Scientific Institute H San Raffaele
Edited by Marco E. Bianchi, Maria Sessa, Laura Tei
Editorial assistants: Angelo Angarano, Diego M. Bertini
Lay-out by Roberto Cremonesi. Co
Printed by Grafiche Parole Nuove, Brugherio
Scientific Direction
Biological mass spectrometry
Biology of myelin
Biomolecular NMR laboratory
Cell adhesion
Cerebral cortex development
Chromatin dynamics
Developmental neurobiology
Dynamics fluorescence spectroscopy
in biomedicine
Genetics of common disorders
Genomics for human disease diagnosis
Human inherited neuropathies
Leukocyte biology
Molecular basis of Polycystic Kidney
Disease (PKD)
Molecular genetics
Molecular genetics of behaviour
Molecular genetics of membrane traffic
Molecular histology
Molecular oncology
Protein transport and secretion
Proteome biochemistry
Proteomics of iron metabolism
and clinical transplantation
Metabolic unit
Bone metabolism
Gastroenterology and digestive endoscopy
Nephrology and hypertension
Hematology and bone marrow transplantation
Unit of lymphoid malignancies
Cancer Immunotherapy and Gene Therapy
Program (CIGTP)
Antigen discovery
Antigen presentation improvements
Host/tumor interaction
Effector function improvements
Tumor targeting
Clinical immunology and rheumatology
Viral Immunology Program (VIP)
Non viral infectious diseases
SARS research
HIV/AIDS pathogenesis
Antiviral therapy and immune reconstitution
Vaccine research
Viral hepatitis
Viral oncogenesis
Emerging bacterial pathogens
General medicine, diabetes, endocrinology
and metabolic diseases
Telethon/JDRF centre for beta cell replacement
Cerebrovascular diseases
Movement disorders
Multiple sclerosis
Neuromuscolar disorders
Biosignal analysis center
Epilepsy and EEG
Neurophysiology of sensory-motor system
Pain medicine center
Sleep disorders
Imaging of brain development
Spectroscopy and functional MR
Neurointensive care
Psychology and psychotherapy
Cellular and molecular neurobiology
Cellular neurophysiology
Developmental neurogenetics
Experimental neuropharmacology
Human molecular genetics
Neurobiology of learning
HSR-TIGET (Telethon Institute for
Gene Therapy)
Gene therapy for primary immunodeficiencies
Gene therapy for leukodystrophies
Molecular and cellular biology of gene transfer
Modulation of immune response
Gene and stem cell therapy for muscular
SCRI (Stem Cell Research Institute)
Mesoderm stem cell
Neural stem cell
Cystic fibrosis
Angiogenesis and tumor targeting
Cardiac surgery
Cardiovascular anaesthesia and intensive care
Clinical cardiology
Cardiovascular epidemiology and informatic unit 136
Coronary care unit
CCVB, (Clinical Cardiovascular Biology Research
Respiratory medicine
Thoracic surgery
Vascular surgery
General and digestive surgery
General, pancreatic, and endocrine surgery
General and hepatobiliary surgery
Gynaecology and obstetrics
Ophthalmology and visual sciences
Anaesthesiology and intensive care
Molecular imaging
Nuclear medicine
Functional neuroimaging
YAC Screening Centre
Clinical chemistry
Diagnostic clinical laboratory
Haemostasis and thrombosis
Medical physics
Human values are the driving force of all the theoretical and practical advances of
each academic discipline, but, above all, of medicine and biomedical research.
contents of this volume are the expression of this fundamental principle,
which inspires everyone who works with faith and dedication at San Raffaele.
scientific research can never be divorced from equally serious clinical
practice. The aim of both is the creation of a product in which quality always comes
before quantity.
this is not enough if our cultural heritage is not passed down to the new
generations. The Ateneo Vita-Salute San Raffaele was conceived to do just this.
Departmentalization was thus devised to fulfil the need to integrate health care,
research and teaching in one structure, the department, which provides the perfect
environment for harmonious integration.
To all my beloved Raphaellians: many thanks and carry on your good work full of
Don Luigi Maria Verzè
President and Chancellor
San Raffaele Institute was established in 1971 and represented one of the first examples of a
fully independent private hospital in Italy. Shortly after its foundation San Raffaele got affiliated
with the public University of Milan Medical School and was granted the status of IRCCS (Research
Hospital), making it a site for clinical research, originally specialized in diabetes and metabolic disorders.
In 1992 San Raffaele expanded further with the inauguration of DIBIT, a building dedicated to basic science, with a research space of about 12,000 square meters. Within 2007, the DIBIT2
project will expand the floor space for research almost three-fold. At present San Raffaele employs
250 people in basic and clinical research, including scientists, technicians and administrative personnel, along with additional 160 fellows, trainees and graduate students. DIBIT is part of the
largest biomedical science park in Italy, which includes San Raffaele Hospital with 1100 beds,
Science Park Raf, created to support the Foundation’s development objectives, and the University.
The University Vita-Salute San Raffaele began in 1996 with a degree course in Psychology followed, in 1998, by the Faculty of Medicine and Surgery and, in 2002, by the Faculty of Philosophy.
The teaching activities comprise also international Ph.D. programs in Cellular and Molecular
Biology, and in Molecular Medicine.
Much effort and many resources have been invested in basic preclinical and clinical research.
Our scientific production continues to experience quite an impressive progress for both the number of publications and the overall quality. In 2004, 600 scientific papers were published, with a
total impact factor of 3100. These figures confirm the Institute’s leading position in Italy and
improved the ability to attract support from public (Ministry of Health, Superior Institute of
Health, Ministry of Education, University and Research, European Community and, for a small
amount, CNR) and private sources (mainly Telethon and the Italian Association for Cancer
Research). Telethon is funding two research centers at San Raffaele: the San Raffaele-Telethon
Institute for Gene Therapy of Genetic Disease (HSR-TIGET) which is pioneering the clinical
application of gene transfer technology, and the Stem Cell Research Institute (SCRI), which studies the biology and the potential clinical applications of stem cells: a field in which San Raffaele
Institute is one of the leaders in the world.
In the last few years biomedical technologies have undergone an impressive development
and diversification, opening the doors to a new type of medicine: “molecular medicine”. The complete sequencing of the human genome has radically modified the scientific scenario. In the near
future researchers’ efforts will be dedicated to the elucidation of the mechanisms of regulation and
expression of the genes and the definition of the functions of their products, the development of
presymptomatic diagnostic tools and the design of appropriate therapeutic approaches. San
Raffaele Scientific Institute has the merit of having advocated from the beginning this new type of
medicine, in which basic and clinical researchers and physicians operate together with the goal of
translating basic research into medical practice, and, in 2001, it was acknowledged, by the Health
Minister, as the IRCCS for Molecular Medicine.
The Scientific Report 2004 is organized in sections (Functional Genomics; Diabetes and
Metabolic Diseases; Oncology, Immunology, and Infectious Diseases; Neurosciences; Gene and
Stem Cells Therapy; Cardiothoracic and Vascular Department; Surgery; Imaging), which reflect
the ongoing departmental reorganization of the Institute. The Core Facilities of the Institute are
grouped in a separate section.
Thanks to the effort and enthusiasm of scientists, physicians, students, and of all the people
working at San Raffaele, our new molecular medicine will provide, in the immediate future, either
new or less disruptive diagnostic and therapeutical approaches, ready to use within the public
Italian health system.
Claudio Bordignon
Scientific Director
Structure and function of nucleoside hydrolases
B. Giabbai, I. Bruno, P. Tornaghi, M. Savoldi Boles, J. Steyeart, W. Versées, V. L. Schramm, M. Degano
Nucleoside Hydrolase (NH) activities are encoded by nucleosidase enzymes throughout bacterial and eukaryotic genomes, albeit not present in mammals. Their function is well established in purine auxotrophic protozoan parasites, but still undefined in bacteria and higher eukaryotes. The structural features defining NH specificity and activity are yet unresolved. We determined the crystal structures of the E. coli YeiK and YbeK NHs
to atomic resolution, both in apo and ligand-bound forms, to establish the framework determining their enzymatic activity. Through a combination of site directed mutagenesis and structural studies on mutated proteins
we aim at dissecting the catalytic mechanism of this widespread family of important enzymes.
Nucleoside hydrolases as novel tools for cancer suicide gene therapy
C. Minici, B. Giabbai, P. Tornaghi, M. Bellone, A. Mondino, A. Cestari, P. Rigatti, M. Degano
Nucleoside hydrolases catalyze the excision of the N-glycosidic bond in nucleosides, yielding free base and ribose. We identified bacterial and yeast enzymes that efficiently convert the prodrug 5-fluorouridine (FUR) to 5fluorouracil (FU), a widely used chemotherapic. The FUR molecule is more bioavailable and less toxic than
FU, thus more amenable for therapeutic usage, but its conversion to the active form is inefficient in human
cells. We transfected tumor cell lines (cervical or prostate adenocarcinoma) with NH genes followed by administration of FUR. Our results show that this approach results in a 1000-fold decrease in the dose required for efficient killing of cancer cells. We aim at engineering specific enzyme mutants with highest therapeutic potential,
as well as demonstrating the efficacy in preclinical models.
The structural basis for NKT cell activation
B. Giabbai, F. Giannese, G. Casorati, A. Bachi, Y. Sanchez-Ruiz, M. Kronenberg, G. Casorati, P. Dellabona,
M. Degano
NKT cells are the paradigm for regulatory lymphocytes, being able to secrete large amounts of regulatory cytokines within hours of activation. The NKT-secreted regulatory factors, such as IL-4 and IFN-gamma, can
modulate the immune response towards antigen from an inflammatory to a protective, tolerance-inducing reaction. The inflammatory response is crucial for the spontaneous rejection of tumors, while the protective response downmodulates autoimmunity in several systems, including type 1 diabetes and multiple sclerosis. The
central event in NKT cell activation is the recognition of lipid antigens bound to the CD1d molecule by the
NKT cell receptor. We determined the structure to 2.8 Å of the mouse CD1d molecule bound to phosphatidyl
choline, and crystallized the human CD1d molecule. The structure revealed how lipid antigens bind to CD1d,
and demonstrated how the ligand-binding cavity is suited for selection of specific lipids. The role of the hydrophilic headgroup in modifying the chemical character of the CD1d surface is crucial for NKT cell activation. We are currently designing altered lipid ligands with different affinities for CD1d to modulate NKT cell
Computational analysis of enzymatic catalysis and protein-protein interactions
B. Giabbai, M. Savoldi Boles, I. Bruno, A. Curioni, W. Andreoni, M. Degano
We employ a combination of X-ray crystallography and ab initio calculations to evaluate the forces at active
sites of enzymes and receptors in order to finely understand the correlation between protein structure and functional features. In silico screening is used to assess the effects of specific amino acidic mutations on the stability
and function of proteins and macromolecular complexes of medical relevance. Moreover, we apply molecular
dynamics simulations combined with docking methods to screen the interaction of potential drugs with target
proteins. This approach aims at complementing the functional studies on the target proteins to provide new
knowledge and tools for modulating their activities in vivo.
