni M., p. 48 Magni S., p. 49 Maiorana M.V., p. 50 Majorini M.T., p. 51 Manganelli S., p. 52 Mantovani C., p. 53


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Marelli G., p. 54 Marzo S., p. 55 Mastrototaro G., p. 56 Molgora M., p. 57 Moro F., p. 58 Nicoletti R., p. 59 Novelli R., p. 60 Palagano E., p. 61 Papa S., p. 62 Parente R., p. 63 Pinessi D., p. 64 Piovani D., p. 65 Piras R., p. 66 Pontarini E., p. 67 Rasile M., p. 68 Rigoni R., p. 69 Romeo M., p. 70 Sanese P., p. 71 Sharma K., p. 72 Stravalaci M., p. 73 Sukubo N.G., p. 74 Tacconi C., p. 75 Tolomeo D., p. 76 Tomirotti A., p. 77 Torselli I., p. 78 Triulzi T., p. 79 Trolese M.C., p. 80 Tuccitto A., p. 81 Uboldi S., p. 82 Villa S., p. 83 Villani C., p. 84

abstract book phd meeting 2015 - open university · [phd students meeting] [2015 ] 1 ... emanuela...

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