biometra - unimi.it · giovanni francesco castino 1*, nina cortese 1, fabio grizzi , marco erreni ,...

workshop

BIOMETRA

September 26th, 2016

Aula Magna - LITA, Segrate

Book of Abstracts

Workshop BIOMETRA 2016 Program

2

Program

9.30-9.45 Welcome and introduction

9.45-10.45

SESSION 1: Experimental Oncology & Genomics

G. Castino

"PD1+ Tumor infiltrating T lymphocytes accumulate in highly metabolic tumors

in pancreatic adenocarcinoma"

C. Vigo

"Feasibility of lifestyle interventions in breast cancer survivors"

L. Abdel Hadi

"Multicellular origin and cancer-promoting effects of Sphingosine-1-phosphate in

human glioblastoma"

L. Ferrari

"Molecular mechanisms involved in variable expressivity of RASopathies:

dissecting functional and genetic features of Noonan syndrome and

Neurofibromatosis type 1"

E. Palagano

"Atypical mutations in the diagnosis of human osteopetrosis: an additional layer

of complexity"

10.45-11.30

SESSION 2: Cell Biology

D. Schiumarini

"Role of the plasma membrane sphingolipid composition in inflammatory response

to Pseudomonas Aeruginosa infection in Cystic Fibrosis"

E. Borroni

"New perspectives on the biological function of the Atypical Chemokine Receptor

2 revealed by SILAC-based phosphoproteomic mapping of its signaling

properties"

C. Vanetti

"Sex-dependent properties of male and female human umbilical vein endothelial

cells (HUVECs): focus on eNOS"

M. Samarani

"Lysosomal dysfunction leads to cell damage altering the plasma membrane

sphingolipid composition"


3

11.30-11.45 Coffee break

11.45-12.45

SESSION 3: Methods and Technologies for Biomedical Science

F. Giavazzi

"Endocytic re-awakening of motility of jammed epithelial"

R. Lanfranco

"Rapid analytical methods for environmental monitoring based on invisible

plastics"

A. Gamba

"Why the same disease can be originated by different genes? Protein complexes as

cause of locus heterogeneity"

L. Ponzoni

"Chronic Nicotine exposure through electronic or standard cigarettes affects the

rewarding properties of Δ-9-tetrahydrocannabinol (THC)"

F. Calcaterra

"MSF (Migration Stimulating Factor)-driven lymphatic differentiation of

endothelial colony-forming cells isolated from adult peripheral blood"

12.45-14.00 Lunch

14.00-15.00

INVITED LECTURE

Matteo Caleo (CNR Neuroscience Institute, Pisa)

"Plasticizing the injured brain: new approaches to stimulate functional recovery

after stroke"

15.00-16.15

SESSION 4: Neuroscience

S. Grassi

"Identification of the antigen recognized by rHIgM22, a remyelination-promoting

human monoclonal antibody"

A. Frasca

"Neural stem cell-based therapy for Rett syndrome"

S. Di Lascio

"Study of the role of lncRNAs in the pathogenesis of Congenital Central

Hypoventilation Syndrome"


4

E. Vezzoli

"Effects of lactate deficiency in the formation of long-term memory and on the

structure of excitatory synapses in mice hippocampus"

L. Pizzamiglio

"New role of ATM in hippocampal neurons during development"

M. Busnelli

"Bivalent ligands boost G-protein coupling of dimeric Oxytocin receptors and

promote social behaviors"

16.15-16.30 Coffee break

16.30-17.30

SESSION 5: Immunology and Immunobiology

E. Rottoli

"Characterization of signaling and metabolic reprogramming in T cells by

purinergic P2X7 receptor"

R. Parente

"Crosstalk between the long pentraxin PTX3 and the complement system in the

immune response to Aspergillus Fumigatus"

E. Pontarini

"NK cells recruitment to the salivary glands regulates early viral control but is

dispensable for the formation of inducible tertiary lymphoid structures"

L. Drufuca

"Multistep regulation of Toll Like Receptor 4 signaling by IL-10-dependent

microRNAs"

E. Focchi

"Prenatal exposure to Poly I:C increases susceptibility to epilepsy in the adult

offspring"

17.30-18:00 Best talk award

Workshop BIOMETRA 2016

5

Oral presentations

Workshop BIOMETRA 2016 Invited lecture

6

INVITED LECTURE

PLASTICIZING THE INJURED BRAIN: NEW APPROACHES TO STIMULATE

FUNCTIONAL RECOVERY AFTER STROKE

Matteo Caleo

CNR Neuroscience Institute, Pisa; email: [email protected]

Ischemic injuries within the motor cortex results in functional deficits that profoundly impact

activities of daily living in patients. Current rehabilitation protocols achieve only limited

recovery of motor abilities. The brain reorganizes spontaneously after injury, and it is

believed that appropriately boosting these neuroplastic processes may restore function via

recruitment of spared areas and pathways. In this presentation, I will describe our recent work

on robot-assisted motor training to rehabilitate motor function in a mouse model of focal

stroke. Second, I will report on the role of GABAergic inhibition in limiting motor

improvements after cortical stroke. Finally, I will present experimental approaches in which

robotic therapy is coupled with delivery of plasticising drugs that render the remaining,

undamaged pathways more sensitive to experience-dependent modifications. These

combinatorial strategies hold promise for the definition of more effective rehabilitation

paradigms that can be translated into clinical practice.

Workshop BIOMETRA 2016 Experimental oncology & Genomics

7

PD1+ TUMOR INFILTRATING T LYMPHOCYTES ACCUMULATE IN HIGHLY

METABOLIC TUMORS IN PANCREATIC ADENOCARCINOMA

Giovanni Francesco Castino1*

, Nina Cortese1, Fabio Grizzi

1, Marco Erreni

1, Daoud Rahal

2,

Giovanni Capretti3, Cristina Ridolfi

3, Francesca Gavazzi

3, Paola Spaggiari

2, Massimo

Roncalli2, Alessandro Zerbi

3, Massimo Locati

1,4, Federica Marchesi

1,4

1Dipartimento di Immunologia e Infiammazione, Humanitas Clinical and Research Center,

Rozzano, Italy 2Dipartimento di Anatomia Patologica, Humanitas Clinical and Research Center, Rozzano,

Italy 3Sezione di Chirurgia Pancreatica, Dipartimento di Chirurgia, Humanitas Clinical and

Research Center, Rozzano, Italy 4BIOMETRA, Università degli Studi di Milano, Milan, Italy

*e-mail: [email protected]

The pathways that regulate immune cell function and metabolism are tightly linked and in

many pathologic states, including cancer, metabolic dysfunction can severely impact on the

efficacy of the immune response. On these premises, here we investigated the association of

tumor metabolic activity and immune infiltration in Pancreatic Ductal Adenocarcinoma

(PDAC), a microenvironment characterized by a strong immunosuppression.

We performed immunohistochemical evaluation of metabolic and immune infiltrate markers

in paraffin-embedded tissue specimens from 40 PDAC patients, surgically operated at

Humanitas Clinical and Research Center. The growth and metabolic activity of PDAC cells

in vivo was analyzed by optical 3D tomography and the immune infiltrate by multicolor flow

cytometry.

In the cohort of patients analyzed, PDAC tumors with high density of GLUT-1 displayed a

significantly higher density of PD-1 positive T cells (PD1-TILs). A similar association

between the tumor metabolic state and PD1-TIL infiltration was confirmed in preclinical

models of PDAC, in which highly glycolytic PDAC cells injected orthotopically in the

pancreas recruited a higher amount of PD1+ effector cells compared to low glycolytic PDAC

cells.

Both in human and preclinical models of PDAC, GLUT-1 expression closely associated to

the density of PD1-TILs, suggesting a reciprocal regulation of glucose metabolism, T cell

recruitment and activation.

Keywords: immunology, cancer, metabolism


8

FEASIBILITY OF LIFESTYLE INTERVENTIONS IN BREAST CANCER

SURVIVORS

Chiara Vigo1,2

, Mara Malacarne1,2

, Fabio Tosi1,2

, Roberto Sala1,2

, Daniela Lucini1,2

1BIOMETRA, Università degli Studi di Milano, Milan, Italy

2Exercise Medicine and Functional Conditions Section, Humanitas Clinical and Research

Center, Rozzano, Italy

Ample epidemiological data show that behavioral cardiovascular risk factors, such as

sedentariness and overweight or obesity are higher in Breast Cancer Survivors (hereupon,

Patients) as compared to the normal population. Numerous investigations show also that

changing lifestyle might reduce cardiometabolic risk profile and, concurrently, recurrences in

Patients. In this investigation we evaluated the feasibility of an individual dietary and

physical activity educational intervention program performed during standard medical

examination in Patients.

Methods

In this feasibility study we examined 72 Patients (all females, age 51± 9 yrs) attending our

Exercise Medicine unit. Patients at the initial medical examination received an individual

dietary and physical activity program, inclusive of a follow up appointment. The first

encounter lasted nominally 45 min, while follow ups lasted 30 min. At every encounter

patients received a physical examination and were interviewed on adherence to the program

(outcomes bodyweight, waist circumference and self reported METs/min/week).

Results

Weight loss was observed in 77.8% at the first follow-up (after about 40 days). Average

peak reduction was 4.2 ± 4.1 Kg ( corresponding to Δ% of 5.8 ±5.5, p < 0.001). Their waist

circumference was reduced by 3.9 ± 5.7 cm (p < 0.001). Simultaneously physical activity

increased from 230.56 ± 420.34 to 732.86 ± 657.67 METS/min/week (p<0.001). At the end

of the ongoing intervention (after 4±2 encounters in 7±6 months) 67.9 % of patients

maintained a significant reduction in weight (Δ% 6.9±4.8).

Conclusions

Our data show the feasibility of introducing an individual dietary and physical activity

educational intervention program into the routine of an outpatient life style clinic. This

intervention obtained significant improvements in elements of cardiometabolic risk and could

represent a simple and low cost approach to optimize cardiometabolic risk profile in breast

cancer survivors.

Keywords: breast cancer survivor, cardiometabolic risk, lifestyle program, exercise

medicine, weight management


9

MULTICELLULAR ORIGIN AND CANCER-PROMOTING EFFECTS OF

SPHINGOSINE-1-PHOSPHATE IN HUMAN GLIOBLASTOMA

Loubna Abdel Hadi

1*, Cristina Tringali

1 and Laura Riboni

1



Glioblastoma (GBM) is the most frequent and lethal brain tumor, and is characterized by not

only the presence of cancer cells but also a considerable amount of parenchymal cells.

Among them, microglia and endothelial cells are recognized as crucial for both tumor growth

and spread. However, the signals regulating the interplay between different cells in the GBM

niche are little known. The sphingolipid metabolite sphingosine-1-phosphate (S1P) has

emerged as a crucial factor in promoting GBM growth, invasion, and drug-resistance,

through interaction with its specific receptors. Notwithstanding, its cellular origin in the

GBM niche remains only partially known. In this study we investigated the capacity of

microglia and endothelial cells of the GBM niche to act as source and/or target of S1P. We

found that different cells of the GBM microenvironment, including GBM-derived tumor

cells, stem cells, and endothelial cells, as well as microglia are all able to rapidly synthesize

and secrete S1P. Among different cell types, GBM stem cells and GBM-derived endothelial

cells were found to be particularly effective in releasing newly synthesized S1P

extracellularly. Further experiments revealed that after co-culture, GBM and parenchymal

cells exhibit enhanced expression of S1P receptors, and of sphingosine kinase (leading to

increased S1P secretion), respectively. In addition, we found that extracellular S1P is able to

induce multiple effects on different cells, by promoting growth, stemness and survival of

tumor cells, migration and vasculogenesis of endothelial cells, and inflammatory properties

of microglia. In conclusion, our data demonstrate that different cell types of the GBM niche

and their cross-talk contribute to the S1P enrichment of the GBM microenvironment, where

S1P prompts multiple processes which favor GBM progression and malignancy.