Analysis of protein networks
Y. Sanchez-Ruiz, A. Cattaneo, A. Bachi
The systematic analysis of networks of protein-protein interactions is likely to give functional information
about biological processes. To optimize this strategy we are making use of specific purification methodologies,
such as double epitope tagging or immunoprecipitation techniques, that allow the simultaneous purification of
all protein partners, followed by their identification with MALDI-TOF or multidimensional nanoLC tandem
mass spectrometry which is able to analyze and identify hundreds of proteins in a single experiment. We are applying this methodolgy to study the protein composition of synaptic vescicles under physiological conditions.
Quantitative proteomics
F. Ferron, Y. Sanchez-Ruiz, A. Bachi
To improve the efficiency of protein complexes identification, and to reach a comprehensive analysis of
macromolecular complexes including the dynamics involved in a biological process we are using the SILAC
method (Stable Isotopes Labeling with Amino Acids in Cell Culture) for quantitative proteomics.
Post-translational modifications of proteins
V. Matafora, S. Camerini, L. Polci, A. Bachi
Post-Translational Modification (PTM) of proteins is a key event in several cellular processes as signaling, metabolism and targeting and is, at the end, responsible of the correct protein activity. To understand how and
where a protein is modified we have set up specific and sensitive methods for the enrichment and the isolation
of phosphorylated peptides present in ‘in gel’ separated proteins that allow us to identify and localize the site of
modification. Another field of interest of the laboratory is the assessment of the oxidative status of cysteine
residues. We have recently developed a new method, based on selective enrichment and identification of protein containing oxidatively modified cysteines and S-nitrosylated proteins, that provides us a new tool for the
study of thiols mediated redox regulation.
Laminin receptors in nerves and neuropathies
L. Feltri, L. Wrabetz, S. Occhi, D. Zambroni, A. Quattrini, C. Berti, F. Court, A. Nodari
Schwann Cells (SC) myelinate peripheral nerves, and contribute to organization of axonal domains and neuromuscular junctions and to regeneration. Laminins and their receptors are required for many of these functions, and mutations in genes coding for laminins or components of laminin receptor complexes cause hereditary neuropathies (Merosin Deficient Congenital Muscular Dystrophy and Charcot-Marie-Tooth 4F). We used
the Cre/LoxP system in mice to identify alpha6beta1 integrin and dystroglycan as the laminin receptors that allow SC to radially sort axons and to organize axonal domains (Nodes of Ranvier), respectively. Now, we show
that both SC dystroglycan and laminins cluster sodium channels at nodes of Ranvier, that alpha6beta4 integrin
is required to maintain long term integrity of myelin sheaths, and preliminarily that beta1 integrin in perysinaptic SCs favors re-innervation at the neuromuscular junction. Thus, laminin receptors have important and diverse functions in peripheral nerves, which are relevant to the pathogenesis of neuromuscular diseases.
Transgenic mouse models of hereditary neuropathy
L. Wrabetz, G. Dati, M. Pennuto, E. Tinelli, M. D’Antonio, C. Ferri, A. Quattrini, S. C. Previtali, L. Feltri
We have engineered seven transgenic mouse models of myelin protein zero-related Charcot Marie Tooth
(CMT) hereditary neuropathy. Mice overexpressing wildtype P0 develop a congenital hypomyelination; mice
expressing P0 with a myc-epitope tag at the amino terminus unexpectedly model two more severe subtypes of
CMT1B neuropathy; and mice expressing P0 with any of five known human MPZ mutations model CMT1B or
Congenital Hypomyelination Neuropathy. All mutations produce diverse gain of function effects, suggesting
that variability MPZ neuropathies reflects diverse types of gain of P0 function. For comparison, we are producing mice by homologous recombination with targeted mutations of P0 associated with axonal and other phenotypes, in order to understand the cell biology of CMT neuropathies. In one mouse, MpzS63del, we have identified endoplasmic retention of the mutant P0 and activation of protein quality control pathways. How these intracellular signals produce myelin sheath alterations is under study.
Axo-glial interactions
L. Wrabetz, S. Campanella, C. Ferri, C. Taveggia, A. Pizzagalli, A. Quattrini, L. Feltri, M. Bozzali
We exploited transgenic mice containing the proximal MBP promoter fused to the lacZ reporter gene to
show in optic nerve axotomy experiments that lacZ expression, like endogenous MBP expression, is axonally
dependent. Surprisingly, lacZ expression was not rescued in the Ola strain, in which axons remain after optic
nerve axotomy, but did remain normal after intraocular tetrodotoxin injections, which blocks electrical activity
in optic nerve. These data show that MBP expression is dependent on a dynamic characteristic of the axons and
distinguish the axonal signal that regulates myelin gene expression from those that regulate oligodendrocyte
proliferation and survival in development. Identification of these signals is underway in oligodendrocyte neuronal co-cultures from this mice. We have also identified an upstream enhancer of the MBP gene that is robustly activated in Schwann cells co-cultured with dorsal root ganglia neurons. Thus, axonal signals also target distant enhancers of the myelin genes. This enhancer activates a heterologous myelin promoter 30-fold in transgenic mice, producing a useful tool to target high-level expression of genes specifically to Schwann cells.
Myelinating glia enriched DNA-binding activity
L. Wrabetz, S. Campanella, I. Nisoli, C. Taveggia, A. Pizzagalli, A. Bachi, L. Feltri, M. Bozzali
We have identified Myelinating Glia Enriched DNA-Binding Activity (MEBA), that is important for tissuesspecific activation of the proximal MBP promoter only in myelinating oligodendrocytes or Schwann cells. MEBA may function by competing with nuclear factor 1(NF1)—a transcription factor present ubiquitously that represses the MBP promoter. We showed that the presence of MEBA is topographically and temporally correlated with MBP mRNA expression in the central nervous system during development. Affinity chromatography
purification separated MEBA from NF1 (they bind a common sequence) and has revealed that MEBA contains
at least two proteins of 38 and 40 kD. Their amino acid sequences have been identified by mass spectrometry,
and they are both members of a family of transcription factors regulated by calcium. Functional analysis of promoter mutants that can bind only NF1 or MEBA show that MEBA is the primary determinant of activation of
the proximal MBP promoter; NF1 has little role. Experiments are underway to determine whether MEBA mediates axonal signals that regulate MBP transcription.
Solution structure determination of the first PHD finger of AIRE1, the protein involved in Autoimmune
Polyendocrinopathy Ectodermal Dystrophy
M. Bottomley, M. Sattler, A. Akhtar, D. Pennacchini, G. Musco
Mutations in the autoimmune regulator protein AIRE1 cause a monogenic autosomal recessively inherited
disease: autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. AIRE1 is a multidomain protein
harboring two plant homeodomain (PHD)-type zinc fingers. The first PHD finger of AIRE1 is a mutational
hot-spot, to which several pathological point mutations have been mapped. Using heteronuclear NMR spectroscopy, we determined the solution structure of the first PHD finger of AIRE1 (AIRE1-PHD1), and gave a
structural rationale of disease causing mutations. PHD fingers have a zinc dependent fold, similar to the RING
finger domain, which can function as E3 ligases in the ubiquitination pathway. Based on this fold similarity, we
verified if the AIRE PHD finger could also work as E3 ligase. At variance to a previous report, we could not
find any evidence that AIRE1-PHD1 has an intrinsic E3 ubiquitin ligase activity. Consistently, we show that the
AIRE1-PHD1 structure is clearly distinct from the RING finger fold. Our results point to a function of the
AIRE1-PHD1 domain in protein-protein interactions, which is impaired in some APECED mutations.
Computational studies on membrane bound proteins: applications to Domain C2 of Coagulation Factor V
L. Mollica, F. Fraternali, G. Musco
Proteins that interact with cell membranes are of fundamental importance in biological systems. One of the
largest hurdles to the structural study of membrane bound proteins is the expression and purification of sufficient amounts of protein in order to perform crystallization trials. Hence, in silico studies based on homology
modeling and molecular dynamics simulations constitute a valid alternative. We applied molecular dynamics
simulations (10 ns) to characterize at atomic level the interaction of domain C2 of coagulation Factor V with the
phospholipid membrane which is known to be essential for generation of full procoagulant activity of Factor V.
During the membrane simulation the protein,initially positioned on the surface of the membrane, is progressively attracted towards the center of the membrane until an equilibrium distance of the center of mass is
reached. Positively charged residues are in favourable orientation to create stable electrostatic interactions with
the polar head groups of the membrane and solvent exposed tryptophans undergo conformational changes optimizing the van der Waals interactions with the lipid interior of the membrane.
Structural studies on HMGB1 (High Mobility Group Box 1 protein) and identification of Glycyrrhizic
Acid as new inhibitor of the pro-inflammatory activity of HMGB1
L. Mollica, R. Palumbo, M. Zamai, W. Andreoni, A. Curioni, G. Musco, M. E. Bianchi
The HMGB1 protein has a pivotal role as intracellular and extracellular mediator: inside the cell it is a transcription and growth factor, outside it acts as cytokine for lethal systemic inflammation, arthritis and local inflammation. We have identified glycyrrhizic acid (GL) as a new modulator of the cytokine activity of HMGB1.
By NMR chemical shift mapping and fluorescence we have shown that GL and its derivative carbenoxolone interact directly with HMGB1 with micromolar affinity. The interaction sites are mainly clustered on the first helix of the two HMG boxes, involving highly conserved residues in the HMG family. Moreover, cell migration
and proliferation studies showed that GL and derivatives inhibit the HMGB1 induced migratory response and
proliferation of endothelial cells and mesoangioblasts. We are currently characterizing at molecular level the interaction of GL and HMGB1 by means of NMR,molecular dynamics simulations and ab initio calculations.
The long acidic tail of High Mobility Group Box 1 (HMGB1) protein forms an extended and flexible
structure that interacts with specific residues within and between the HMG boxes
S. Knapp, S. Müller, G. Digilio, T. Bonaldi, M. E. Bianchi, G. Musco
HMGB1 is composed of two similar DNA binding domains (HMG box A and box B) linked by a short basic
stretch to an acidic C-terminal tail of 30 residues. The acidic tail modulates the DNA binding properties of
HMGB1, and its length differentiates the various HMGB family members. We synthesized a peptide (T-peptide) that corresponds to the acidic tail in HMGB1 and studied its interactions with the rest of the protein. CD
spectroscopy showed that T-peptide stabilizes significantly the rest of the protein, and that the complex has an
identical thermal stability as full length HMGB1. Calorimetric and NMR data showed that T-peptide binds
with a dissociation constant of 9 microM to box A and much more weakly to box B. 1H-15N HSQC spectra of
full-length HMGB1 and of the tailless fragment are very similar; the small chemical shift differences that exist
correspond to those residues of the tailless fragment that were affected by the addition of the T-peptide. Thus,
the T-peptide mimics closely the acidic tail; the basic stretch and the acidic tail form an extended and flexible
segment that interacts with specific residues in the boxes and shields them from other interactions.