Keywords: GBM, GBM niche, sphingolipid metabolism, S1P


10

MOLECULAR MECHANISMS INVOLVED IN VARIABLE EXPRESSIVITY OF

RASOPATHIES: DISSECTING FUNCTIONAL AND GENETIC FEATURES OF

NOONAN SYNDROME AND NEUROFIBROMATOSIS TYPE 1

Luca Ferrari1*

, Cristina Battaglia1 and Paola Riva

1



Introduction: RASopathies are a class of developmental disorders caused by germline

mutation in several genes encoding proteins for Ras/MAPK pathway. They share phenotypic

features that include postnatal reduced growth, facial dysmorphisms, cardiac defects, mental

retardation, skin defects, musculo-skeletal defects, short stature and cryptorchidism. Among

them, the two most frequent syndromes are Neurofibromatosis type 1 (NF1; 1:2000-5000)

and Noonan Syndrome (NS; 1:1000-2500). Despite the allele and/or locus heterogeneity and

a certain genotype/phenotype correlation, the RASopathies are characterized by substantial

variable expressivity and incomplete penetrance and 10-20% of patients remains without

molecular diagnosis.

Aims: the long lasting interest in this research field has allowed us to develop suitable

approaches for genetic diagnosis of RASopathies with the aim of identifying both new

pathogenic variants and new causative genes. Moreover, we aim to identify the molecular

mechanisms affecting penetrance and expressivity of specific traits. At this purpose we are

investigating the presence of Expression Quantitative Trait Loci elements and the effects of

Differential Allelic Expression of causative genes in NS and the presence of possible

modifier genes and/or genomic rearrangements in a selected cohort of NF1 patients with

peculiar phenotype.

Methods: both diagnosis and research projects are developed by using conventional

molecular genetics methods such as PCR and Sanger sequencing, by Next Generation

Targeted Resequencing, by RNA sequencing and by CGH+SNP array.

Results: during these years we have identified several new mutations affecting known

Rasopathies genes and we recently identified also new genes possibly implicated in NS by

taking advantage of NGS. The study of the mechanisms implicated in NS and NF1 genetic

heterogeneity will address a genotype-phenotype correlation and provide new insights on

pathogenesis of these syndromes.

Keywords: RASopathies, variable expressivity, Next Generation Sequencing


11

ATYPICAL MUTATIONS IN THE DIAGNOSIS OF HUMAN OSTEOPETROSIS:

AN ADDITIONAL LAYER OF COMPLEXITY

Eleonora Palagano1,2*

, Lucia Susani2,3

, Ciro Menale

2,3, Paolo Uva

4, Domenico Mavilio

1,2,

Paolo Vezzoni2,3

, Anna Villa2,3

, Cristina Sobacchi2,3


2Humanitas Clinical and Research Institute, Rozzano, Italy

3CNR-IRGB, Milan Unit, Milan, Italy

4CRS4, Science and Technology Park Polaris, Pula, Italy


Human Autosomal Recessive Osteopetrosis (ARO) is a rare skeletal disease characterized by

increased bone density. ARO is genetically and phenotypically heterogeneous, with

mutations identified in at least 7 genes and a range of severity and clinical manifestations. In

the most severe forms, ARO is often lethal if left untreated, and a precise molecular

classification is relevant for the patients’ management. Here we describe the identification of

atypical mutations in two well-known mutated genes in ARO, TCIRG1 and CLCN7.

The first mutations were found in a deep intronic region in the TCIRG1 gene. More in detail,

we identified 4 different intronic single nucleotide changes in intron 15 in 5 patients from 4

unrelated families. These novel mutations were found exactly in the middle of a long intron

(more than 300 bp), far from the canonical splice sites; for this reason, they were missed by a

standard protocol for gene amplification and sequencing, focused on exons and exon-intron

boundaries, and went ignored by exome sequencing. Despite their position, these mutations

were predicted to impact on the splicing process of the neighboring exons. Indeed for two

variants, by cloning and sequencing a number of independent cDNA clones covering exons

14 to 17, we demonstrated a greatly reduced splicing efficiency.

The second particular type of mutations is represented by two synonymous variants in the

TCIRG1 and CLCN7 genes, which we found in two unrelated ARO patients, respectively. In

silico analysis predicted an impact on the splicing process in both cases, and indeed we

confirmed a disruptive effect exploiting the minigene technology, for what pertains to the

variant in the TCIRG1 gene, and through cloning of the RT-PCR product and sequencing of

independent clones, for what pertains to the variant in the CLCN7 gene . By these means we

provided evidence that the synonymous changes investigated were not silent and were

responsible for the disease in these affected individuals. Thus, we highlight the possibility

that at least in some cases ARO is due to synonymous changes, erroneously considered silent.

Keywords: genetics, bone, Next Generation Sequencing, molecular biology, cellular biology

Workshop BIOMETRA 2016 Cell biology

12

ROLE OF THE PLASMA MEMBRANE SPHINGOLIPID COMPOSITION IN

INFLAMMATORY RESPONSE TO PSEUDOMONAS AERUGINOSA INFECTION

IN CYSTIC FIBROSIS

Domitilla Schiumarini1*

, Massimo

Aureli1, Nicoletta Loberto

1, Rosaria Bassi

1, Giulia

Mancini1, Maria Cristina Dechecchi

2, Sandro Sonnino

1


2Laboratory of Molecular Pathology, University Hospital of Verona, Verona, Italy


Cystic fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane

Conductance Regulator gene and is characterized by progressive chronic infection of

airways. Sphingolipids play a regulatory role in the airways inflammation in CF. In

particular, the modulation of enzymes involved in their catabolism such as the plasma

membrane (PM) glucocerebrosidase-GBA2 induces a reduction of IL-8 secretion after P.

aeruginosa (PAO-1) infection. However, it is accepted that, in addition to GBA2, several

enzymes involved in sphingolipids catabolism are associated with PM. These enzymes could

be directly involved in in-situ sphingolipids PM modifications resulting in the activation of

inflammatory response occurring in CF, even if their possible involvement is still unknown.

To address this issue, we investigated the effect of PAO-1 on the sphingolipid composition

and glycohydrolases associated with specialized regions of the cell plasma membrane

involved in the regulation of the signal transduction called lipids rafts.

We isolated lipid rafts from non CF (NuLi-1) and CF (CuFi-1) human bronchial epithelial

cells subjected or not to PAO-1 infection. In CuFi-1 cells PAO1 infection causes an increase

in glucosylceramide and ceramide, and a reduction in GM3. We found also an increased

activity of β-glucocerebrosidase (GBA1), β-galactosidase (β-gal) and β-hexosaminidase (β-

hex). Conversely, no changes were found in the sphingolipid composition of lipid rafts of

NuLi-1 cells. These data suggest that, in CF cells, PAO infection causes a recruitment of PM

glycohydrolases into lipid rafts. The concomitant presence of the enzymes and their

substrates induces local changes of sphingolipids composition and PM conformation that,

together with ceramide formation, is responsible for the activation of the inflammatory

response.

Supported by: Italian Cystic Fibrosis Research Foundation (grant FFC #24/2014)

Keywords: sphingolipids analysis, Cystic Fibrosis, cell signaling, inflammation


13

NEW PERSPECTIVES ON THE BIOLOGICAL FUNCTION OF THE ATYPICAL

CHEMOKINE RECEPTOR 2 REVEALED BY SILAC-BASED

PHOSPHOPROTEOMIC MAPPING OF ITS SIGNALLING PROPERTIES

Alessandro Vacchini1,2

, Cinzia Cancellieri1,2

, Andrea Ferraro2, Armando Negri

3,4, Gabriella

Tedeschi3,4

, Massimo Locati1,2

, Elena Borroni1,2


2Humanitas Clinical and Research Center, Rozzano, Italy

3Department of Veterinary Science and Public Health, University of Milan, Milan, Italy

4Fondazione Filarete, Milan, Italy.

ACKR2 is a prototypic atypical chemokine receptor with an essential role in the control of

inflammation and in the proper development of the adaptive immune responses. As all

ACKRs, it is structurally uncoupled from G proteins and therefore does not activate the

conventional signaling pathways leading to directional migration of expressing cells.

Conversely, we and other have demonstrated that ACKR2 plays a key role in generating

chemokine gradients in tissues by means of its ability to scavenge a broad range of

inflammatory chemokines. We have previously demonstrated that ACKR2 activates β-

arrestin1-dependent signaling pathways which reorganize the actin cytoskeleton and support

the receptor scavenger function. To define the signalling pathways activated downstream

ACKR2, both in constitutive and ligand-stimulated conditions, we carried out a large-scale

mass spec-based quantitative phosphoproteomic analysis by SILAC technique. ACKR2 and

its conventional counterpart CCR5 have been expressed in HEK293T cells using a

tetracycline-inducible system and stimulated with the CCL3L1, an inflammatory chemokine

recognized by both receptors. Using computational approaches, we performed a comparative

system-based analysis of the ACKR2- and CCR5-mediated phosphoproteomes. Gene

Ontology analysis revealed a complex cytoskeletal reorganization signaling network based on

the dynamic interplay between actin and microtubules for both receptors. The analysis also

revealed a unique phosphorylation signature for the two receptors. In particular, ACKR2-

responsive phosphosites were enriched for signaling pathways involved in mRNA

transcription and protein translation networks and in components of the mTOR signaling

cascade, signatures not observed in the CCR5 phosphoproteome. In conclusion, our study

provides the first system-wide mapping of phosphorylation events downstream conventional

and atypical chemokine receptors and suggests the involvement of ACKR2 in unpredicted

complex biologic events.

Keywords: chemokine receptor, signalling


14

SEX-DEPENDENT PROPERTIES OF MALE AND FEMALE HUMAN UMBILICAL

VEIN ENDOTHELIAL CELLS (HUVECs): FOCUS ON eNOS

Claudia Vanetti1*

, Francesco Bifari1, Lucia M. Vicentini

1, Maria Grazia Cattaneo

1



Atherosclerosis and cardiovascular diseases (CVDs) are classical examples of diseases where

sex/gender differences have been described. A significant body of evidence suggests that

CVDs are less prevalent in women than men until midlife, and the female advantage has been

attributed to estrogens, which are lost with menopause.

Since the earliest event in the onset of atherosclerosis and CVDs is endothelial dysfunction

(ED) - a reduced release of nitric oxide (NO) coupled with an increase in Reactive Oxygen

Species (ROS) in the vascular wall - many in vitro studies have been focused on endothelial

cells (ECs). However, the sex of ECs has not been consistently reported in these studies.

To investigate inborn sex differences in ECs, we focused on the role of eNOS and of its

product NO since this gaseous mediator plays a key role not only in CVD onset and

development, but also in angiogenesis, by stimulating EC proliferation, migration and

differentiation. We found that female HUVECs constitutively expressed an higher amount

of eNOS both at mRNA and protein level. Moreover, female HUVECs possess greater

migratory and 3-D spheroid sprouting properties in comparison to male cells. The increased

migratory and angiogenic capabilities observed in female HUVECs were counteracted by the

pretreatment with the NO synthesis inhibitor L-NAME.

These results suggest that the constitutive higher expression of eNOS observed in female

HUVECs might contribute to the protection against CVDs characteristic of the younger

female population. We will carry out further studies on ECs from different sources and ages

to determine if the increase in eNOS expression observed in female HUVECs is preserved

during lifetime and in ECs obtained from different vascular bed.