Mechanisms for Rac-mediated cell motility
L. Za, S. Paris, O. Botrugno, I. de Curtis
We have identified GIT1/p95-APP1 as part of a protein complex interacting with GTP-Rac proteins. Biochemical analysis has indicated a strong association of the complex with membranes. The SH3 domain of PIX
is essential for the localization of GIT1, since a monomeric PIX-SH3 mutant interferes with this localization. A
PAK1 fragment including the PIX-binding region prevents the recruitment at membranes. PIX is a limiting factor for the formation of the endogenous complexes, and PIX binding correlates with a conformational change
in GIT1. This represents a novel mechanism for the regulation of the function of GIT1-PIX. We have also
analysed the dynamic behaviour of A431 transformed cells by motogenic stimuli with EGF. ArfGAP mutants
unable to leave endocytic membranes abolish EGF-induced lamellipodia, and induce the formation of GIT1positive large vesicles and cell retraction. These effects are negligible in EGF-stimulated cells expressing the
full-length protein or fragments lacking the PIX-binding domain. Our work indicates that the p95-complex is
an important link between membrane trafficking and cytoskeleton during cell motility, fundamental for the formation of metastases.
Molecular mechanisms of neural development
S. Corbetta, S. Gualdoni, S. Paris, C. Albertinazzi, A. Bolis, I. de Curtis
The Rho family of small GTPases is implicated in neuronal differentiation. We have identified the
Rac3/Rac1B gene, a Rho family member expressed in the nervous system, implicated in neuronal development.
We have recently obtained knock out mice for the Rac3 gene. These mice develop normally, are fertile, and do
not show any obvious morphological defect. On the other hand, Rotarod experiments show a clear and significant effect in mice lacking the Rac3 protein, which suggest improved learning of motor tasks, and improved
memory of these abilities. In situ hybridization on wild type animals has shown accumulation of Rac3 mRNA in
the nervous system, with particularly strong signals in Dorsal Root Ganglia (DRG), in the CA1-CA3 region of
the hippocampus, and in some layers of the developing cortex. Current analysis includes more behavioural tests
aimed at identifying other sensory or behavioural phenotypes, and fine morphological analysis of the developing central nervous system, with particular emphasis on the synapses at regions of highest Rac3 expression.
Mechanisms of Rac3-mediated neuronal differentiation
S. Gualdoni, S. Corbetta, I. de Curtis
Based on our previous studies on the role of Rac3 in neuronal development in vitro, and on the high and specific expression of the Rac3 transcript in the CA1-CA3 regions of the hippocampus, we have started the analysis of hippocampal neuronal cultures obtained from wild-type and Rac3 knock-out animals. Preliminary analysis shows that Rac3 deficiency results in the partial disorganization of the dendritic tree of cultured neurons,
and in reduced synaptogenesis, as detected by immunostaining with markers of synaptic sites. This analysis is
aimed at identifying the role of Rac3 in the developing neuron, in a setting where the effects of Rac3 depletion
may be enhanced when compared to the “in vivo” environment. Given the functional interactions recently
identified by us between Rac3 and the GIT/p95 complex, this culture system will be fundamental to further explore the relationships between the function of Rac3 and GIT1/PIX during neuronal development. Given the
implication of PIX and other Rac regulators and effectors on mental retardation, this analysis, with the analysis
performed in vivo, is important to establish possible molecular mechanisms responsible for cognitive defects.
Characterization of the Liprin - p95-APP1 complex
M. De Marni, I. de Curtis
GIT1/p95-APP1 is a complex protein, which has both a scaffolding function, by recruiting together a number of proteins in a stable complex, and enzymatic activity, by acting as an ArfGAP. We have identified the
adaptor protein Liprin-alpha as a new component of the GIT1/p95-APP1 complex, with the aim of clarifying
new functions of this ubiquitous protein. Liprins are a family of adaptor proteins involved both in the assembly
of synapses in developing neurons, and in the organization of focal adhesions in non-neuronal cells. We have
now identified the site of interaction of the ubiquitously expressed Liprin-alpha1 on the GIT1/p95-APP1
polypeptide, and we are in the process of clarifying the function of the p95-APP1 / Liprin complex in cells. Co-
expression of p95 with Liprin-alpha1 in fibroblasts induces the relocalization and colocalization of the latter
with p95 at endocytic membranes, thus implicating p95 in the recruitment of Liprins at specific sites in the cell.
Moreover, we are identifying the effects of Liprin-p95 interactions on the organization of integrins and focal adhesions.
Foxg1 confines hippocampal morphogenesis to the dorso-medial pallium
L. Muzio, A. Mallamaci
Molecular mechanisms controlling areal-laminar specification of neurons within the developing cerebral cortex are largely obscure. In this context, of special interest is the recent report that Foxg1, encoding for a transcription factor expressed within the telencephalon along a rostral/lateral/high-to-caudal/medial/low gradient,
is necessary to cortical neuroblasts to switch from early generation of primordial plexiform layer to late production of cortical plate. We reanalyzed Foxg1(-/-) mutants and found that overproduction of Cajal-Retzius neurons characterizing them does not specifically arise from blockage of laminar histogenetic progression of neocortical neuroblasts but rather reflects lateral-to-medial repatterning of their cortical primordium. Even if lacking a neocortical plate, Foxg1(-/-) embryos give rise to structures, which, for molecular properties and birthdating profile, are highly reminiscent of hippocampal plate and dentate blade. Remarkably, in the absence of
Foxg1, further inactivation of the medial cortical fates promoter Emx2, while not suppressiing cortical specification, conversely rescues overproduction of Reln(on) neurons.
Emx2 regulates the areal growth profile of the embryonal cortical primordium
L. Muzio, J.-M. Soria-Lopez, M. Pannese, S. Piccolo, A. Mallamaci
It has been shown that canonical Wnt signalling active within the dorso-medial cortical primordium, stimulates transcription of Emx2, which promotes development of occipital cortex and hippocampus. We found
that, within the same region, Emx2 is in turn necessary for proper activity of the canonical Wnt pathway. Collapse of the resulting positive regulatory loop occurring in Emx2 null mutants results in severe, area-specific
distortion of cortical neuroblasts proliferation-differentiation profile, due to misregulation of cell cycle-,
proneural- and lateral inhibition-molecular machineries, and leads to dramatic and selective size-reduction of
occipito-hippocampal cortex. In the same mutants, reactivation of the Wnt signalling pathway corrects a subset
of Emx2(null)-specific molecular abnormalities and partially rescues the areal kinetic phenotype.
Generation of mutant mouse models for the study of aetiopathogenesis of human Williams-Beuren
M. Pannese, S. Capossela, L. Muzio, L. Pintonello, A. Mallamaci
Williams-Beuren Syndrome (WBS) is a complex developmental disorder affecting about 1 in 20,000 people.
It includes cerebral cortex dysplasias, mental retardation, and a peculiar cognitive-behaviourial profile, with
relative strength in auditory memory and weakness in visual-spatial cognition. Most WBS patients are hemizygous for a common deletion in 7q11.23, including almost twenty genes; their contribution to the genesis of this
profile is currently subject of debate. We inactivated two WBS-specific genes in ES cells, Fzd9, encoding for a
Wnt-receptor specifically expressed in the embryonic caudal-medial cortex, and Wbscr1, encoding for an ATPase/RNAhelicase expressed at high levels in the adult hippocampus. Moreover we generated mice knock-out
for the latter one. Their phenotype, including reduced body weight, sterility and peculiar neurological traits, is
currently under investigation. Moreover, to score contributions of other genes in the WBS canonical deletion to
the origin of the disease, we are preparing a set of nested deletions in the murine genomic region synthenic to
human 7q11.23.
Interaction of transcription factors during gene transcription
A. Agresti, P. Scaffidi, A. Riva, V. R. Caiolfa, M. E. Bianchi
Most nuclear proteins reside on a specific chromatin site only for seconds or less. The hit-and-run model of
transcriptional control maintains that transcription complexes are assembled in a stochastic fashion from such
freely diffusible proteins; this contrasts to models involving stepwise assembly of stable holo-complexes. However, the chances of forming a productive complex improve if the binding of one factor promotes the binding of
its interactors. We have shown that in living cells the glucocorticoid receptor and HMGB1 interact only within
chromatin and not in the nucleoplasm, and decrease each other’s mobility. Thus, the formation of a GRHMGB1-chromatin complex is more likely than one would expect from independent binding to chromatin of
GR and HMGB1. Remarkably, such complex is potentially stable, and its disassembly is effected by active,
ATP-consuming processes. We propose that kinetic cooperativity among transcription factors in chromatin
binding may be a common feature in transcription and DNA transactions.
Extracellular HMGB1 induces stem cell migration and proliferation
R. Palumbo, M. Sampaolesi, F. De Marchis, R. Tonlorenzi, S. Colombetti, A. Mondino, G. Cossu, M. E. Bianchi
High mobility group box 1 (HMGB1) is an abundant chromatin protein that acts as a cytokine when released
by necrotic and inflammatory cells. Extracellular HMGB1, binding to its receptor RAGE (receptor for advanced glycation end products), induces both migration and proliferation of stem cells. In particular, vessel-associated stem cells (mesoangioblasts) respond most vigorously, but bone marrow derived stem cells respond
too. In vitro, HMGB1 induces migration and proliferation of both adult and embryonic mesoangioblasts, and
disrupts the barrier function of endothelial monolayers. In living mice, mesoangioblasts injected into the
femoral artery migrate close to HMGB1-loaded heparin-Sepharose beads implanted in healthy muscle, but are
unresponsive to control beads. Interestingly, alpha-sarcoglycan null dystrophic muscle contains elevated levels
of HMGB1; however, mesoangioblasts migrate into dystrophic muscle even if their RAGE receptor is disabled.
This implies that the HMGB1–RAGE interaction is sufficient, but not strictly necessary, for mesoangioblast
homing; a different pathway might coexist. Thus, injected HMGB1 may be used to promote tissue regeneration.
Chromatin and cell death
M. E. Bianchi, L. Trisciuoglio, A. A. Manfredi
HMGB1, a very mobile chromatin protein, leaks out from necrotic cells and signals to neighbouring cells that
tissue damage has occurred. At least one receptor for extracellular HMGB1 exists, and signals to different cells
to divide, migrate, activate inflammation or start an immune response. Remarkably, apoptotic chromatin binds
HMGB1 irreversibly, thereby ensuring that it will not diffuse away to activate responses from neighbouring
cells. Thus, dying cells use their own chromatin to signal how they have died. Nuclear events happening during
apoptosis serve to control the molecular signals that dying cells send out.
See: Gene and Stem Cell Therapy
A Drosophila model for DRPLA
I. Nisoli, S. Frontini, B. Charroux, S. Kerridge, H. McNeill, M. Fanto
Dentatorubropallydoluisian Atrophy (DRPLA) is a dominantly inherited neurodegenerative disease very similar to Huntington’s and due to polyglutamine expansion in the atrophin-1 gene. In Drosophila there is only
one Atrophin, Atro, a transcriptional co-repressor required for several developmental processes. Atro has also a
role in the cytoplasm where it binds to atypical cadherins and is involved in cell polarity and cell adhesion. We
are undertaking several lines of research to address the multiple functions of Atro in flies and also to build a
Drosophila model for DRPLA by generating transgenics with different forms of human Atrophins and also by
expanding the polyglutamine stretches found in Atro itself. Given that Atro is the best characterised fly equivalent of a polyglutamine disease protein, it will be possible for the first time to combine loss and gain of function
genetic approaches to study neurodegeneration by polyglutamine in Drosophila. Once the model will be extensively characterised genetic and genomic screenings will be set up to identify genes that interact with Atro and
that are transcriptionally regulated by it.