15

LYSOSOMAL DYSFUNCTION LEADS TO CELL DAMAGE ALTERING THE

PLASMA MEMBRANE SPHINGOLIPID COMPOSITION

Maura Samarani1*

, Nicoletta Loberto1, Simona Prioni

1, Paola Giussani

1, Alessandro Prinetti

1,

Rosaria Bassi1, Massimo Aureli

1, Sandro Sonnino

1



Lysosomal homeostasis is fundamental for cell viability. Alteration in the endolysosomal

function together with the accumulation of uncatabolized molecules are common features of

several diseases. Nevertheless, the molecular mechanism linking lysosomal dysfunction to

the onset of cell damage is still unknown.

To address this issue, we generated an artificial model of lysosomal impairment represented

by cell cultures of human fibroblasts subjected to sucrose loading. We found that sucrose

accumulation within the lysosomes causes the nuclear translocation of the Transcription

Factor EB, a master regulator of lysosomal function, which in turn leads to an increased

lysosomal biogenesis and the consequent accumulation of impaired lysosomes. Since the

sphingolipid catabolism occurs mainly in lysosomes, in sucrose-loaded cells the lysosomal

impairment is associated with an increased content of complex sphingolipids not correctly

hydrolyzed. In addition, we found an augmented fusion of impaired lysosomes with the cell

plasma membrane. This event is responsible for: i) release of undegraded molecules in the

extracellular milieu, ii) increase of sphingolipid-hydrolases at the cell surface, and iii)

enrichment of complex sphingolipids uncatabolized within the lysosomes. In this way, the

coexistence of sphingolipids (substrates) and their catabolic enzymes at the plasma

membrane level results in the ectopic production of ceramide, which in turn leads to the

activation of cell death pathways such as apoptosis and autophagy. These events unveil a new

molecular pathway in which sphingolipids and lysosomes are the main players in the onset of

cell damage.

Keywords: lysosomal dysfunction, sphingolipid metabolism, cell damage, plasma

membrane, neuronal differentiation and senescence

Workshop BIOMETRA 2016 Methods and technologies for biomedical science

16

ENDOCYTIC RE-AWAKENING OF MOTILITY OF JAMMED EPITHELIAL

Fabio Giavazzi1*

, Chiara Malinverno3, Salvatore Corallino

3, Martin Bergert

2, Aldo Ferrari

2,

Giorgio Scita3,4

, Roberto Cerbino1


2Laboratory of Thermodynamics in Emerging Technologies, Zurich, Switzerland

3IFOM-FIRC Institute of Molecular Oncology, Milan, Italy

4Dipartimento di Oncologia e Emato-Oncologia, Università degli Studi di Milano, Milan,

Italy.


Dynamics of epithelial monolayers has recently been interpreted in terms of a jamming or

rigidity transition. How cells control the proximity to that transition is, however, unknown.

Here we show that elevation of RAB5A, a member of a three-genes family that is frequently

hijacked by different epithelial-like tumors to promote their dissemination, is sufficient to

induce large-scale, coordinated motility, ballistic motion and large cell volume fluctuations in

otherwise kinetically-arrested monolayers. Our findings suggest that the increased

fluctuations and reawakening of motility are the result of globally enhanced endosomal

trafficking and macropinocytic internalization. These variations lead to an increase in

junctional tension and traction forces exerted on the substrate and promote the extension of

persistent cell protrusions which align with local velocity. To rationalize our findings, we

propose a simple model where, in addition to cell-cell adhesion and cortical tension, we

consider an active reorientation mechanism for the velocity of self-propelled cells. The model

explains the observed reawakening of RAB5A motility in terms of a combination of large-

scale directed migration and a local unjamming. These changes in multicellular dynamics

allow collectives to migrate under physical constrains and may be exploited by tumors for

interstitial dissemination.

Keywords: endocytosis, collective cell migration, jamming transition, cell-cell adhesion,

active matter


17

RAPID ANALYTICAL METHODS FOR ENVIRONMENTAL MONITORING

BASED ON INVISIBLE PLASTICS

Roberta Lanfranco1*

, Fabio Giavazzi1, Matteo Salina

2, Marco Buscaglia

1


2Proxentia S.r.l., Segrate (MI), Italy


Techniques that enable the identification and recognition of various molecules in a liquid

sample usually require complex instrumentations and qualified personnel to conduct the

experiments and are time consuming. Examples of common approaches are Liquid

Chromatography or Mass Spectrometry. An effective device, easy-to-use and possibly

autonomous, that can discriminate different classes of molecule in real time is of great

interest in various fields, and in particular in environmental monitoring. In this framework,

we propose a novel method to discriminate harmful molecules based on the different

characteristic of adsorption on an isorefractive-to-water fluorinated plastic. This kind of

material was already exploited in the shape of planar surface for the study of various bio-

molecular interactions, as the antibody-antigen binding [1] or DNA hybridization [2]. In this

work, we use the same planar material without any surface treatment to detect polluting

molecules in water, as different surfactants, paraffin and bio-molecules. In this case, the

selectivity is possible because the adsorption process strongly depends on the hydrophobic

moiety of the molecules: both the equilibrium and the kinetics constants are order of

magnitude different for these molecular classes [3]. In order to realize a portable, disposable

and autonomous sensor, we realized different invisible micro-porous plastic media, as micro-

porous membrane and chromatography column, integrated into microfluidic devices. We

tested these substrates for molecular adsorption proving their real applicability.

[1] F. Giavazzi, M. Salina et al, PNAS USA, 110, 15633 (2013)

[2] G. Nava, E. Ceccarello et al. Phys. Chem. Chem. Phys,18, 13395-13402 (2016)

[3] R. Lanfranco et al. PRApplied 5, 054012 (2016)

Keywords: molecular detection, fluorinated materials, environmental monitoring.


18

WHY THE SAME DISEASE CAN BE ORIGINATED BY DIFFERENT GENES?

PROTEIN COMPLEXES AS CAUSE OF LOCUS HETEROGENEITY

Alessio Gamba1*

, Laura Cantù1, Mario Salmona

2, Gianfranco Bazzoni

2


2IRCCS Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy


Complex diseases, including neurodegenerative disorder, are due to mutations in more than

one gene. Hence, it can be useful to study the interactions among the corresponding proteins,

such as those occurring within Protein Complexes (PC). In particular, we hypothesize that

membership of more disease gene proteins (DGP) in the same PC might account for “Locus

Heterogeneity” (LH), a condition whereby different genes (or loci) cause the same

pathology. The aim of this study is to analyze to which extent PC are related to LH.

We derived our data from manually curated databases: OMIM reports diseases, genes and

Phenotypic Series (PS, a list of DGP causing the same disease); HPRD, CORUM and

CHPC2012 databases report PC characterized in human experimental systems.

The first step aims at relating one PS with its associated DGPs and with one or more PC and

consists of generating a set of triplets, each composed of one PS, one DGP and one PC (see

figure). The second step aims at subdividing (by means of a Python script) the triplets into

the following four categories, or outputs. Output 1 is the condition whereby two (or more)

different DGPs (black dots) cause similar diseases (i.e., belonging to the same PS) and are

found in the same PC. The resulting PS-to-PC relations are then prioritized using the Jaccard

index. In addition, outputs 2 and 3 are the conditions whereby different DGPs are found in

the same PS (but in a different PC) and in the same PC (but in a different PS), respectively.

The first output is key to proving our hypothesis of a direct correlation between PS with PC,

whereas outputs 2 and 3 allow integrating our results with information coming from

additional databases (BioGrid and HPO) to look for PC-independent causes of LH.

In conclusion, we have found several instances of LH due to a shared membership of the

relevant DGP into the same PC. Detailed results and representative examples will be

provided in the presentation.

Keywords: protein complexes, protein interaction, molecular medicine, systems

biochemistry


19

CHRONIC NICOTINE EXPOSURE THROUGH ELECTRONIC OR STANDARD

CIGARETTES AFFECTS THE REWARDING PROPERTIES OF Δ9-

TETRAHYDROCANNABINOL (THC)

L. Ponzoni1*

, M. Moretti1,2

, G. Cannazza3, M. Zoli

4, M. Sala

1,2, C. Gotti

2, D. Braida

1


2CNR, Institute of Neuroscience, Milan, Italy

3Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy

4Section of Physiology and Neurosciences, Department of Biomedical, Metabolic and Neural

Sciences, University of Modena and Reggio Emilia, Modena, Italy


Among adolescent tobacco

smokers who also smoke

marijuana, the frequency of

marijuana use is associated

with greater levels of nicotine

addiction (1). Many smokers

have recently switched from

standard to electronic cigarettes

(e-cig) as an alternative to

tobacco despite the contrary

recommendation of the World

Health Organization (2008).

Recently we have validated a

new mouse model of smoke

exposure (2) and the results show that chronic intermittent exposure to e-cig vapour or

tobacco smoke (cig) for 7 weeks has the same effects on nAChR up-regulation, brain nicotine

and cotinine levels. Tobacco smoke led to more severe mecamylamine-precipitated

withdrawal (WDW) and more evident cognitive deficit 24 hours after cig cessation whereas

e-cig vapour elicits more severe anxiety. Based on these results, the aim of the present work

was to test whether e-cig or standard cig exposure could reinforce the subsequent effects of

Δ9-Tetrahydrocannabinol (THC). Thus, 2 or 60 days after exposure, animals were injected

i.p. with a low dose (0.01 mg/kg) of THC or vehicle and submitted to Conditioned Place

Preference task. The second aim was to to correlate the behavioural findings with possible

neurochemical and neurobiological changes (variations in nicotinic and cannabinoid

receptors level or subtypes and changes in lipid neurotransmitters/neuromodulators). Mice

exposed to either e-cig or cig showed a higher sensitivity to THC, compared to control group,

in terms of increased time spent in the drug-associated compartment. The increased

sensitivity persists up to 60 days from nicotine withdrawal. Preliminary results indicate that

CB1 cannabinoid receptor function is not affected at least in the Nucleus Accumbens.

In conclusion, our results show that standard tobacco cigarette and e-cigarette exposure

induce altered response to THC-induced CPP probably through multiple neurotransmitters

involvement.

1. Rubinstein et al. 2014 Drug and Alcohol Dependence 141:159–162

2. Ponzoni et al. 2015 Eur Neuropsycopharmacol 25(10):1775-86

The study was supported by Fondazione Umberto Veronesi and Zardi Gori Foundation

Keywords: behaviour, addiction, electronic cigarette, reward

mailto:[email protected]


20

MSF (MIGRATION STIMULATING FACTOR)-DRIVEN LYMPHATIC

DIFFERENTIATION OF ENDOTHELIAL COLONY-FORMING CELLS

ISOLATED FROM ADULT PERIPHERAL BLOOD

Francesca Calcaterra1,2*

, Claudia Carenza1, Barbara Bottazzi

3, Ilaria Laface

3, Domenico

Mavilio1,2

, Silvia Della Bella1,2


2Laboratory of Clinical and Experimental Immunology, Humanitas Clinical and Research

Center, Rozzano (MI), Italy 3Laboratory of Immunopharmacology, Humanitas Clinical and Research Center, Rozzano

(MI), Italy


Background: Endothelial colony-forming cells (ECFCs) are endothelial progenitor cells

(EPCs) endowed with the unique ability to sustain adult blood vasculogenesis. Lymphatic

EPCs (LEPCs) may similarly sustain lymphovasculogenesis, and their identification would

be relevant to the pathogenesis of pathological lymphangiogenesis, as occurring in cancer

microenvironment. In this context, peritumoral expression of fibronectins is associated with

increased lymphangiogenesis in several human cancers. MSF is an oncofetal fibronectin

overexpressed in the tumor microenvironment, whose possible role in promoting

lymphangiogenesis has never been investigated, so far.