Analysis of Atrophin’s role in ommatidial symmetry and Notch signalling
I. Nisoli, M. Mlodzik, M. Fanto
The compound eye of Drosophila melanogaster is made of 800 units, the ommatidia, made each of 8 photoreceptor neurones (R1-R8) all arranged in a stereotypical trapezoid pattern, which set up an internal asymmetry
between the anterior and the posterior half of each ommatidium. The cells R3 and R4 are the ones responsible
for establishing this asymmetry. A molecular cross-talk between these two neighbouring cells where the opposite cell fates of R3 and R4 are determined involves the Notch receptor. However different formats of the Notch
pathway exist and it is debated how the R3/R4 cell fate choice is achieved. We have data that confirm our initial
proposal that this is a typical lateral inhibition paradigm where a signal sent by the R3 inhibits the adoption of
the R3 cell-fate in R4. In characterising the pathway responsible for this cross-talk we are focusing in particular
on Atro, which appears to act as a negative regulator of Notch. We are testing the possibility that Atro acts in
the nucleus to repress genes normally activated by Notch or that it may promote the post-endocytic degradation of the receptor itself.
Drosophila chorein gene family
S. Montrasio, L. Rapoldi, V. Broccoli, M. Fanto
Chorea achantocytosis (CHAC) is a recessive neurodegenerative disease leading to extensive atrophy of the
striatum and resembling Huntington with an additional star-shaped erythrocytic phenotype in the blood. The
disease is caused by the chorein gene, whose function is unknown, but whose yeast and Dictostelium counterparts may be implicated in intracellular vesicular sorting. Chorein is one of four highly related proteins, all of
which are evolutionarily preserved in Drosophila. We have identified a series of small chromosomal deficiencies
and P-element insertions in all four genes, and are now trying to address the precise function of the proteins. A
mouse model for CHAC as been recently produced, displaying only slight behavioural alterations despite increased apoptosis in the striatum. We plan to build a fly model of CHAC, to be used in parallel to the mice
Dynamics and oligomerization of membrane proteins in living cells by single molecule fluorescence
fluctuations and time resolved spectroscopy: uPAR, a GPI-anchored receptor
G. Malengo, M. Zamai, A. Andolfo, F. Blasi, N. Sidenius, V. R. Caiolfa
Many adhesion receptors are anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI)
moiety and are thereby partitioned into membrane rafts. Rafts can facilitate hierarchical interactions and
oligomerization of the GPI-anchored receptors leading to the assembly of adhesion complexes. It has been
demonstrated that the urokinase plasminogen receptor (uPAR) undergoes dimerization (Cunningham et al.,
2003; Sidenius et al., 2002). We are studying the dynamics of uPAR monomers and dimers in living cells and the
effect of specific ligands. Using our novel FCS platform combined with FLIM, we have demonstrated that the
distribution of receptor dimers is heterogeneous. We also measure different motilities (from 1 ms to more than
100 ms) for both uPAR monomers and dimers, which indicate the presence of the receptor in rafts and non-raft
membrane domains.
The genetics of human mental retardations
P. D’Adamo, C. Sala, F. Cardone, M. Goegan, A. Sirri, D. Toniolo
The group is involved in the genetic characterization of common disorders. Progress were made toward the
functional analysis of human Mental Retardation (MR), a highly heterogeneous disorder affecting >2% of children. A gene for MR identified by our group, GDI1, encodes a protein controlling the activity of the Rab GTPases in intracellular trafficking. We have analyzed the phenotype of a mouse knock out of Gdi1, that was viable and fertile. Mice were normal in many behavioral and cognitive tasks, but they were impaired in tasks requiring formation of short term memory. EM analysis of synapses of WT and KO mice showed that the number
of reserve synaptic vesicle in the KO is reduced and that the mouse seem to have a defect in biogenesis of
synaptic vesicle that may explain the behavioral phenotype.
The genetics of Premature Ovarian Failure (POF)
C. Sala, S. Bione, F. Rizzolio, M. Goegan, S. Alboresi, S. Gilli, D. Toniolo
One of the aim of the work of the group is the genetic dissection of X-linked ovarian failure, POF, a form of
hypergonadotropic amenorrhea affecting 1% of women. The analysis of a large number of X;autosome balanced translocations in the POF “critical region” has shown that few genes were interrupted by the rearrangements and has suggested that translocations may cause a position effect on flanking X-linked or autosomal
genes. This was verified by chromatin immunoprecipitation (ChIP) and gene expression analysis that identified
a number of genes flanking the breakpoints that were affected and may be responsible for ovarian failure. The
role of candidate genes is sistematically verified by mutation analysis in a large collection of POF patients with
normal caryotype. Association of POF to SNPs in the DIAPH2 gene was demonstrated. The DIAPH2 gene is
the first indication that risk factors and not genes inherited as mendelian factors may be a common cause of
Genetics of migraine
P. Carrera, C. Montrasio, S. Balan, S. Battistini, J. Striessnig, M. Ferrari
We are involved in the identification and functional characterization of CaV2.1 gene variants, associated with
Familial Hemiplegic Migraine. Searching for mutations in the CACNA1A gene is performed using DHPLC
and direct sequencing. Functional studies are carried out in collaboration with Prof. J. Striessnig in the frame of
the Network on Neuronal Calcium Channels in Disease (http://calcium.ion.ucl.ac.uk/). Moreover, our group
developed a specific database for FHM accessible on the web (fhmdb.org). The FHMdb aims at collecting
published clinical and genetic data of affected family members. Moreover, electrophysiological data on the various mutations are collected. Such comprehensive and standardized data collection will allow a more efficient
and accurate genotype-phenotype correlation. To investigate the role of the CaV2.1 as a predisposing factor in
common Migraine, we set up a genetic association study in a patient-control format. As a pilot study, we ana-
lyzed on a DNA bio-chip platform, two coding SNPs located in the LII-III domain. Preliminary results indicate
that these variants do not represent a predisposing factor for Migraine.
Molecular analysis of lamin A/C associated pathologies
S. Benedetti, I. Menditto, S. C. Previtali, C. Rodolico, E. Bertini, D. Toniolo, P. Carrera, M. Ferrari
Mutations in the nuclear envelope protein lamin A/C have been associated to a variety of pathologies affecting mainly muscular, nervous and adipous tissues, but no clear genotype-phenotype correlation has been established. The analysis of 140 patients with different phenotypes revealed 20 alterations, 14 of which had not been
previously described, including a conservative substitution leading to a splicing mutation confirmed at the
RNA level both in silico and in vitro. However, discrepancies in the results of theoretical and experimental
RNA analysis were evidenced for other conservative variants identified, underlining the need of a careful interpretation of molecular data. Our analysis suggests that lamin A/C alterations are not only associated to a variety
of clinical presentations, but also to a diverseness of genetic variants even in the same familiy, challenging the
elucidation of pathogenetic mechanisms. Histopathological, in vitro and proteomic studies now underway on
lamin A/C mutated samples will help identifying differences in direct interactors and pathways modulated by
the mutant protein.
Development of advanced technologies for mutational scanning and detection in disease genes
M. Ferrari, L. Cremonesi, B. Foglieni, N. Soriani, S. Stenirri, I. Fermo
Conditions were set up for DHPLC scanning of 50 exons of the ABCA4 gene involved in Stargardt macular
dystrophy, resulting in the identification of 26 mutations in 29 out of the 30 patients screened, including16 mutations never reported before. We set up a microchip for the identification of mutations within IRE structure of
the ferritin-L gene, where 30 mutations causing Hereditary Hyperferritinemia Cataract Syndrome (HHCS)
have been reported. This represents a unique model to evaluate the influence of different parameters such as
probe and stabilizer design. Conditions to detect 24 mutations, including 4 deletions from 2 to 28 nucleotides
and 20 substitutions among which 7 conservative transversions were developed. The same cartridge could be
serially hybridized with all the 24 probe sets allowing correct genotyping till the end of the analysis. No crosshybridization was found even for mutations affecting the same nucleotide. Blind validation on 200 samples gave
total concordance of results. This represents a first step towards the development of a diagnostic microchip for
high-throughput analysis and screening of mutations associated with iron disorders.
Fetal DNA in maternal plasma: physiological aspects of fetomaternal transfer
L. Cremonesi, S. Galbiati, M. Ferrari, L. Valsecchi, A. Ferrari, M. Smid
Since the identification of fetal DNA in maternal plasma we focused on assessing whether maternal plasma
could be used to develop noninvasive prenatal tests. In our study on 1837 pregnancies we showed that fetal
DNA can be retrieved in all gestations with a close to 100% accuracy in fetal gender detection by 6 weeks. Our
data showed that placental pathological conditions associated with preeclampsia, Intra Uterine Growth Restriction (IUGR) and trisomy 21 substantially increase fetal DNA concentration in maternal plasma, indicating that
fetal DNA quantification is a potential marker for monitoring these diseases. These findings definitively indicate that fetal DNA in maternal plasma may represent an optimal source of fetal genetic material for noninvasive prenatal testing. Our main goals is to set up advanced methodologies for noninvasive prenatal diagnosis of
genetic disorders. To this aim we are developing microchip-based tests to detect mutations causing beta-thalassemia which will be applied to the analysis of maternal plasma in couples at risk for this disease. This will be
the basis for developing additional assays to identify mutations in other genetic disorders.
Mouse models of the Charcot-Marie Tooth type 4B1 neuropathy
A. Bolis, S. Coviello, G. Dina, S. C. Previtali, U. Del Carro, L. Feltri, A. Quattrini, L. Wrabetz, A. Bolino
Charcot-Marie-Tooth type 4B (CMT4B1) disease is a severe autosomal recessive peripheral neuropathy with
childhood onset, characterised by myelin outfoldings in the peripheral nerve and azoospermia. We first demonstrated that CMT4B1 is caused by loss of function mutations in the Myotubularin-related 2 gene, MTMR2.
MTMR2 is a ubiquitously expressed phosphatase whose preferential activity is toward PI(3,5)P2 a key regulator of vacuolar homeostasis and vesicle transport. We generated Mtmr2-null mice which reproduce the
CMT4B1 pathology. These mice develop a progressive neuropathy with myelin outfoldings which predominantly arise from paranodal myelin and defects in spermatogenesis. We also produced two conditional mutant
mice in which Mtmr2 is specifically inactivated in either Schwann cells or motorneurons. Only when Mtmr2 is
disrupted in Schwann cells a peripheral neuropathy is observed. This information is useful to design a therapeutical approach aimed at replacing the enzyme in the nerve of Mtmr2-null mice.