Aims: to investigate 1) the lymphatic differentiative potential of ECFCs; 2) the ability of

MSF to promote ECFC lymphatic differentiation.

Methods: ECFCs were isolated using a protocol we previously optimized. The expression of

lymphatic markers (PROX-1, podoplanin, LYVE-1 and VEGFR-3) and the production of

lymphatic-associated molecules (CCL21 and PTX3) were analyzed in ECFCs either

unstimulated or stimulated with VEGF-C and MSF.

Results: The expression of all the lymphatic markers was detected in basal conditions and

upregulated by VEGF-C and MSF. CCL21 in the supernatants was increased by VEGF-C

and reduced by MSF.

Conclusions: ECFCs may act as putative LEPCs and this finding should be considered when

using ECFCs in regenerative medicine. ECFCs may represent a useful tool for the study of

lymphangiogenesis. The ability of MSF to promote ECFC lymphatic differentiation may

suggest that in the tumor microenvironment MSF may act as a lymphangiogenic factor.

Further studies investigating the mechanisms involved in this effect of MSF may provide

novel insights into the comprehension of tumor lymphangiogenesis.

Keywords: endothelial progenitor cells, lymphangiogenesis, tumor immunology, extra-

cellular matrix

Workshop BIOMETRA 2016 Neuroscience

21

IDENTIFICATION OF THE ANTIGEN RECOGNIZED BY rHIgM22, A

REMYELINATION-PROMOTING HUMAN MONOCLONAL ANTIBODY

Sara Grassi1*, Simona Prioni

1, Yana Zorina

2, Livia Cabitta

1, Sandro Sonnino

1, Alessandro

Prinetti1


2Acorda Therapeutics, Inc., Ardsley, NY, USA


Recombinant human IgM22 (rHIgM22) binds to myelin and to oligodendrocytes, and

promotes remyelination in a mouse model of multiple sclerosis. rHIgM22 preferentially

reacts with sulfatide-positive (O4-positive) oligodendrocytes. Moreover, binding of rHIgM22

is abolished in CNS tissue slices from Cst(-/-) mice, suggesting that its binding to myelin

requires the presence of a product of cerebroside sulfotransferase, possibly sulfatide,

abundantly expressed in oligodendrocytes and myelin. However the exact identity of the

antigen recognized by this antibody remains to be elucidated.

We have tested the binding of rHIgM22 to purified lipids and to lipid extracts prepared from

mouse brain, brain myelin, mixed glial cultures, and O4-positive oligodendrocytes using TLC

immunostaining and SPR using liposomes and lipid monolayers with different composition.

Our preliminary results show that rHIgM22 binds to sulfatide in vitro, while it does not bind

to other myelin sphingolipids, including galactosylceramide and sphingomyelin, suggesting

that sulfatide at the oligodendrocyte surface might be important for the binding of rHIgM22

to the surface of these cells and to myelin. However, rHIgM22 does not bind structures

expressing sulfatide outside the nervous system, thus additional factors are likely relevant for

the immunoreactivity of rHIgM22 in CNS. Indeed, we have observed in lipid extracts from

different sources another lipid molecule selectively recognized by rHIgM22, whose identity

is still under investigation. Remarkably, this lipid is also present in the extracts from mixed

glial cultures, which do not contain mature O4-positive oligodendrocytes, suggesting that

other glial cells in addition to oligodendrocytes might be important in the response to

rHIgM22.

Keywords: Multiple Sclerosis, sulfatide, sphingolipids


22

NEURAL STEM CELL-BASED THERAPY FOR RETT SYNDROME

Angelisa Frasca1*

, Silvia Ferrari1, Francesco Bedogni

2, Nicoletta Landsberger

1


2San Raffaele Rett Research Unit, Division of Neuroscience, San Raffaele Scientific Institute,

Milan, Italy


Astrocytes, besides neurons, are nowadays considered active and integral components of Rett

syndrome (RTT), a neurodevelopmental disorder caused for 95% of cases by mutation in

Mecp2 gene. A functional role of astrocytes in the pathology is supported by the in vitro

evidence that Mecp2 null astrocytes and their conditional medium fail to support normal

neuronal growth of both wild-type (WT) and mutant neurons, suggesting that the loss of

Mecp2 from astrocytes negatively influences neurons in a non-cell autonomous manner. Re-

expression of Mecp2 in astrocytes in null mice mainly stabilizes symptoms. Neural stem cell

(NSC) therapy can be used to promote neuroprotection. When transplanted into a diseased

CNS, NSCs migrate toward and engraft within injured areas and adapt their fate and

functions to specific environmental needs. They might differentiate to neurons, astrocytes

and oligodendrocytes or they might maintain a stem cell phenotype and promote

neuroprotection by secreting a plethora of molecules, by the so-called bystander effect. We

postulated that NSC can partially compensate the pathological astrocytic failure in RTT, by

differentiating to astrocytes and/or exerting a bystander effect.

We used a co-culture system between NSCs seeded on transwell inserts, and WT and Mecp2

null neurons, prepared from E15 cortexes after in utero electroporation at E13 of a plasmid

driving green fluorescent protein (GFP). Transwell inserts with attached NSCs were

transferred above neurons and co-culture was maintained from DIV1 up to the morphological

analysis. Results indicate that NSCs promoted dendritic branching maturation in Mecp2 null

neurons at DIV4, while at DIV8, they induced a recovery in nuclear area and slightly

improved dendritic morphology of null neurons. These results inform about the intrinsic

capability of NSCs to improve morphological phenotype of Mecp2 null neurons and

encourage a preclinical study in RTT animal models.

Keywords: Rett syndrome, astrocytes, stem cells


23

STUDY OF THE ROLE OF lncRNAs IN THE PATHOGENESIS OF CONGENITAL

CENTRAL HYPOVENTILATION SYNDROME

Simona Di Lascio1*, Roberta Benfante

1,2, Diego Fornasari

1


2CNR, Neuroscience Institute, Milan, Italy


Heterozygous mutations in the coding region of PHOX2B gene are associated with

Congenital Central Hypoventilation Syndrome (CCHS), a rare disorder characterized by a

broad variety of symptoms of autonomic nervous system dysfunction including inadequate

control of breathing. PHOX2B is a transcription factor that plays a crucial role in autonomic

nervous system development. In vivo and in vitro studies suggest that a loss of function

mechanism, combined with a dominant-negative effect and/or toxic gain of function of the

mutated proteins, is responsible for the entire disease spectrum. We have recently reported

that several mutant proteins interfere with the transcriptional activity of the wild-type protein

in a promoter-specific manner. Of note, we showed that PHOX2B is capable of controlling

its own transcription by binding its own promoter, and PHOX2B mutants can negatively

interfere with the expression of the normal allele, thus further reducing the amount of normal

PHOX2B protein. Nevertheless, so far, very few studies aimed at understanding how

PHOX2B gene expression is regulated. Many studies are showing that long noncoding RNAs

(lncRNAs) play important and diverse roles in gene expression regulation and several

lncRNAs have been implicated in the molecular pathogenesis of some neuro-developmental

disorders. Bioinformatics analyses of the PHOX2B locus region predict an antisense

transcript that partially overlaps the first exon of PHOX2B. Natural antisense transcripts

(NATs), a class of >200 nucleotide long non-coding RNAs (lncRNAs), are RNA molecules

that are transcribed from the opposite DNA strand with respect to sense transcripts, partially

overlap sense RNAs, and positively or negatively regulate gene expression at different levels

by means of multiple mechanisms. Our data provide evidence of the expression of an

antisense transcript that negatively influences PHOX2B gene expression, and suggest that

PHOX2B mutations alter antisense transcription.

Keywords: molecular biology, transcriptional regulation, long noncoding RNAs, autonomic

nervous system, sympathetic neuron


24

EFFECTS OF LACTATE DEFICENCY IN THE FORMATION OF LONG-TERM

MEMORY AND ON THE STRUCTURE OF EXCITATORY SYNAPSES IN MICE

HIPPOCAMPUS

Elena Vezzoli1,2*

, Luisa Ponzoni1, Norma Lattuada

1, Corrado Calì

3, Daniela Braida

1, Paola

Viani1, Pierre J. Magistretti

3, Andrea Falqui

3, Mariaelvina Sala

4, Maura Francolini

1


2Department of Pharmacological and Biomolecular Sciences, Università degli Studi di

Milano, Milan, Italy 3King Abdullah University of Science and Technology (KAUST), Biological and

Environmental Sciences and Engineering Division (BESE), Thuwal, Kingdom of Saudi

Arabia 4National Research Council (CNR), Institute of Neuroscience, Milan, Italy


The inactivation of

glycogen metabolism,

and the consequent

decrease of lactate

production in

hippocampal astrocytes,

lead to an impairment of

long-term memory and

defects in long-term

potentiation (LTP). These

effects were rescued by

L-lactate, that is an

energy source for

neurons and it is crucial

to mediate astrocyte-

neuron functional

connections (Suzuki et

al., 2011). As LTP

defects are associated with alterations in dendritic spines morphology and density, we studied

if anatomical differences in hippocampal excitatory synapses were associated to defects in

long term memory in mice treated with the inhibitor of glycogen phosphorylase, 1,4-dideoxy-

1,4-imino-D-arabinitol (DAB) and if these defects were rescued by L-lactate administration.

We evaluated the effect of DAB administration on short and long-term memory, with two

memory paradigms (novel object recognition (NOR) and passive avoidance (PA)) and we

demonstrated that while DAB did not affect long-term memory evaluated with the PA test,

the treatment impaired both short and long-term episodic memory evaluated with the NOR.

L-lactate treatment reversed DAB-induced memory impairment. Regardless of the memory

paradigm used, 24 hours after treatment and training, hippocampal neurons from mice treated

with DAB, showed a marked reduction in dendritic spine density compared to mice injected

with vehicle. Through Serial-Block Face Scanning Electron Microscopy (SBFSEM) and

volume reconstruction, powerful techniques to study large volume of tissue at ultrastructural

level, we studied the role of the astrocyte-neuron lactate shuttle on the 3D architecture and

number of excitatory synapses through its inhibition with DAB and the combined effects

DAB and Lactate.

Keywords: lactate, short-long term memory, serial-block face scanning electron microscopy


25

NEW ROLE OF ATM IN HIPPOCAMPAL NEURONS DURING DEVELOPMENT

Lara Pizzamiglio1*

, Elisa Focchi1,3

, Luca Murru

2, Matteo Tamborini

3, Maria Passafaro

2,

Elisabetta Menna2,3

, Michela Matteoli2,3

and Flavia Antonucci1,2


2Institute of Neuroscience, C.N.R., Milan, Italy

3Humanitas Clinical and Research Center, IRCCS Rozzano, Italy


ATM (Ataxia Telangiectasia mutated) is a serine/threonine protein kinase linked to DNA

damage response. Despite its activity in dividing cell has been largely investigated, a high

number of evidences are demonstrating new roles of ATM in adult neurons, such as its

involvement in adult neurogenesis, its crucial function in oxidative stress response and its

interaction with two synaptic vesicle proteins, VAMP2 and synapsin-I. Coherently, the

neurodegenerative condition associated to genetic mutations in Atm gene, the Ataxia

Telangiectasia (A-T), exhibits a variable phenotype with impairment in cognition. Thus,

ATM may impact brain functions through distinct mechanisms, in different brain regions and

cell populations. To directly address these new and unclear aspects of ATM in neurons, we

prepared hippocampal neuronal cultures starting from ATM heterozygous mice. Here we

found a significant excitatory/inhibitory unbalance toward inhibition as indicated by the

higher frequency of miniature inhibitory postsynaptic current events, an increased number of

GABAergic synapses and a more precocious development of inhibitory system (GABA

switch). In vivo, the enhanced inhibition still persists and, even if a higher excitation is also

present, a reduced neuronal excitability is shown as indicated by the lower action potential

frequency generated in response to high-current intensity stimuli. Finally, we found an

elevated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in heterozygous

hippocampi associated with lower expression levels of PP1 phosphatase. These data unveils

an unexpected role of ATM in the maintenance of the appropriate GABAergic development

and transmission in hippocampal formation, laying the basis for a more clear comprehension

of cognitive defects occurring in A-T and opening to novel therapeutic strategies.