Role of Dlg1/SAP97 in myelinating Schwann cells
A. Bolis, S. Bussini, S. Coviello, G. Dina, L. Feltri, V. Broccoli, A. Quattrini, L. Wrabetz, A. Bolino
We found that Mtmr2 phosphatase interacts with Dlg1/SAP97, a scaffolding molecule belonging to the
MAGUK (Membrane Associated Guanilate Kinase – like) family of proteins. Dlg1 homologues have been located in several types of cellular junctions and play role in cell polarity and membrane addition. We first
demonstrated Dlg1 expression in Schwann cells. Dlg1 is enriched at paranodal myelin, a region flanking the
node of Ranvier, where thight, adherens, and gap junctions are located between adjacent loops of Schwann cell
membrane thus establishing cell polarity. In Mtmr2-null mouse nerve fibers Dlg1 is not detected at paranodal
myelin. Our project is now aimed at defining the molecular partners of Dlg1 in Schwann cells to understand the
molecular mechanism at the basis of CMT4B1 pathology. We will also disrupt Dlg1 specifically in Schwann
cells to elucidate the role of this molecule in the nerve.
Adhesion and the onset of genetic programs in normal and neoplastic cells
R. Pardi, G. Rotondo, G. Rossetti, M. Savio, S. Putignano
Most somatic cells require adhesion to substrate for progression through the cell cycle and the onset of DNA
replication in response to growth factors. This phenomenon is known as “anchorage-dependence” (AD), and is
thought to be largely dependent on active signaling by surface integrins, a highly conserved family of adhesion
receptors involved in cell-extracellular matrix, as well as cell-cell adhesion. Notably, reduced anchorage dependence is a feature of cancer cells, suggesting that adhesion-regulated processes are constitutively active in
transformed cells. We have recently identified a protein, JAB1 (Jun Activation domain Binding protein 1),
which interacts both in vitro and in vivo with the cytoplasmic domain of beta-1 and beta-2 integrin subunits.
JAB1 may therefore represent a novel intracellular adaptor relaying integrin-dependent signals to the control of
gene expression and the ubiquitin-mediated degradation of critical effectors involved in cell cycle progression.
We have generated cellular as well as animal models to validate the hypothesis that JAB1 and its interacting
partners are required to effect integrin-mediated events controlling the onset og genetic programs.
Integrin dynamics in migrating leukocytes
R. Pardi, M. Fabbri, R. Molteni, S. Di Meglio
Inflammatory cell migration is a central process in the pathogenesis of chronic inflammatory diseases, including arthritis, atherosclerosis and asthma. Identifying novel targets for anti-inflammatory drugs is a formidable
intellectual challenge requiring a multi disciplinary approach. Cell migration implies the existence of mechanisms that move adhesion receptors from the cell rear toward the cell front, where they are available to form
new protrusions and adhesions. One such mechanism may involve regulated endo-exocytosis of integrins, a
family of heterodimeric transmembrane receptors mediating cell-cell or cell-extracellular matrix adhesion. By
using a combination of quantitative imaging and biochemical analysis we have demonstrated that the endo-exocytic cycle of integrins is required to establish and maintain the polarized phenotype of directionally migrating
leukocytes. We further established a novel role for beta-arrestins in sustaining the signaling pathways triggered
by chemotactic cytokines. These findings identify potential molecular targets for novel and more specific antiinflammatory drugs.
Polycystin-1 Signaling
A. Casanova, G. Distefano, A. Boletta
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is characterized by bilateral renal cyst formation.
Polycystin-1 (PC-1), the gene product of PKD1 responsible for 85% of all cases, is a large (520kDa) non-tyrosine kinase receptor, whose ligand(s) and function are largely unknown. We showed that PC-1 induces activation of the PI3kinase/Akt pathway thereby mediating resistance to apoptosis. We are currently investigating the
mechanism through which PC-1 activates this pathway. We have identified two highly conserved putative
polyproline domains in the intracellular C-terminus of PC-1. One is predicted with high probability to bind to
the SH3 domain of the p85 subunit of PI3kinase. In order to test whether this interaction occurs and/or other
SH3-domain-containing molecules bind to PC-1, we have carried out an overlay assay approach to screen for
152 proteins containing SH3 domains using PC-1 C-terminus as a probe. We have identified 16 molecules
diplaying strong binding capacity. We have confirmed by GST pull-down that one of these molecules is interacting with wt, but not mutant PC-1, and are currently investigating the physiological role of such interaction.
Studies on the function of polycystin-1 in renal epithelial cells
M. Boca, D. Deflorio, A. Boletta
Cyst formation in the renal tubule is a common feature of many diseases. It is believed that this phenotype results from perturbing of the cell polarity properties of renal epithelial cells. Autosomal Dominant Polycystic
Disease (ADPKD) is the most common cause of renal cystogenesis. Polycystin-1 (PC-1), the product of the
PKD1 gene mutated in the disease, is postulated to be involved in cell-cell/matrix interactions. We have shown
that PC-1 expression in renal epithelial cells determines reduced growth rates, resistance to apoptosis and
spontaneous tubulogenesis. We now find that PC-1 induces profound cytoskeletal re-arrangements, cell scattering and reduced cell-cell adhesion. We performed time-lapse videomicroscopy of wound healing assays to assess the migratory properties of PC-1 expressing cells. Interestingly, while controls move forward as a compact
unit PC-1 expressing cells acquire a polarized migratory shape and have faster migration. Concomitantly, Ecadherin appears to be quickly internalized. PI3kinase inhibitors prevent cell migration, but not reduced cellcell adhesion. We are investigating the role of downstream effectors of PI3 kinase in PC-1 induced migration.
Characterization of the developmental defects in the embryonic kidneys of Pkd1-/- mice
I. Rowe, C. Wodarczyk, A. Boletta
Several different mice lines carrying homozygous truncations of the Pkd1 or Pkd2 genes (the mouse orthologues of the two genes mutated in Autosomal Dominant Polycystic Kidney Disease) were reported to die in
utero at day E15.5 and to have a dramatic renal cystic phenotype. During embryonic renal development a series
of successive steps take place resulting in generation of the permanent kidney or metanephric kidney starting at
day 10.5-11.5 of gestation in the mouse. At this stage the Ureteric Bud (UB), a small epithelial outgrowth from
the wolfian duct, is induced to invade the metanephric blastema (or metanephric mesenchyme, MM) by secretory signals released by the latter. Upon invasion of the metanephric blastema the UB is induced to branch and
elongate by signals released by the MM, while each tip of the UB induces the mesenchyme to condensate and
undergo a program of mesenchymal-epithelial transition eventually leading to the formation of the glomerulus.
Using a series of ex vivo and in vivo approaches we are characterizing the different stages of renal development
in a mouse model carrying null alleles for the Pkd1 gene to understand its function in this process.
The embryonal phenotype of the Prep-1 hypomorph mice
F. Blasi, E. Ferretti, D. Penkow, P. Di Rosa, J. C. Villaescusa, E. Longobardi, A. Mondino, L. Fernandez,
G. Ferrari
We studied the phenotype resulting from two different mutations in the murine Prep1 gene. The first mutation knocks out the gene expression and gives a lethal phenotype at E7.5. This makes it difficult to study any
Prep1 related function. The second mutation does not knock out the gene, it only reduces its expression down
to 2-8% of the wt expression level. This hypomorphic mouse shows several phenotypes including embryonic
lethality, edema, pallor, small liver and alterations in angiogenesis and hematopoiesis, all characterized by a variable penetrance. In particular, the hematopoietic effect consists in a strong reduction of LTR-HSC and in a reduced number of the circulating T cells. Prepi/i embryos die variably between E16.5 –19.5 with a 4% of survival.
We studied in embryo and in adult the thymic phenotype of the Prep-1 hypomorph mouse. The Prep1i/i
mice that survive the embryonic lethal phenotype live at least 8 months and are apparently normal, with no obvious phenotype. However, CD4+ and CD8+ T cells are strongly decreased in hypomorph mice compared to
wt and heterozygous mice. In T cell maturation in thymus, mutant mice present some increased subpopulations
of double negative cells, while the single positive cells are decreased. Moreover, we observed a delay in TCR rearrangement and an enrichment for the gamma-delta T cells. From a molecular point of view, there is a complete absence of Pbx proteins in the thymus of the hypomorph mice, with no DNA binding activity of Pbx and
Prep complexes. Therefore, Prep1 seems to be involved in different steps of T cell maturation, even in the positive selection.
Revisitation of the role of the PM and PH sites in HoxB1 expression
F. Blasi, E. Ferretti, E. Longobardi, R. Krumlauf
Prep-1 protein forms a ternary complex with Pbx proteins and HoxB1. This is an essential complex for
HoxB2 but not for HoxB1 expression. We have revised the HoxB1 enhancer structure and demostrated that it
is more complex than previously described. In fact, the enhancer contains sequences that can activate or inhibit the formation of the ternary complex. Furthermore, there are at least two sequences that can activate ternary
complex formation, so the mutation of only one does not have any effect on HoxB1 expression. These two sequences can drive in vivo expression of a reporter protein in the 4th rhombomer. Finally, we have demonstrated that the mutation of both sequences blocks in vivo HoxB1 expression. All these data demonstrate the essential role of the Prep1-Pbx-HoxB1 ternary complex on the HoxB1 gene expression in human and chicken.
Identification of an integrin binding site in the urokinase receptor
F. Blasi, B. Degryse, R. P. Czekay, D. Loskutoff, M. Resnati, I. Pallavicini
We have demonstrated that the chemotactic activity of vitronectin requires the presence of the urokinase
recptor, uPAR, and avb3 integrin. We identified an internal sequence of uPAR that mediates the chemotactic
function of vitronectin. A peptide derived from this sequence promotes cell migration in several cell lines in a
manner different from the uPA-uPAR-activated pathway. Mutations in this sequence eliminate the chemotactic
activity and transform this peptide in an inhibitor of the vitronectin-induced chemotaxis. This finding has been
submitted for a patent.
The linker region between domain D1 and domain D2 of uPAR is highly susceptible to endoproteolysis, and
after cleavage it is endowed with potent chemotactic activity. Fragments of cleaved uPAR are found in blood,
urine and tissues of patients with several diseases, including leukemias and cancer. In order to identify and
quantitate the presence of these fragments and eventually to block their biological functions (i.e. chemotaxis),
we have generated monoclonal antibodies specific for the N-terminal AVTY sequence that is required for the
chemotactic activity.
These antibodies recognize only fragments containing at their N-termini the AVTY epitope in several assays,
including ELISA, immuno-precipitation, cytofluorimetry and immuno-blotting. We have also developed an immunofluorimetric method to quantitatively assess the presence of fragments of uPAR in blood and urine samples. Finally we have identified two antibodies that inhibit uPA-induced chemotaxis in cultured cells. These data have been used for a patent.
Characterization of MNase-resistant chromatin fragments in the minimal promoter region of the human
uPA gene reveals the interaction of the enhancer with a single nucleosome population and looping of the
intervening sequence
M. Crippa, F. Blasi, C. Ferrai, C. Doglioni, L. Pecciarini, M. G. Cangi
Using chromatin immunoprecipitation (ChIP) coupled with micrococcal nuclease (MNase) digestion, we
studied the interaction between the minimal promoter (MP) and the enhancer (E) of the human urokinase gene
in PC3 cells. We first demonstrated that antibodies against Sp1, that specifically binds the MP, and p300 immunoprecipitate both E and MP sequences. MNase digestion of cross-linked chromatin from PC3 cells allowed
us to identify a number of large, cleavage-resistant fragments in the region of the MP, but not in the E region.
Furthemore, we show that these fragments possess specific chromatin compositions, which might be associated
with different transcription states of the uPA gene. In particular, a specific MNase resistant fragment contains
the elongating form of RNA Polymerase II (RNAP II), phosphorylated in serine 2 of the CTD. Interestingly,
only a nucleosome core-size fragment (145 bp) that spans the enhancer shares the same exact chromatin composition, HMGN protein and RNAP II content of the structure on the MP. These results provide evidence on
the proximity of the enhancer and the minimal promoter of the uPA gene by looping of the intervening sequence. Furthermore they support the presence of multiple chromatin populations of the MP engaged in different steps of transcription.
Striatal creb plays opposite roles in striatum-dependent habit formation and drug-reinforced learning
S. Fasano, R. Brambilla
The striatum participates in several forms of behavioral adaptation, including habit learning and the response
to addictive drugs. The CREB family of transcription factors is implicated in various forms of learning and in
addiction, though its role in striatum-dependent habit learning has not previously been explored. We examined
the role of striatal CREB in habit learning and drug-reinforced learning using transgenic mice. We found that
striatal CREB modulates them in opposite ways. Reversible inhibiton of CREB-family transcription factors in
the striatum of transgenic mice produces a long-term deficit in two striatum-dependent learning tasks, active
and passive avoidance. In contrast, inhibition of striatal CREB facilitates cocaine-reinforced conditioned place
preference and enhances locomotor sensitization to cocaine. These findings implicate CREB as a positive regulator of striatum dependent habits but a negative regulator of drug-reinforced memories. Cocaine has previously been shown to lead to increased activation of striatal CREB; since CREB is a negative regulator of some drug
responses, this may represent a homeostatic response to repeated drug exposure.
Ras-grf1 regulates striatum-dependent behavioural plasticity and long term synaptic signaling
S. Fasano, A. D’Antoni, R. Brambilla
The striatum is a collection of several nuclei involved in a variety of behaviors and brain diseases, including
drug addiction, Huntington’s and Parkinson’s diseases. Synaptic mechanisms governing neural adaptations in
the striatum are still poorly understood. We have recently demonstrated that ERK pathway is a major determinant of long-term plasticity and memory formation in this brain region. However, the molecular links between
glutamatergic and dopaminergic receptors and intracellular signalling in the striatum are at present not characterized. By means of genetic manipulation in the mouse striatum we show that the neuronal-specific Ras exchange factor RasGRF1 is necessary for both glutamate- and dopamine-dependent ERK activation and immediate early gene expression. Consequently, by modulating RasGRF1 activity in vivo we observe alterations in
the process of long term memory formation, as revealed by striatum-specific learning tests. This phenotype is
consistent with a clear involvement of RasGRF1 in striatal long term potentation (LTP). Our results suggest a
crucial function of RasGRF1 in modulating ERK signaling in the striatum.
Animal models to study cognitive disorders associated to the neurofibromatosis type 1 (nf1) disease
I. Formentini, M. Martignoni, S. Fasano, R. Brambilla
Neurofibromatosis type 1 (NF1) is one of the most common dominantly inherited diseases, affecting approximately 1 in 3500 individuals. A large percentage (30-45%) of NF1 patients manifest mental retardation and
learning deficits, suggesting dysfunction in some, yet to be identified, brain structures. NF1 protein, also
known as neurofibromin, is a GTPase activating protein (GAP) specific for Ras proteins. We have addressed
the implication of neurofibromin-dependent signalling in the learning process by means of conditional mutagenesis using the CRE-Lox system in the mouse. CRE mediated recombination driven exclusively in excitatory
neurons of the central nervous system, leads to the expression of an oncogenic, constitutively active form of KRas (G12V), the major substrate of neurofibromin. We demonstrated that K-Ras G12V is activated in most part
of the forebrain. Moreover, these mice show significant impairments in associative learning and memory tasks.
These data indicate that an hyperactivation of Ras signalling is sufficient to recapitulate the cognitive symptoms
observed in NF1 patients and could represent a valid experimental model for the neurofibromatosis type 1 disease.
Dissecting the molecular basis of ocular albinism type 1: OA1 is a canonical GPCR exclusively localized to
intracellular organelles
G. Innamorati, R. Piccirillo, P. Bagnato, I. Palmisano, M. V. Schiaffino
The protein product of the gene responsible for ocular albinism type 1, named OA1, is a pigment cell-specific glycoprotein, displaying structural features of G Protein-Coupled Receptors (GPCR) and exclusively localized to intracellular organelles, i. e. melanosomes and lysosomes. To investigate the molecular function of OA1,
we have utilized a heterologous expression system commonly exploited to study receptor signaling. In these experimental conditions, OA1 displayed a significant and spontaneous capacity to activate heterotrimeric G proteins and the associated signaling cascade. Moreover, OA1 was phosphorylated and showed the ability to functionally interact with arrestins, well-established regulators and adaptors of GPCRs. In fact, arrestins were co-localized and co-precipitated with OA1 and were found to efficiently inhibit its signaling. These findings demonstrate that OA1 behaves as a canonical GPCR and support the hypothesis that it triggers a signal transduction
cascade at the melanosomal/lysosomal membranes to direct proper organelle biogenesis. Our results imply that
GPCR-mediated signal transduction systems can also work at the internal membranes in mammalian cells.
Exploring the biology of beta-cells: role of heterotrimeric G proteins on insulin secretory granules
I. Palmisano, P. Bagnato, G. Innamorati, R. Piccirillo, M. V. Schiaffino
Heterotrimeric G proteins, best known as transducers of plasma membrane receptors, are also associated
with internal organelles, suggesting that analogous signaling systems operate at these locations. In ß-cells, G-α-i
has been identified on insulin secretory granules and has been implicated in insulin secretion. To investigate the
role of G proteins localized to these organelles, we generated a protein chimera consisting of a resident protein
of insulin granules, IA-2, fused with a constitutively active form of G-α-i. This approach allowed us to enrich
the target organelles with the relevant G protein and selectively activate its downstream pathway at the intracellular location. Expression of the IA-2/ G-α-i fusion protein in beta-cells resulted in the perinuclear aggregation
of insulin granules, suggesting a role of G proteins in controlling organelle transport to the cell periphery. In
contrast, a protein chimera containing a constitutively inactive form of G-alpha-i behaved as IA-2 alone. Further studies are in progress to identify the molecular machinery involved in the aggregation process and determine the effects of this phenomenon on insulin secretion.
A dynamic podosome-like structure of epithelial cells
L. Spinardi, J. Rietdorf, L. Nitsch, M. Bono, C. Tacchetti, M. Way, P. C. Marchisio
Focal contacts and hemidesmosomes are cell-matrix adhesion structures of cultured epithelial cells. While focal contacts link the extracellular matrix to microfilaments, hemidesmosomes make connections with intermediate filaments. We have analyzed hemidesmosome assembly in 804G carcinoma cells. Our data show that
hemidesmosomes are organized around a core of actin filaments that appears early during cell adhesion. These
actin structures look similar to podosomes described in cells of mesenchymal origin. These podosome-like
structures are distinct from focal contacts and specifically contain Arp3 (Arp2/3 complex), cortactin, dynamin,
gelsolin, N-WASP, VASP, Grb2 and src-like kinase(s). The integrin a3b1 is localized circularly around F-actin
cores and co-distributes with paxillin, vinculin and zyxin. We also show that the maintenance of the actin core
and hemidesmosomes depends on actin polymerization, src-family kinases and Grb2, but not on microtubules.
Video microscopy analysis reveals that assembly of hemidesmosomes is preceded by recruitment of b4 integrin
subunit to the F-actin core prior to its positioning at hemidesmosomes.
The biological function of p27BBP/eIF6 and the control of ribosome biogenesis
S. Biffo, P. C. Marchisio, A. M. Barbieri, S. Tognin, C. Gaviraghi, C. Gorrini, M. Ceci, L. A. Sala, A. Donadini
The ribosome biogenesis control factor p27BBP/eIF6 was originally cloned and sequenced in this laboratory.
The gene and the protein have been earlier characterized and found to be overxpressed in human carcinomas.
In the last year it has been found that p27BBp is part of a large molecular complex that is required to control
ribosomal subunit traffic between the nucleus and the cytoplasm and this event occurs in a phosphorylation-dependent manner. A crucial role is played by protein kinase C and its receptor RACK1, which suggests that a
crucial step in protein synthesis may be controlled by signaling pathways common to adhesion and cell survival.
The laboratory is also involved in defining the structure of the human p27BBP gene and its promoter, as well as
in producing a mouse in which the gene for p27 is inactivated by homologous recombination.
Identification of ferroportin 1 ligands
L. Mascheroni, E. Pignatti, A. Pietrangelo, P. C. Marchisio, S. Biffo
Fp1 protein is mutated in a form of non-HFE inherited hemochromatosis. Fp1 is a transmembrane protein
whose precise topology, and mechanism of action in iron transport are unknown. Current evidence suggests
that it is targeted at the basolateral membrane of duodenal enterocytes and that is highly expressed in reticuloendothelial macrophages, but the factors (proteins) responsible of its targeting, the molecular basis for its
function as well as possible intracellular or extracellular partners are unknown. The goal of the project is to purify the Fp1 protein from cells and tissues in order to define its postranslational modifications, and interactorsthat result in its intracellular processing/targeting and iron-export function, thus providing a framework to understand the pathogenesis of Fp1 mutations. We have devised efficient procedures for the purification of Fp1
in native form and we are currently defining putative interactors.
Overexpression of p27BBP in head and Neck carcinomas and their lymphnode metastases
P. Rosso, F. Sanvito, B. Di Benedetto, A. Donadini, S. Biffo, P. C. Marchisio
p27BBP is a regulator of ribosome assembly and an essential nuclear and cytoplasmic component of eukaryotes.We investigated the immunochemical distribution of p27BBP in head and neck carcinomas, in the associated normal mucosa, and in regional lymph nodes.p27BBP is detectable in mucosal cells but is overexpressed
in carcinomas, highly concentrated in large polymorphous nucleoli, and even larger and more evident in lymph
node metastatic foci. Western blotting confirms increased p27BBP in carcinomas versus normal mucosa and also in metastatic versus normal lymph nodes. The overexpression of p27BBP corresponds to mRNA upregulation in carcinomas. Unexpectedly, a 52-kDa band specifically reacting with antibodies to p27BBP was observed
in several carcinomas. Conclusions: p27BBP alterations are common events in the transition to malignancy and
are probably involved in squamous carcinoma progression. Immune reagents raised to p27BBP may provide
additional diagnostic tools for surgical pathology of tumor boundaries and lymph nodes. The 52-kDa band may
represent an abnormal form of p27BBP expressed by transformed airway epithelia.
Molecular mapping of regulatory domains in the EGF-R molecule
L. Beguinot, M. Riolfo, A. Sorkin
By molecular genetic analysis we have identifed three major regulatory regions in the EGF-R C-terminus tail.