Keywords: electrophysiology, live imaging, biochemistry, cellular biology



26

BIVALENT LIGANDS BOOST G-PROTEIN COUPLING OF DIMERIC OXYTOCIN

RECEPTORS AND PROMOTE SOCIAL BEHAVIORS

Marta Busnelli1,2*

, Gunnar Kleinau3, Markus Muttenthaler

4,5, Stoytcho Stoev

6, Maurice

Manning6, Lucka Bibic

7, Lesley A. Howell

7, Peter J. McCormick

7 , Simona Di Lascio

2,

Daniela Braida2, Mariaelvina Sala

1,2, G.Enrico Rovati

8, Tommaso Bellini

2, Bice Chini

1



3Institute of Experimental Pediatric Endocrinology, Charité-Universitätsmedizin Berlin,

Germany 4Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

5Department of Biochemistry and Cancer Biology, University of Toledo, Toledo, OH USA

6School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich, UK

7Department of Pharmacological and Biomolecular Sciences, Università degli Studi di

Milano, Milan, Italy


Oxytocin is a hypothalamic neuropeptide that regulates a number of socio-emotional

behaviors, through the activation of a specific G-protein coupled receptor (OXTR) which

can exist as monomers and dimers.

To identify and target specific dimeric conformations of the OXTR we designed bivalent

ligands consisting of two identical oxytocin-like ligands connected by carboxylic spacers of

different lengths. We probed this bivalent series using a BRET G-protein activation assay,

receptor mutagenesis and interference experiments with synthetic peptides that mimic

transmembrane helices. Biphasic activation responses provided evidence for the presence of a

dimeric receptor arrangement in which only two bivalents, with defined spacer length, fit

within the channel-like structure that connects the two protomers of the dimer. These

bivalents induced a three order magnitude boost in G-protein signaling in vitro, and a 100-

and 40-fold gain in potency in vivo in the social behavior of mice and zebrafish as compared

to the monovalent and endogenous peptides control groups.

Therefore, OXT bivalent ligands are very promising as a new tool for targeting dimeric

OXTR in neurodevelopmental and psychiatric disorders and, in general to untangle the

functional activity of G-protein coupled receptor dimers.

Keywords: oxytocin receptor, GPCR dimer, drug-design, social behavior, animal models

Workshop BIOMETRA 2016 Immunology and immunobiology

27

CHARACTERIZATION OF SIGNALING AND METABOLIC REPROGRAMMING

IN T CELLS BY PURINERGIC P2X7 RECEPTOR

Elsa Rottoli1*

, Fabio Grassi1



Adenosine triphosphate is an ubiquitous extracellular messenger, which activates purinergic

receptors in the plasma membrane of eukaryotic cells termed P2 receptors. We showed that T

effector/memory (TEM) cells express high levels of P2rx7 encoding for the ATP-gated

ionotropic P2X7 receptor subtype. The deletion of the gene in these cells results in increased

survival and proliferation rate both in vitro and in in vivo. P2rx7-/-

TEM cells are

characterized by a bioenergetic advantage with respect to wild-type (WT) cells.

Morphometric analysis of mitochondria revealed altered cristae and increased mitochondrial

mass in P2rx7-/-

TEM cells. Consistent with these morphometric changes, Western Blot

analysis showed an impairment of the autophagic flux in p2rx7-/-

TEM cells by a decrease in

both lipidated form of LC3 protein, namely LC3-II and LC3-I and accumulation of p62

protein. Microarray gene expression analysis showed that P2rx7-/-

TEM cells clustered

together and separately from WT cells. Among differentially expressed genes we identified

cyclin-dependent kinase inhibitor 1A (Cdkn1a), encoding for p21Waf1/Cip1

, as a transcript

upregulated in WT cells. P21 regulates progression through G1 to S phase in mammalian

cells. To address whether P2X7 signaling directly regulated Cdkn1a expression we

stimulated WT TEM cells with BzATP as a selective P2X7 agonist. This resulted in

significant increase in Cdkn1a transcripts with respect to unstimulated cells and this increase

was abrogated by the selective P2X7 antagonist A-438079. These results suggest that P2X7

activity limits expansion of TEM cells in ATP-rich microenvironment (e.g. during

inflammation), thereby controlling potential T cell mediated tissue damage.

Keywords: purinergic signaling, T cell activation, autoimmunity, cell death, senescence


28

CROSSTALK BETWEEN THE LONG PENTRAXIN PTX3 AND THE

COMPLEMENT SYSTEM IN THE IMMUNE RESPONSE TO ASPERGILLUS

FUMIGATUS

Raffaella Parente1*

, Francesca Petroni1, Marina Sironi

1, Sonia Valentino

1, Barbara Bottazzi

1,

Alberto Mantovani1,2

, Antonio Inforzato1,3


2Humanitas University, Rozzano, Italy



Aspergillus fumigatus (AF) is the major etiologic agent of Invasive aspergillosis, a life-

threatening infection amongst immunocompromised individuals. The innate immune system

plays a key role in the host resistance to AF. In this regard, the Alternative Pathway (AP) of

Complement has been described to cooperate with the long pentraxin PTX3 in the

complement-mediated reaction to AF. PTX3 exerts an opsono-phagocytic activity towards

AF: it enhances recognition, phagocytosis and killing of fungal conidia by immune cells via

Complement and Fc receptors pathways. Based on this, we hypothesized that PTX3 can

establish multiple interactions with conidia, Complement and effector cells, thus acting as a

molecular bridge in bringing together target microbes and immune components.

In this study, we found that PTX3 inhibits the interaction of AF conidia with factor H (FH,

the major soluble inhibitor of AP), which is a critical immune evasion strategy adopted by

AF. This is due to competition for common binding sites on the conidial wall and is

recapitulated by the N-terminal domain of PTX3 (N_PTX3) and a Cys/Ser substitution

mutant of this domain (N_PTX3C47S/C49S/C103S) that forms dimers, as opposed to

N_PTX3 that assembles into tetramers. This indicates that the minimal quaternary structure

that is required for PTX3 to bind AF conidia and displace FH is the dimer. Consistent with

these findings, the cofactor activity of FH on AF conidia (i.e., the factor I-mediated cleavage

of C3b to iC3b) is inhibited by PTX3, N_PTX3 and N_PTX3_3S. Moreover, we observed

that PTX3 forms a stable complex with C3b in an FH-dependent manner, and recruits C3b

onto AF conidia.

We propose that PTX3 enhances C3b deposition onto AF conidia via downregulation of the

FH cofactor activity and a novel FH-dependent mechanism that leads to C3b recruitment

onto the fungal wall. Given that C3b is the prototypic opsonin of Complement (i.e., that is

recognized by the phagocytic receptor CR1), our findings might explain the PTX3-dependent

enhancement of AF phagocytosis and, possibly, killing by innate immune cells.

Keywords: innate immunity, pentraxins, complement, invasive aspergillosis


29

NK CELLS RECRUITMENT TO THE SALIVARY GLANDS REGULATES EARLY

VIRAL CONTROL BUT IS DISPENSABLE FOR THE FORMATION OF

INDUCIBLE TERTIARY LYMPHOID STRUCTURES

Elena Pontarini1, Davide Lucchesi

2, Liliane Fossati-Jimack

2, Cristina Croia

2, Michele

Bombardieri2, Domenico Mavilio

1,3

1Clinical and Experimental Immunology, Humanitas Clinical and Research Centre, Milan,

Italy 2Experimental Medicine and Rheumatology, Queen Mary University of London, London,

UK 3BIOMETRA, Università degli Studi di Milano, Milan, Italy

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.

Keywords: NK cells, Sjogren’s Syndrome, autoimmunity, ectopic lymphoid structures, viral


30

MULTISTEP REGULATION OF TOLL LIKE RECEPTOR 4 SIGNALING BY IL-10-

DEPENDENT MICRORNAS

Graziella Curtale1,2

, Tiziana Renzi1,2

, Lorenzo Drufuca1,2

, Flavia Bazzoni3, Marzia Rossato

3,

Massimo Locati1,2



3Department of Pathology, University of Verona, Verona, Italy

MicroRNAs (miRs) are small non-coding RNAs with a post-transcriptional effect with an

emerging role in several biological functions. To identify miRs potentially involved in the

regulation of the inflammatory response, we analyzed the miR expression profile regulated

by the Toll Like Receptor 4 agonist LPS in human monocytes. We confirmed induction of

know pro-inflammatory miRs (miR-155, miR-146a) and identified a set of previously

unknown miRs, including the cluster miR-125a~99b~let-7e. As expression of this miR

cluster was significantly higher at late time points, when the IL-10-dependent anti-

inflammatory loop is usually detected, we measured the expression of these miRs in

monocytes stimulated with IL-10, in the presence or not of LPS, showing a potentiating

effect of IL-10 on LPS-induced cluster miR-125a~99b~let-7e expression. We found a

STAT3 consensus site and demonstrated the binding of STAT3 to the miR-cluster promoter

in condition of IL-10 stimulation. These data were consistent with the decrease of cluster

expression levels in the presence of blocking antibody against IL-10R and JAK-STAT

inhibitors. An in silico analysis was performed to assess the potential role of cluster miR-

125a~99b~let-7e in the context of inflammation; notably we found that the TLR pathway was

potentially regulated by these miRs at multiple steps of the signaling cascade. We validated

TLR4 and CD14 receptors, the adaptor proteins MyD88 and IRAK1 and a set of pro-

inflammatory cytokines as direct targets. Furthermore, the enforced expression of miR-

cluster in LPS stimulated monocytes led to an extensive down-regulation of pro-

inflammatory cytokines. In conclusion, we have identified an IL-10-responsive miR cluster

with anti-inflammatory activity in monocytes, able to target multiple components of the

TLR4 pathway, candidating miRs as new feedback modulators of LPS response involved in

the resolution of inflammation. A cluster miR-125a~99b~let-7e floxed animal is at present

being crossed with a LyM-CRE animal for selective deletion of this miR cluster in monocyte-

macrophages.

Keywords: chemokine, chemokine receptor, leukocyte mobilization


31

PRENATAL EXPOSURE TO POLY I:C INCREASES SUSCEPTIBILITY TO

EPILEPSY IN THE ADULT OFFSPRING

Elisa Focchi1,2*

, Marco Rasile3, Flavia Antonucci

1, Raffaella Morini

3, Elisabetta Menna

2,3,

Davide Pozzi3, Irene Corradini

2,3, Michela Matteoli

2,3


2CNR, Istituto di neuroscienze, Milan, Italy



In the last years, evidences have accumulated showing a direct connection between epilepsy

and brain inflammation. Indeed, epilepsy is associated to enhanced inflammation, while

activation of the immune response consequent to infections strongly increases the risk of

seizures.

By using the Poly I:C (polyinosinic-polycytidylic acid) mouse model of inflammation, we

demonstrated that a single Poly I:C injection at gestation day 9 (GD9) is able to increase

susceptibility to kainate-induced seizures in the offspring at postnatal day 90. As a control,

mice injected with Poly I:C in the adult life show no increased susceptibility to kainate-

induced seizures, thus providing the evidence that the higher susceptibility to seizures

consequent to prenatal Poly I:C exposure is the consequence of a neurodevelopmental

process.