The very C-terminus with five autophosphorylation sites positively affects EGF-mediated transformation. Upstream of the autophosphorylation sites we identified a region of 30 aminoacids with an inhibitory activity on
transformation and required to phosphorylate two non-SH2 substrates. Further upstream we mapped a principal domain mediating EGF-dependent receptor internalization. Coated pits accumulation and rapid internalzation also require autophosphorylation on specific tyrosines and we have defined the specific tyr-inhibtors able
to block the process. We defined the single aminoacids involved in adaptin interaction, which allows EGF-R localization in coated pits and internalization on its way to rapid degradation. We also determined that there is at
least one more EGF-R internalization domain able to allow rapid internalization independently of adaptin interaction located in the C-terminus upstream the autophosphorylation sites. We have also identified the pathway of EGF-R internalization mediated by adhesion in complete absence of EGF.
EGF-R and cell adhesion
L. Beguinot, M. Riolfo, L. Moro, P. Defilippi
Receptor activation does not occur “in vacuo” in vivo, since cells are bound to each other and to the extracellular matrix. To study the role of cell adhesion on the signaling activity of the EGF-R, we have employed cells in
suspension, and plated on specific extracellular matrixes. We showed that in complete absence of EGF cell adhesion stimulates EGF-R autophosphorylation and activation of its signal transduction pathway by SHC phosphorylation and MAP-kinase activation. We used our large collection of EGF-R mutants(in endocytosis, cell
transformation and signaling properties) previously generated and characterized to analyze this phenomenon in
detail. We established that four EGF-R tyrosines are phosphorylated in response to the adhesive stimulus and
identified that c-src is activated upon adhesion. C-src phosphorylates the EGF-R on a novel tyrosine, Y845
within the kinase domain while the EGF-R TK autophosporylates on 3 canonical sites in the C-terminus. The
845F EGF-R mutant is less tyr-phosphorylated after cell adhesion and EGF treatment. It is now being studied
in its biological properties and signalling pathway.
Characterization of a novel cytosolic tyrosine phosphatase
L. Beguinot, M. Mariotti, J. Majer
We have recently partially cloned a novel member of the cytosolic tyrosine phosphatase family (HD-PTP)
abundant in human breast carcinoma tissue and carcinoma cell lines lsuch as Hela KB and ECV cells. It is also
expressed in normal and transformed endothelial cells where it is modulated by Tat and by activation of the
FGF receptor. HD-PTP possesses a C-terminus with a PEST domain and several consensus phosphorylation
sites for TK-R, PKC and MAPK, a single phoshatase domain and 2 novel Bro and His domains at the N-terminus. It is a large 190 Kda cytosolic protein which is phosphorylated and associated with EGF-R in human carcinoma cells. This finding suggests that it may be involved in the EGF-R/Erb-2 signal transduction pathway and
we are currently addressing its role and biochemical activity.
Mechanisms of B cell differentiation and myeloma sensitivity to proteasome inhibitors
R. Sitia, S. Cenci, C. Fagioli, S. Masciarelli, S. Nerini, L. Oliva, A. Orsi, E. Pasqualetto, E. Ruffato, S. Cozza
B lymphocytes are small, long-lived cells that express antigen receptors and do not secrete IgM. Upon encounter with antigen, they differentiate into plasma-cells; after few days of intense Ig secretion plasma cells undergo apoptosis, thus ending the antibody response. How is plasma cell lifespan controlled? We observed that
in the last phases of B cell differentiation proteasomal activity decreases, which correlates with the accumula-
tion of polyubiquitinated proteins and stabilization of proteasomal substrates, including Xbp1 and Bax. We are
now determining whether and how these events generate ER stress, hypersensitivity to proteasome inhibitors
(PIs) and apoptosis. PIs are providing promising results in the therapy of multiple myeloma, though their
mechanisms of action remain largely unknown. A few lines of evidence indicate a possible contribution of oxidative stress. Our results suggest a role for glutathione metabolism in determining PIs effectiveness on human
myeloma cells. We are now trying to identify some of the signalling steps that lead from proteasome inhibition
to changes in environment and oxidative stress.
Protein folding and quality control in the endoplasmic reticulum and early secretory pathway
R. Sitia, T. Anelli, L. Bergamelli, S. Ceppi, C. Fagioli, G. Bertoli, M. Otsu, S. Nerini
The Endoplasmic Reticulum (ER) is the factory where proteins destined to the secretory pathway or extracellular space are manufactured: retaining aberrant products, Quality Control (QC) ensures that only native products are dispatched to their final destination. The formation of disulfide bonds is an essential step in ER protein
folding and is controlled by highly integrated protein networks. We characterized the function and localization
of the pivotal element, Ero1a, and the role of its interactions with PDI and ERp44, in coupling disulfide bond
formation and quality control. We are now dissecting the timing and location of these differential interactions.
We had previously established that ERp44 is involved in thiol-mediated retention. Recent findings reveal that
ERp44 is enriched in the ERGIC compartment, suggesting a compartimentalization of QC in the early secretory pathway possibli finalized to optimise the production of complex, oligomeric molecules. We are now trying
to better characterize the mechanisms involved in QC and thiol-mediated retention.
Estrogenic suppression of bone-wasting inflammatory genes
S. Cenci, E. Benasciutti
Estrogen (E)-mediated suppression of pro-inflammatory cytokines is crucial to mantain bone homeostasis. E
inhibits inflammation both downstream, by reducing the levels of pro-inflammatory cytokines, and upstream,
e.g. by decreasing the expression of the MHC Class II Transactivator (CIITA). However, the molecular mechanisms by which E represses inflammatory genes are currently unclear. In particular, while chromatin dynamics
are known to mediate activation of gene expression upon interaction of the liganded E receptor with an E Responsive Element (ERE), whether chromatin remodelling occurs in E-mediated gene suppression is unknown.
We are investigating the regulation of CIITA in murine bone marrow monocytes as a paradigm of inflammatory
genes suppressed by E. Our findings reveal a role for chromatin dynamics in this regulatory mechanism, and
point to specific post-translational modifications of histone residues as an E-dependent histone code. We are also testing whether an E-dependent control of chromatin accessibility may represent a general mechanism
whereby E suppresses genes encoding inflammatory cytokines.
Investigating membrane IgE-FcepsilonRI binding
A. G. Siccardi, L. Vangelista, E. Soprana, G. Di Lullo, R. Fucci, O. Burrone, G. Paganelli
Secretory IgE (sIgE), targeted onto tumor cells, shown strong adjuvanticity in mouse tumor vaccination models. Transfection of membrane IgE isoforms (mIgE) would simplify the vaccine, guaranteeing tumor cell mIgE
expression with no circulation of sIgE. The success of this strategy requires that a direct mIgE-FcepsilonRI-mediated cell-to-cell interaction is possible and functionally efficient. We analyzed the interaction by using human
mIgE-transfected mouse B cell lines and the RBL SX38 cell line (rat basophilic leukemia cells transfected with
human trimeric FcepsilonRI). The cell-to-cell contact led to the release of allergy mediators, indicating full activation of the RBL cells. Mediator release was detected exclusively in the presence of mIgE and inhibited either
by the preincubation of the B cells with soluble FcepsilonRI or by the preincubation of RBL SX38 cells with
human sIgE. The interaction is likely to be physiological, since also human peripheral blood basophils and
monocytes interact with mIgE-bearing cells and release mediators.
Immuno-proteomics of cancer: biochemical and immunological characterization of Mena breast carcinoma
L. De Monte, P. Nisticò, F. Dimodugno, M. Alessio
Human Mena antigen, is a new antigen overexpressed in breast cancer identified using SEREX approach by
screening a cDNA espression library from a primary breast tumor with the autologous serum of a long surviving patient. Mena belongs to the Ena/VASP family and plays a role in the control of cell motility and cell-cell
adhesion regulating the actin cytoskeleton. By 2D-Electrophoresis (2DE) analysis we have been able to identify
and discriminate different Mena isoforms, possibly generated by alternative splicing and/or by different protein
post translational modifications. The isoforms are specifically expressed based on the histological origin of the
cell line used (epithelial vs. mesenchimal) and may be involved in the induction of immune response. Isoforms
identity has been confirmed in 2DE by using specific transfectants, and phosphorylation has been detected as
possible cause of different electrophoretic mobility of some isoforms.
Proteomics study of the protein pattern changes in cerebrospinal fluid (CSF): looking for markers of
neurodegenerative diseases
A. Conti, S. Iannaccone, M. Beltramo, M. Alessio
The major challenge in conducting a proteomics analysis of Central Nervous System (CNS) pathologies is to
obtain appropriate samples. The cerebrospinal fluid (CSF) being in contact with the brain contains proteins released directly from the CNS, and samples from patients and controls are routinely available. For these reasons
CSF analysis is very important to understand the pathological processes involved in diseases, and for diagnostic
purpose. We successfully applied differential expression proteomics analysis at human CSF of patients with
traumatic brain injury compared to healthy patients. We found the de novo appearance of fibrinogen degradation products that correlates with patients outcome and may be used as markers. We are currently working on
CSF from patients affected by Amyotrophic Lateral Sclerosis and on CSF from peripheral neuropathies patients with and without pain. To increase the detection sensitivity, a methodological improvement dedicated to
the removal of the most abundant proteins from CSF is ongoing.
Identification of grass pollen allergens by 2D-electrophoresis and serological screening
V. Corti, S. Burastero, C. Paolucci, A. Cattaneo, A. Bachi, M. Alessio
Approximately 50% of allergic patients are sensitized against grass pollen allergens. The characterisation of
specific IgE reactivity to allergen components is fundamental for clinical diagnosis and immunotherapy. Complex allergen extracts are used in diagnostic tests and in immunotherapy preparations, but their composition in
single allergenic molecules is partially known. Diagnostic tests which utilize recombinant or immuno-purified
allergens have been made available allowing to obtain specific profiles of IgE reactivity, but the panel of available molecules is far from complete. We used a proteomic approach in order to detect grass allergens from a
natural protein extract. A five-grass pollen exctract used for diagnosis and immunotherapy was resolved by 2Delectrophoresis, and assayed with sera from pollen-allergic patients whose sensitization profile was known. We
identified 6 out of 8 expected clinically relevant allergens in the natural grass extract. Identity was confirmed by
mass spectrometry analysis. Moreover, we identified different molecular isoforms of single allergens obtaining a
detailed profile of IgE reactivity, and two new putative allergens were identified.
Proteomic analysis of long term potentiation in rat hippocampal neurons
V. Corti, Y. Sanchez-Ruiz, A. Bergamaschi, A. Bachi, A. Malgaroli, M. Alessio
Long-Term Potentiation (LTP) is an activity-dependent change in synaptic efficacy involved in learning and
memory in the mammalian brain. Repeated high-frequency tetanic stimulations results in LTP lasting for
days/weeks in the in vivo animal. The molecular mechanisms underlying maintenance of LTP are far from being understood. Since LTP can last for weeks, how a synaptic change can be maintained for long periods considering that the most of neuronal proteins have a lifetime of few hours-days. LTP is dependent on protein and
mRNA synthesis suggesting that potentiated synapses must differ in molecular component(s) from unpotentiated synapses. These components must be either synthesized at the neuronal soma and then specifically shipped
to potentiated synapses or made in situ at tetanized terminals using locally stored mRNA(s). To investigate this
issue, we generated 2DE reference map for synaptic proteins purified from CA1-CA3 hippocampal cultured
neurons and for protein material corresponding to the somatodentritic compartment (HSS). By mass production of neuronal cultures, subcellular fractionation of synapses, 2DE and mass spectrometry, we have identified
more than 100 proteins.