We also evaluated the inflammatory profile of the Poly I:C-prenatally treated mice and we

found that a single Poly I:C injection in early/mid gestation is able to induce an inflammatory

response in the embryos 6 hours after the injection, as demonstrated by the significant

increase of IL6 and IL1-beta mRNA levels. On the other hand, no significant signs of

inflammation were found in adult mice, not even changes in synaptic protein levels and

synaptic morphology indicating that these features, usually related to epilepsy, are not

directly involved in the susceptibility to seizures in our model. Interestingly, we have found,

both in vitro and in vivo, a higher network hyperexcitability. Although the mechanisms

underlying this phenotype are still unknown, these results highlight the possibility that a

single inflammatory event during pregnancy may alter neurodevelopment, leading to

neurologic disorders in the adult offspring.

Keywords: biochemistry, cellular biology, molecular biology, animal behavior

Workshop BIOMETRA 2016

32

Other abstracts

Workshop BIOMETRA 2016 Other abstracts

33

DOWN-REGULATION OF ATYPICAL CHEMOKINE RECEPTOR ACKR2

EXPRESSION BY HEMATOPOIETIC PROGENITORS PROMOTES MYELOID

CELL MOBILIZATION AND DIFFERENTIATION

Ornella Bonavita1,2

, Nicoletta Caronni1,2

, Benedetta Savino1,2

, Matteo Massara1,2

, Laura

Crisafulli2,3

, Francesca Ficara2,3

, Alberto Mantovani2,4

, Massimo Locati1,2

, Raffaella

Bonecchi2,4



3Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Milan, Italy

4Humanitas University, Rozzano, Italy

The atypical receptor ACKR2 is a scavenger receptor for many inflammatory CC

chemokines and it has been shown to prevent the development of exacerbated inflammatory

reactions. ACKR2 is expressed either by non-hematopoietic cell compartment or by

hematopoietic cells. The aim of this investigation was to gain inside into the role of ACKR2

in myeloid cells trafficking and differentiation. To address the role of ACKR2 in the

proposed research we took advantage from ACKR2-gene targeted mice, FACS analysis,

primary cell cultures and cell line. Monocytes and neutrophils from ACKR2-/-

mice had

increased chemokine-induced mobilization and pharmacological blocking experiments

revealed that it was CXCR4-independent. ACKR2-/-

mice showed increased number of

monocytes confined to bone marrow sinusoids compared to wild type. Bone marrow chimera

experiments showed that the increased mobilization was due to the hematopoietic

compartment. The analysis of hematopoietic progenitors revealed that ACKR2 is expressed

by Lin−Sca-1

+c-Kit

+ cells (LSK) to faint thereafter in more mature myeloid progenitors in

contrast with canonical chemokine receptor CCR2. Moreover myeloid committed progenitors

from ACKR2-/-

mice expressed high levels of CCR1, CCR2 and CCR5, but not of CXCR4

and they had higher proliferation and differentiation rate compared to ACKR2 sufficient

LSK. These data suggest that ACKR2 is not only involved in regulating chemokine gradient

and leukocytes recruitment, but can directly regulate cell activity through the inhibition of

chemokine receptor expression by a mechanism still unclear. Collectively these results

demonstrate that ACKR2 is differentially expressed by hematopoietic progenitors during

myeloid cell maturation and its expression on LSK is involved in controlling their

localization and mobilization.

Keywords: chemokine, chemokine receptor, leukocyte mobilization


34

ROLE OF SEROTONIN 5HT2-TYPE RECEPTORS ON REWARDING AND

BEHAVIOURAL EFFECTS OF METHYLENEDIOXYMETHAMPHETAMINE

(MDMA) AND ITS DERIVATIVES IN ZEBRAFISH

D. Braida1*

, L. Ponzoni1, M. Sala

1,2




The synthetic phenethylamines are recreational drugs known to produce psychostimulant

effects (1). However, their abuse potential and pharmacological effects have not been widely

studied.

In the present study, the effect on reward, social preference and anxiety-like behaviour of

2,5-dimetoxy-4-bromo-amphetamine hydrobromide (DOB) and para-methoxyamphetamine

(PMA), in comparison with MDMA was investigated in zebrafish, an emerging model to

study emotional behaviour in an inexpensive and quick manner. In addition, the role of

serotonin 5-HT2 like- receptors on the above mentioned effects was evaluated.

Zebrafish were treated i.m. with a wide range of doses of DOB (0.05-20 mg/kg), PMA

(0.0005-2 mg/kg) or MDMA (0.25-160 mg/kg). Animals were submitted to a conditioned

place preference (CPP) task for the rewarding properties, to a social preference task to

evaluate social behaviour, and to the novel tank diving and light-dark tests to study emotional

behaviour. Hallucinatory behaviour was evaluated in terms of appearance of a trance-like

effect. The serotonin 5-HT2 subtype receptor antagonist ritanserin (0.025-2.5 mg/kg/i.m.) was

given in association with the maximal effective dose of MDMA, DOB and PMA. MDMA

and its derivatives exhibited dose-dependent CPP, anxiolytic effect and increase in social

preference in a biphasic fashion, being PMA the most potent. These effects were

accompanied, for DOB (2 mg/kg) and PMA (0.1 mg/kg), by a trance-like hallucinatory

behaviour. MDMA, at a high dose as 160 mg/kg, did not induce any hallucinatory behaviour.

Ritanserin significantly blocked all the effects, suggesting the involvement of serotonin

5HT2-like subtype receptors.

Collectively, these findings demonstrate for the first time the rewarding, prosocial and

anxiolytic properties of DOB and PMA accompanied by hallucinatory behaviour and focus

on the mechanisms of their action through the serotonergic-like system. Our results reinforce

zebrafish as an emerging experimental model for screening new hallucinogens.

1. Hill SL, Thomas SH (2011) Clin Toxicol (Phila) 49:705-719

The study was supported by Zardi Gori Foundation

Keywords: zebrafish, hallucinogens, addiction, anxiety, sociability

SOCIAL PREFERENCE ANXIETY

ADDICTION HALLUCINATIONS

MDMA

DOB

PMA

BLOCKED BY

RITANSERIN



35

EVIDENCE FOR A ROLE OF A NEW ANTIOXIDANT COMPOUND IN THE

PREVENTION OF AGE RELATED OSTEOBLAST DYSFUNCTION

Lavinia Casati1*

, Francesca Pagani 1, Valeria Sibilia

1


*e-mail address: [email protected]

Aim & Results: increasing evidence suggests a role for oxidative stress in age-related

decrease both in osteoblast number and function suggesting the potential use of antioxidant

compounds in the prevention/treatment of osteoporosis. This study was undertaken to

investigate whether ghrelin, previously reported to stimulate osteoblast proliferation,

counteracts tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in osteoblastic cells.

For this purpose, MC3T3-E1 cells and rat calvariae osteoblasts were exposed to t-BHP,

250M, for 3 h and a pre-treated with increasing ghrelin concentrations (10-9/ -11

M, 24 h

before). We found that ghrelin (10-9

M) significantly prevented t-BHP-induced osteoblastic

dysfunction and changes in the cytoskeleton organization evidenced by the staining of the

actin fibres with Phalloidin-FITC in MC3T3 cells.

As far as the molecular pathway involved in such ghrelin protective action, we investigated

Wnt/beta catenin pathway. We have shown that ghrelin increases GSK-3β phosphorylation

and β-catenin accumulation in the nucleus (by Western blot). Moreover cAMP/PKA pathway

is activated by ghrelin, since H89, an inhibitor of PKA, is able to abolish the ghrelin effect.

In addition to its anabolic action, ghrelin reduces the osteoclastogenesis, thus we analysed

the transcriptomic and epigenetic profile. When we analysed the OPG/RANKL

transcriptomic profile, we found that Ghrelin (10-9

M) is able to induce OPG expression and

to modify RANKL/OPG ratio, probably by changing the level of the histone modification

H3K4me3 and of its related histone post-translational enzyme, Jarid1b. Moreover we found

that the ghrelin increase the global DNA methylation level, promoting an “anti-aging” action

in osteoblasts.

Conclusions: these findings suggest that ghrelin could be considered a promising compound

to re-equilibrate bone cell activities in condition of impaired bone remodelling, and may have

therapeutic potential in age related osteoblast dysfunction.

Keywords: bone remodelling, oxidative stress, ghrelin, wnt/beta catenin pathway,

epigenetics


36

PTC SUPPRESSION STRATEGY AS “PERSONALIZED MEDICINE” APPROACH

FOR CDKL5 RELATED PATHOLOGIES

Maria Fazzari1*

, Charlotte Kilstrup-Nielsen2, Nicoletta Landsberger

1


2Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy


Rett syndrome (RTT) is a childhood neurological disorder and it is considered one of the

main causes of severe intellectual disability in girls. Classic RTT results from mutations in

MECP2 gene, but atypical forms exist where alterations occur at CDKL5 or FOXG1 loci.

CDKL5 is a serine/threonine kinase characterized by a N-terminal catalytic domain and a

long C-terminus unique to CDKL5.

Alterations of CDKL5 gene comprise a wide range of variants including non sense mutations,

which give rise to premature termination codons (PTCs). PTCs may cause the production of a

truncated protein or the activation of non sense-mediated decay (NMD). However, during

translation, it is possible that a near-cognate aminoacyl-tRNA binds the PTC. This mispairing

may result in the incorporation of amino acid attached to near-cognate aminoacyl-tRNA and

then in the production of full-length protein. This process is known as read-through and can

be facilitated using a wide range of drugs like aminoglycoside and non aminoglycoside

compounds.

In order to test PTC suppression, we have chosen some human CDKL5 non sense mutations

located either in the catalytic N-terminus (R59X, R134X) or in the C-terminus (R550X,

S855X) domain; the latter could be more suitable for this study because read-through might

produce a protein maintaining most of its functions. We introduced the selected mutations in

the pEGFPC1-hCdkl5107 construct and evaluated read-through using aminoglycoside drugs

after transient transfection: full-length proteins were detectable by WB and IF for all mutants

tested. Moreover, while truncated proteins showed an altered subcellular

localisation/organisation, after read-through the cellular phenotype was more similar to wild

type meaning that a rescue could be possible.

In the future, since our PTCs might activate the NMD mechanism, the read-through

efficiency will be tested in presence of NMD inhibitors in patients-derived fibroblasts.

Keywords: Rett syndrome, non sense mutations, read-through


37

HUMAN LIVER-RESIDENT CD56(BRIGHT)/CD16(NEG) NK CELLS ARE

RETAINED WITHIN HEPATIC SINUSOIDS VIA THE ENGAGEMENT OF CCR5

AND CXCR6 PATHWAYS

Hudspeth K1, Di Vito C

1, Zaghi E

1, Donadon M

2, Cimino M

2, Pontarini E

1, Tentorio P

1, Preti

M1, Hong M

3, Bertoletti A

3, Bicciato S

4, Invernizzi P

5, Lugli E

1, Torzilli G

2, Gershwin ME

6,

Mavilio D1,7

1Unit of Clinical and Experimental Immunology, Humanitas Clinical and Research Center,

Rozzano, Italy 2Department of Hepatobiliary & General Surgery, Humanitas Clinical and Research Center,

Rozzano, Italy 3Viral Hepatitis Laboratory, Singapore Institute for Clinical Sciences, Agency of Science,

Technology and Research (A*STAR), Singapore 4Department of Life Sciences, Center for Genome Research, University of Modena and

Reggio Emilia, Modena, Italy 5Liver Unit and Center for Autoimmune Liver Diseases, Humanitas Clinical and Research

Center, Rozzano, Italy 6Division of Rheumatology, Allergy, and Clinical Immunology, University of California

Davis, Davis, CA, USA 7BIOMETRA, Università degli Studi di Milano, Milan, Italy

The liver-specific natural killer (NK) cell population is critical for local innate immune

responses, but the mechanisms that lead to their selective homing and the definition of their

functionally relevance remain enigmatic.

We took advantage of the availability of healthy human liver to rigorously define the

mechanisms regulating the homing of NK cells to liver and the repertoire of receptors that

distinguish liver-resident NK (lr-NK) cells from circulating counterparts.

Nearly 50% of the entire liver NK cell population is composed of functionally relevant

CD56(bright) lr-NK cells that localize within hepatic sinusoids. CD56(bright) lr-NK cells

express CD69, CCR5 and CXCR6 and this unique repertoire of chemokine receptors is

functionally critical as it determines selective migration in response to the chemotactic

stimuli exerted by CCL3, CCL5 and CXCL16. Here, we also show that hepatic sinusoids

express CCL3(pos) Kupffer cells, CXCL16(pos) endothelial cells and CCL5(pos) T and NK

lymphocytes. The selective presence of these chemokines in sinusoidal spaces creates a

unique tissue niche for lr-CD56(bright) NK cells that constitutively express CCR5 and

CXCR6. CD56(bright) lr-NK cells co-exist with CD56(dim) conventional NK (c-NK) cells

that are, interestingly, transcriptionally and phenotypically similar to their peripheral

circulating counterparts. Indeed, CD56(dim) c-NK cells lack expression of CD69, CCR5, and

CXCR6 but express selectins, integrins and CX3CR1.

Our findings disclosing the phenotypic and functional differences between lr-NK cells and c-

NK cells are critical to distinguish liver-specific innate immune responses. Hence, any

therapeutic attempts at modifying the large population of CD56(bright) lr-NK cells will

require modification of hepatic CCR5 and CXCR6.

Keywords: natural killer cells, liver, chemokine receptors, innate immunity.


38

SPHINGOLIPIDS AS ACTORS IN THE STABILIZATION OF CFTR AT PLASMA

MEMBRANE

G. Mancini1*

, N. Loberto1, R. Bassi

1, D. Schiumarini

1, S. Munari

1, M.C. Dechecchi

1, G.

Lippi1, G. Cabrini

1, M.

Aureli

1, A. Tamanini

1, S. Sonnino

1



Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel

expressed at the plasma membrane (PM) of epithelial cells. Mutations in CFTR gene cause

Cystic Fibrosis (CF), an autosomal recessive disease characterized by severe lung disease due

to the loss of CFTR at the cell PM. Many pharmacological agents have been designed to

increase the surface level of mutated CFTR (correctors), as well as its PM stability and

activity (potentiators), even if their efficacy seems to be time-limited, particularly for the

most common CF-causing mutation F508del. Several factors influence the PM CFTR

stability, such as its compartmentalization in the sphingolipid(SL)-enriched lipid rafts and its

interaction with the a component of lipid rafts: the scaffolding protein ezrin, namely its

phosphorylated form. However is known that the phosphorylation state of ezrin depends on

PM levels of ceramide.

Based on these findings, we investigated the effects of corrector (VX-809) and potentiator

(VX-770) on CFTR PM microenvironment.

We analysed the SL composition and the phosphorylation state of ezrin in CF (CuFi-1) and

non-CF (NuLi-1) bronchial epithelial cell lines, treated or not with VX-809 and VX-770.

Moreover, we evaluated the SL pattern of lipid rafts.

In both cell lines we observed a marked reduction of phosphorylated ezrin, after the

combined treatment with corrector and potentiator. Interestingly, even if the cell SL total

content did not change, in treated cells we found a marked increase of all lipid rafts-

associated SL species, in particular ceramide, glucosylceramide and ganglioside GM3, which

could be responsible for the ezrin dephosphorylation.

These preliminary data suggest that the combined treatment induces modification in lipid

rafts organization in terms of proteins and lipids that could limit the stability of mutated

CFTR at PM level. These results could permit the development of new therapeutic strategies

for CF treatment.

Supported by Italian Cystic Fibrosis Research Foundation grant FFC#09/2015

Keywords: biochemistry, sphingolipids, cystic fibrosis, primary cell culture


39

THE HUMAN-RESTRICTED DUPLICATED FORM OF THE α7 NEURONAL

NICOTINIC RECEPTOR, CHRFAM7A: EXPRESSION, TRANSCRIPTIONAL

REGULATION AND ROLE IN THE INFLAMMATORY PROCESS

Maroli A.1*

, Fornasari D.1,2

, Benfante R1,2


2CNR, Neuroscience Institute, Milan, Italy


In the last years, increasing evidence have linked several neurological disorders to

inflammation. Controlling neuro-inflammation has indeed become a promising approach to

treat neuro-degenerative diseases. The Central Nervous System exerts a control on the

peripheral inflammation, through the “cholinergic anti-inflammatory pathway”, in which the

splenic terminals of the Vagus nerve induce an anti-inflammatory response by stimulating the

release of acetylcholine (ACh). The ACh interacts with the α7 Nicotine Receptor (CHRNA7)

expressed by the macrophages, thus inducing down-regulation of pro-inflammatory

cytokines. Recently, the CHRFAM7A gene has been discovered. It is the human restricted

product of a partial duplication and fusion of exons 5-10 of CHRNA7 gene with the novel

FAM7A gene and maps 1.6 Mb apart from CHRNA7 in inverted orientation. The

CHRFAM7A gene undergo alternative splicing, giving rise to two proteins of 46 and 35

KDa, which differ from the α7 conventional subunit for the N-terminus domain. These

proteins seem to have a dominant negative effect on the conventional α7 function. Moreover,

we have previously shown that CHRFAM7A is down-regulated after LPS treatment in THP-

1 cell model and primary monocytes and macrophages by a transcriptional mechanism reliant

on NF-κB. Our purpose is to define the transcriptional regulation of CHRFAM7A to better

understand its role in peripheral and central inflammation. In this study, we characterize the

gene’s regulatory region and its isoforms’ expression in neuronal and immune cell models.

Moreover, we investigate the responsiveness of CHRFAM7A gene to pro- and anti-

inflammatory stimuli, defining a human mechanism modulating the inflammatory response.

Keywords: CHRNA7, CHRFAM7A, inflammation, transcriptional regulation.


40

ATYPICAL CHEMOKINE RECEPTOR 2 PROMOTES TUMOR

METASTATIZATION BY INHIBITING RECRUITMENT AND ACTIVATION OF

ANTI-METASTATIC NEUTROPHILS

Matteo Massara1,2

, Ornella Bonavita1,2

, Benedetta Savino1,2

, Valeria Mollica Poeta1,2

, Elisa

Setten1,2

, Nicoletta Caronni1,2

, Camilla Recordati3, Marina Sironi

2, Alberto Mantovani

1,4,

Massimo Locati1,2

, Raffaella Bonecchi2,4



3Fondazione Filarete, Milan, Italy

4Department of Biomedical Sciences, Humanitas University, Rozzano, Italy

The atypical chemokine receptor 2 (ACKR2) is a scavenger receptor for most inflammatory

CC chemokines, with a protective role in chronic inflammation, autoimmunity and

inflammation-related cancer. The aim of this project is to investigate the role of ACKR2 in

primary tumor development and metastatization in different mouse models. Tumor arising in

ACKR2-/-

NeuT mice, that develop spontaneous breast adenocarcinoma, showed a more

aggressive phenotype when compared to WT NeuT mice. On the contrary, ACKR2-/-

NeuT

mice were protected from spontaneous lung metastasis. Metastasis protection was registered

also using the 4T1 orthotopic model of breast cancer. FACS analysis indicated an increase of

Ly6G+ polymorphonuclear cells and Ly6C

high monocytes in the blood and in the pre-

metastatic lungs of ACKR2-/-

mice. Depletion experiments demonstrated that neutrophils are

implied in metastasis protection in the B16F10 melanoma metastasis models. Moreover,

ACKR2-/-

neutrophils express increased levels of the chemokine receptor CCR2 and have

increased CCR2-dependent cytotoxic activity. These results indicate that ACKR2 expression

has a dual and opposite role in tumor biology, inhibiting primary tumor growth but

promoting lung metastatization trough the inhibition of anti-metastatic neutrophils.

Keywords: chemokine receptor, cancer, metastasis, neutrophil


41

CHARACTERIZATION OF A NOVEL NKp46POS

/V1-RESTRICTED TCR

LYMPHOCYTES RESIDENT WITHIN THE HUMAN INTESTINE

Joanna Mikulak1,2*

, Ferdinando Oriolo1,5,

Kelly L. Hudspeth1, Alessandra Roberto

1, Elena

Bruni1, Paolo Tentorio

1, Federico Colombo

3, Anna Villa

1,2,4 and DomenicoMavilio

1,5

1Unit of Clinical and Experimental Immunology, Humanitas Clinical and Research Center,

Rozzano, Milan, Italy 2Institute of Genetic and Biomedical Research (IRGB), CNR, Milan, Italy

3Flow Cytometry and Cell Sorting Unit, Humanitas Clinical and Research Center, Rozzano,

Milan, Italy 4Telethon Institute for Gene Therapy, Division of Regenerative Medicine, Stem Cells and

Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) San Raffaele

Scientific Institute, Milan, Italy



NKp46 is a member of Natural Cytotoxic Receptors (NCRs) triggering Natural Killer (NK)

cell cytotoxicity and production of inflammatory cytokine against harmful cells. Although

NKp46 had been first identified as a specific NK cell receptors, recent studies revealed that it

is also expressed on other immune cells such as Innate Lymphoid cells, T and NKT

lymphocytes. We previously reported that NKp46 expression is inducible upon in vitro

activation on a subset of human peripheral blood VT cells. Here, we identify a novel

NKp46pos

/ T lymphocytes subset resident in human intestine under homeostatic conditions

within the intraepithelial (IEL) compartment (average of 50% of all T IELs) and, to a

lower degree, in lamina propria (LP) (average of 10% of all T LPLs). This NKp46pos

/ T

IEL subset is characterized by a cytotoxic phenotype expressing CD56, CD8, NKG2D and

NKG2C. Moreover, we found that the expression of NKp46 in intestinal IELs is restricted

to V1 TCR chain repertoire that indeed distinguish their tissue-like phenotype from

peripheral blood T cells which mainly exhibited a V2 TCR distribution. We also

observed that IL-2 and IL-15-stimulation induce a de-novo expression of NKp46 in human

thymic T cell precursors, but not in circulating T cells. Their co-culture with intestinal

epithelial cells, IL-10 and TGF- can modulate the NKp46 expression according to the

activation steady state of these cells. These results suggest an early extra-thymic and gut-

conditioned maturation of NKp46pos

/ T cells likely relevant both in GALT homeostasis and

physiopathology of the gut.

Keywords: T lymphocytes, NKp46, cytotoxicity, gut, homeostasis


42

MECHANISMS OF GENERATION OF LONG-LIVED T MEMORY STEM CELLS

DURING LYMPHOPENIA

Alessandra Roberto1,3*

, Veronica Zanon1, Luca Castagna

2, Stefania Bramanti

2, Roberto

Crocchiolo2, Paolo Tentorio

3, Armando Santoro

2, Emma Gostick

4, Kristin Ladell

4, James

McLaren4, David A. Price

4, Mario Roederer

5, Domenico Mavilio

3,6, Enrico Lugli

1

1Laboratory of Translational Immunology, Humanitas Clinical and Research Center,

Rozzano, Milan, Italy 2Hematology and Bone Marrow Transplant Unit, Humanitas Cancer Center, Rozzano, Milan,

Italy 3Unit of Clinical and Experimental Immunology, Humanitas Clinical and Research Center,

Rozzano, Milan, Italy 4Institute of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK

5Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA



Recent evidence indicated that T memory stem cells (TSCM), the earliest differentiated

memory T cell subset in humans, display superior persistence in vivo as well as effector

functions; however, their mechanisms of generation in vivo remain elusive. In the context of

T-replete haploidentical bone marrow transplantation, we show that TSCM cells of donor

origin dominate the T cell compartment of lymphopenic recipients early after T cell transfer

and precede the expansion of effector cells. Importantly, transferred naïve T cells (TN), but

not TSCM or memory cells (TMEM), preferentially survive post ‐transplant cyclophosphamide,

used as GVHD prophylaxis, thus suggesting that post-transplant TSCM originate from TN cells.

In this regard, TN-derived TSCM cells specific for exogenous and self/tumor antigens

contribute to peripheral reconstitution of lymphopenic patients by differentiating into effector

T cells. Likewise, pathogen-specific TMEM cells generate detectable recall responses, but only

in the presence of the cognate antigen. The genome-wide expression analysis on sorted TN,

TSCM and TMEM cells under different stimulatory conditions revealed gene products

specifically expressed by self-renewing TSCM cells that are shared with somatic stem cells.

These genes are currently being used to induce stem cell-like properties in tumor-specific T

cells to be used in adoptive transfer experiments.

Keywords: Immune-reconstitution, T-cells, bone marrow transplantation, adoptive T cell

transfer


43

STRUCTURAL STUDIES AT THE PROTEOLIPIDOMICS HEDGE:

THE K+-CHANNEL KCV IN BIOMIMETIC MEMBRANES

Valeria Rondelli1*

, Elena Del Favero1, Paola Brocca

1, Anna Moroni

2, Laura Cantù

1


2Department of Biosciences, Università degli Studi di Milano, Milan, Italy


The activities of integral membrane proteins are often affected by the surrounding molecules

within the membrane. An important parameter is the hydrophobic thickness of the lipid

bilayer. In fact, both membrane and proteins may deform to ensure good hydrophobic

matching. Another important parameter for the regulation of membrane proteins functions is

the specific composition of the membrane portion where they are hosted. K+-channels are

transmembrane proteins abundant in virtually all biological systems. We studied a viral K+-

channel, Kcv (1), in interaction with different model membranes. Biochemical and functional

evidences indicate that K+-channels localize in lipid raft microdomains and that changes in

the membrane cholesterol content can directly modulate the ion channel functionality.

Calorimetry and X-ray spectroscopy revealed that the thermotropic and structural properties

of model biomimetic membranes are affected by the presence of Kcv, depending on lipid

composition and on the amount of cholesterol. Results show that the channel protein dictates

its preferred environment, inducing a lateral phase separation in membranes where the

average cholesterol content is different from the favored. Neutron reflectometry (2)(3) has

also been applied to study the structural changes induced by Kcv on single, extended, model

membranes, both in adhering bilayers and in floating membranes prepared by the Langmuir-

Blodgett/Langmuir-Schaefer technique. It evidences that Kcv inserts in the membranes with a

preferential orientation. Moreover, the protein induces cross asymmetric rearrangement of

cholesterol in the two leaflets of the hosting membranes.

(1) C. Pagliuca et al., Biochemistry 46 (2007) 1079-1090.

(2) V. Rondelli et al., Biochimica et Biophysica Acta 1818 (2012) 2860–2867.

(3) V. Rondelli et al., Nature Scientific Reports 6 (2016) 20997.

Keywords: model membranes, potassium channels, protein-lipid interaction, cholesterol


44

LSD1 AND neuroLSD1: THE YIN AND YANG OF EXPERIENCE-DRIVEN

NEURONAL PLASTICITY

Francesco Rusconi

1*, Barbara Grillo

1, Emanuela Toffolo

1, Federica Giona

1, Alessandra

Longaretti1, Elena Battaglioli

1



In mammals, different forms of stress including psychosocial stress can impact on several

aspects of human health, fostering mood and anxiety disorders. However, very little is known

about the mechanisms underlying brain physiology of stress response, hindering development

of new therapeutic strategies. Here we indicate a role for transcriptional corepressor Lysine-

specific demethylase 1 (LSD1) and its dominant-negative splicing isoform neuroLSD1, in the

modulation of emotional behavior. In mouse hippocampus, LSD1 and neuroLSD1 interact

with transcription factor SRF setting chromatin state of SRF-targeted genes egr1 and c-fos.

Deletion or reduction of neuroLSD1 in mutant mice induce decreased levels of activating

histone marks at egr1 and c-fos promoters, which dampen their psychosocial stress-induced

transcription, and result in low anxiety-like behavior. SAHA administration to

neuroLSD1KO mice restores egr1 and c-fos promoter chromatin structure, and behavioral

phenotype. These findings indicate LSD1 as a molecular transducer of stress stimuli as well

as a stress-response modifier. Indeed, LSD1 expression itself is acutely increased by

psychosocial stress at transcriptional and splicing levels suggesting involvement of LSD1 in

adaptive response to stress.

Keywords: epigenetics, neuroscience, anxiety, transcription, memory


45

A COMPOSITE AUTONOMIC INDEX AS UNITARY METRIC FOR HEART RATE

VARIABILITY: A TRANSLATIONAL APPROACH

Roberto Sala1,2

, Mara Malacarne1,2

, Fabio Tosi1,2

, Chiara Vigo1,2

, Daniela Lucini1,2




Introduction: Here we assess whether a unitary cardiac autonomic nervous system index

(ANSI), obtained combining multiple metrics from Heart Rate Variability (HRV) into a radar

plot could provide an easy appreciation of autonomic performance in a clinical setting.

Methods: We standardize data using percentile ranking of autonomic proxies from a

relatively large reference population (n= 1593, age 39±13 years ) composed of individuals

that are either healthy, or with various levels of putative autonomic dysregulation as

consequence of increased arterial pressure, Body Mass Index or smoking.

Autonomic indices are obtained from autoregressive spectral analysis of (ECG derived) HRV

at rest and during standing up. An ordinal scale is developed in order to define a gradual

level of cardiometabolic risk.

Results: With growing risk level there is a significant negative trend of various, HRV

derived, autonomic indices towards lower ranks (test for linearity p0.001 for all indices, but

LFnu p=0.005). Furthermore, the rank value of ANSI, quantified individually by the radar

plot area, is markedly reduced in moving from low to high risk level (linearity test, p<0.001).

A similar information is provided also by ANSI indices obtained from a reduced set of only 3

autonomic proxies.

Conclusions: Our observational study shows the feasibility of testing simpler metrics of

cardiac autonomic regulation based on a multivariate unitary index in a preventive setting.

Keywords: autonomic nervous system, personal prevention, cardiometabolic risk,

hypertension, BMI, smoke


46

MICRORNA miR-135b CONTROLS MACROPHAGE POLARIZED ACTIVATION

Grazia Naths Sukubo1,2

, Matthieu Pesant1,2

, Massimo Locati1,2



MicroRNAs (miRs) are small non-coding RNAs with a post-transcriptional effect. Recent

evidence indicate that miRNAs are involved in the inflammatory response, but their role in

the activation of macrophages in response 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. Monocyte-derived macrophages were polarized to

classic (M1; LPS + INFγ) or alternative (M2; IL-4) cells and 732 miRNAs were profiled

using TaqMan MicroRNA Arrays. Out of 732 miRNAs investigated, 74 resulted differentially

expressed among the polarized macrophages. Among these, miR-135b was strongly

associated with M1 polarization. Although miR-135b expression profile was 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, its overexpression had no effect neither on

resting nor in M1 macrophages. Conversely, in silico analysis using the Ingenuity Pathway

Analysis software identified miR-135b as a potential modulator of the alternative activation

pathway through direct targeting of the M2-related key transcriptional factors c-Myc 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 (CCL18 and CCL22) when its function was investigated in M2

cells. Finally, miR-135b proved to be responsible for preventing the expression of a set of M2

genes in M1 macrophages tested under repolarization settings. These results indicate that

macrophage phenotypes are characterized by differential expression of miRNA profiles and

identified miR-135b as an M1-associated miRNA targeting the transcription factors c-Myc

and KLF4, thus limiting macrophage plasticity. Altogether these data indicate miR-135b as a

pro-inflammatory miRNA possibly involved in chronicization of the inflammatory response.

Keywords: macrophage, microRNA


47

AUTONOMIC CARDIAC REGULATION IN ELITE ATHLETES.PRELIMINARY

OBSERVATIONS WITH A UNITARY AUTONOMIC INDEX ON THE NATIONAL

ITALIAN OLYMPIC COMMITTEE TEAM

Fabio Tosi1,2

, Roberto Sala1,2

, Mara Malacarne1,2

, Chiara Vigo1,2

, Daniela Lucini1,2




Introduction: Long term endurance training, as occurring in elite athletes, is associated to

cardiac neural remodeling in favor of cardio-protective vagal mechanisms, resulting in

bradycardia at rest and an augmented contribution of cardiac parasympathetic nerve activity.

Hence we hypothesize that intensity of individual work might be significantly correlated to

autonomic regulation.

Methods: We studied 711 (23.6 ± 6.2 years) elite athletes that took part to the selection

procedure for the 2016 Rio Olympic Games for the National Italian Olympic Committee

(CONI). Indices from Heart Rate Variability HRV obtained at rest, during standing up and

during recovery from exercise test were used to compute a percent ranked unitary autonomic

index for sport (ANSIs), taken as proxy of quality of autonomic regulation.

Results: Within the observed wide range of energy expenditure, ANSIs appears significantly

correlated to individual and discipline specific training work-loads (r=0.25, p<0.001 and

r=0.78, p<0.001, respectively). ANSIs is also positively linked to metabolic profile.

Conclusions: Estimated intensity of physical activity correlates with quality of cardiac

autonomic regulation, as expressed by a novel unitary index of cardiac autonomic regulation:

ANSIs.

Keywords: exercise, elite sports, competition, vagal drive, Heart Rate Variability

Workshop BIOMETRA 2016 Author index

48

AUTHOR INDEX

A

Abdel Hadi L., p. 9

B

Bonavita O., p. 33

Borroni E., p. 13

Braida D., p. 34

Busnelli M., p. 26

C

Calcaterra F., p. 20

Caleo M., p. 6

Casati L., p. 35

Castino G.F., p. 7

D

Di Lascio S., p. 23

Drufuca L., p. 30

F

Fazzari M., p. 36

Ferrari L., p. 10

Focchi E., p. 31

Frasca A., p. 22

G

Gamba A., p. 18

Giavazzi F., p. 16

Grassi S., p. 21

H

Hudspeth K., p. 37

L

Lanfranco R., p. 17

M

Mancini G., p. 38

Maroli A., p. 39

Massara M., p. 40

Mikulak J., p. 41

P

Palagano E., p. 11

Parente R., p. 28

Pizzamiglio L., p. 25

Pontarini E., p. 29

Ponzoni L., p. 19

R

Roberto A., p. 42

Rondelli V., p. 43

Rottoli E., p. 27

Rusconi F., p. 44

S

Sala R., p. 45

Samarani M., p. 15

Schiumarini D., p. 12

Sukubo N.G., p. 46

T

Tosi F., p. 47

V

Vanetti C., p. 14

Vezzoli E., p. 24

Vigo C., p. 8

biometra - unimi.it · giovanni francesco castino 1*, nina cortese 1, fabio grizzi , marco erreni ,...

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