Study on the protective role of mitochondrial ferritin in mitochondria iron overloaded yeast model
A. Campanella, P. Santambrogio, A. Cozzi, P. Arosio, G. Isaya, H. O’Neill, S. Levi
Mitochondrial Ferritin (MtF) is an iron storage protein, which is structurally and functionally similar to the
cytosolic ferritins. MtF is abundant in the erythroblasts of patients with Sideroblastic Anemia, where it is
thought to protect the mitochondria from the damage caused by iron loading. Mitochondria iron overload occurs also in cells deficient in frataxin, a mitochondrial protein involved in iron handling and implicated in
Friedreich ataxia. We expressed human MtF in frataxin-deficient yeast cells, a well-characterized model of mitochondrial iron overload and oxidative damage. The human MtF precursor was efficiently imported by yeast
mitochondria and processed to form functional ferritin that actively sequestered iron in the organelle. MtF expression rescued the yeast respiratory deficiency, caused by the lack of frataxin. Moreover it protected the activity of iron-sulfur enzymes, prevented the development of mitochondrial iron overload, preserved mtDNA integrity and increased cell resistance to H2O2. We deduce that MtF can substitute frataxin for most functions in
yeast, suggesting that frataxin is directly involved in mitochondrial iron binding and detoxification.
Biochemical and structural characterization of mitochondrial ferritin
P. Santambrogio, A. Cozzi, T. Granier, P. Arosio, S. Levi
Mitochondrial ferritin is a recently identified protein precursor encoded by an intronless gene. It is specifically taken up by the mitochondria and processed to a mature peptide, which assembles into functional ferritin
shells. We produced the full mature recombinant protein and its S144A mutant to study their structural and
functional properties. They yielded high quality crystals from Mg(II) solutions which diffracted up to 1.38 Å
resolution. The 3D structures of the two proteins resulted very similar to that of human H ferritin, to which
they have high level of sequence identity (~80%). Mitochondrial ferritin showed biochemical properties remarkably similar to those of human H ferritin, except for a higher difficulty to yield the ferritin shell in renaturation experiments and for a reduced (~41%) rate in ferroxidase activity. The latter property was partially rescued by the substitution of the bulkier Ser144 with Ala, which occurs in H ferritin. Present data define the
structure of human mitochondrial ferritin and provide new data on the iron pathways within the H-type ferritin
Studies on the physiological role of cytosolic ferritins
A. Cozzi, P. Santambrogio, A. Campanella, P. Arosio, S. Levi
Ferritins are the major iron storage proteins; their physiological role is not completely understood. In human,
two subunit types compose cytosolic ferritins: H and L, which co-assemble in a 24 subunit heteropolimer. We
use RNA interfering technology to down regulate H- and L-ferritin levels in HeLa cells, and evaluate their effects in comparison to those obtained by up regulating the ferritin via cDNA transfection. The results show that
the modifications of L-ferritin levels did not affect iron availability in HeLa cells, while positively affected cell
proliferation rate in an iron-independent manner. In addition, we found that the transient down regulation of
H-ferritin modified cellular iron availability and resistance to oxidative damage, while the stable reduction of
H-ferritin by siRNA in HeLa cell clones do not increase cellular iron availability, but made cells less resistant to
iron supplementation and chelation. In conclusion, the data indicate that the L-ferritin, while has no direct effects on cellular iron homeostasis it has new iron-unrelated functions. Furthermore, the data support the notion
that H-ferritin primary function is to act as an iron buffer.
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M.159. Knapp, S; Muller, S; Digilio, G; Bonaldi, T; Bianchi, ME;
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M.162. McKinnell, IW; Makarenkova, H; de Curtis, I; Turmaine,
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M.164. Mondino, A; Blasi, F. UPA and uPAR in fibrinolysis, immunity and pathology. Trends Immunol.: 2004; 25(8): 450-455
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Since the San Raffaele’s construction diabetes and metabolic diseases have been a major patient and research
theme. The San Raffaele Hospital is recognized as the reference center for the study and care of diabetes in the
Lombardy Region, pioneering over the years the development and application of many innovative therapeutic
approaches, including implantable pumps for insulin administration, pancreas transplantation, islet transplantation and the prevention of type 1 diabetes. The major research activities currently ongoing at the San Raffaele
Institute regarding diabetes are autoimmunity, prediction and prevention of type 1 diabetes, islet transplantation and tolerance induction, gene therapy; stem cell and b-cell biology; genetics and pathophysiology of insulin
resistance and cardiovascular complications, diabetic neuropathy and nephropathy, intermediary metabolism.
Recently, the human islet transplantation program and the research activities in the areas of immunology of diabetes, immune tolerance and metabolism have been united under a Center program at San Raffaele funded by
Telethon and the Juvenile Diabetes Research Foundation (the Telethon-JDRF Center for Beta Cell Replacement) to develop and apply tolerogenic therapies to allogeneic islet transplantation. Moreover, the San Raffaele
is an international center of TrialNet, a NIH-funded network of cooperative research groups for the conduct of
studies on natural history and clinical trials to prevent Type 1 diabetes.
The TrialNet clinical center
E. Bosi, A. G. Ziegler, E. Bonifacio, P. Grogan, L. Falqui, S. Martinenghi, M. R. Pastore, F. Meschi, R. Bonfanti,
M. G. Roncarolo, G. Chiumello
TrialNet is a NIH-funded network of cooperative clinical research groups to create a stable, high quality infrastructure for studies on natural history and effective clinical trials to preserve beta cell function and prevent
Type 1 diabetes. Established in 2001 as a continuation of the network which performed the DPT-1 (Diabetes
Prevention Trial-1) and initially based in U.S. and Canada, since 2003, with the support of the JDRF, the infrastructure has been extended to four other international clinical centers: Bristol, UK; Turku, Finland; Melbourne, Australia; and Milan, Italy/Germany based at the San Raffaele. The overall purpose of this initiative is
to expedite the translation of research on prevention of Type 1 diabetes from the laboratory to the clinic. Current studies under implementation are the natural history study of development of Type 1 diabetes, the metabolic assessment of residual insulin secretion in patients with type 1 diabetes and the Mycophenolate
Mofetil/Daclizumab clinical trial in patients with type 1 diabetes of recent onset.
A novel electrotherapy for the treatment of painful diabetic neuropathy
E. Bosi, G. Galimberti
The largely unsatisfactory results of pharmacological treatment of diabetic neuropathy encourage the searching for alternative therapies. We recently considered a novel transcutaneous electrotherapy frequency modulated electro-magnetic neural stimulation (FREMS) as a possible treatment of painful diabetic neuropathy.
FREMS is an electrotherapy characterized by sequences of high voltage and low pulse duration electrical stimuli which vary in both frequency and duration. With the purpose of establishing safety and efficacy of this innovative treatment, we conducted a multicentre, randomised, double-blind, crossover FREMS vs placebo clinical
trial in patients with either type 1 or type 2 diabetes and painful neuropathy. The results showed that FREMS
induced a significant reduction of day time and night time pain, an increase in sensory tactile perception and
foot vibration perception and an increase of motor nerve conduction velocity; these beneficial effects, with the
addition of an improvement of quality of life, were maintained for at least 4 months, with no significant side effects. These data indicate that FREMS is a safe and effective therapy of neuropathic pain in patients with diabetes and is able to modify some parameters of peripheral nerve function. Further studies on experimental
models of neuropathy will be performed in the future in order to clarify the mechanism of action of FREMS.
Insulin resistance and endothelial dysfunction in type 2 diabetes and cardiovascular disease
L. D. Monti, E. Galluccio, E. Setola, P. Piatti
This line of investigation is focused on the study of endothelial dysfunction and insulin resistance in type 2 diabetes and cardiovascular disease. The overall goal is to understand the common metabolic, vascular and genetic soil linking type 2 diabetes and cardiovascular disease and to identify innovative treatments to prevent and
cure endothelial dysfunction and type 2 diabetes mellitus. This matter is approached along three lines: 1- identification of genes and underlying molecular mechanisms involved in early phases of endothelial activation. An
example is the demonstration of a significant association between eNOS gene polymorphisms and hyperinsulinemia, insulin resistance and type 2 diabetes, thus indicating a genetic background common to both cardiovascular disease and type 2 diabetes. 2- sorting out the molecular interplay between endothelial dysfunction, insulin resistance and oxidative stress within the metabolic syndrome. An example is the demonstration of hyperinsulinemia and other markers of reduced insulin sensitivity as predictors of coronary restenosis in patients undergoing coronary revascularization. 3- Search for innovative treatments for reverting endothelial dysfunction
and insulin resistance. An example is the demonstration of amelioration of insulin sensitivity after long-term administration of L-arginine, an essential amino acid precursor of nitric oxide.
Inflammation and tissue remodelling in type 1 diabetes and islet transplantation
L. Piemonti, V. Sordi, A. Mercalli, M. L. Malosio, R. Melzi, F. Bertuzzi, E. Bonifacio
Inflammation accompanies autoimmunity, beta cell death and islet transplantation. Our objective is to modulate inflammation so that it improves islet beta cell survival. We have demonstrated that human pancreatic islets
release cytokines and chemokines that promote inflammation and tissue remodelling in the surrounding environment. Some of these, e.g. CCL2, are potent chemoattractants for macrophages that can have both harmful
(MI) and beneficial (M2) effects on beta cell survival. Our aim is to identify agents which promote the beneficial effects of inflammation in order to improve islet engraftment. We have also shown that islets release
chemokines which promote the migration of human mesenchymal stem cells in the pancreas and bone marrow,
and attract bone marrow cells with the capacity to expand within islets. We are examining the potential of these
mesenchymal stem cells to assist the regeneration of beta cells in a transplant model. Finally, we have demonstrated that modulation of dendritic cells by mannose receptor binding molecules such as MUC1 or PAM-1
MAb yields a regulatory DC phenotype that favours expansion of regulatory T cells.
Natural history of type 1 diabetes.
E. Bonifacio, A. G. Ziegler, P. Achenbach, K. Kozwara, V. Lampasona
Together with the Diabetes Research Institute in Munich we have examined factors that influence the development of islet autoimmunity and subsequent progression to diabetes. We have determined that by combining
HLA DR/DQ genotypes and the extent of family history, the risk for developing multiple islet autoantibodies
during childhood can be discretely stratified from less than 1% to above 50%. Multiple islet autoantibodies
were preceded by high affinity proinsulin-reactive insulin antibodies, whereas children who had lower affinity
proinsulin non-reactive antibodies did not progress to other antibodies. Progression from multiple antibodies
to diabetes could be further stratified on the basis of titre, subclass and/or epitope of insulin and IA-2 antibodies. These findings have led to the proposal of an antigen-based primary prevention trial in high risk neonates.
Modulation of autoimmunity in patients with type 1 diabetes by islet allo-graft
E. Bonifacio, P. Monti, M. Scirpoli, N. Ghidoli, M. Falcone, P. Maffi, F. Bertuzzi, A. Secchi
Allogeneic islet transplantation in patients with type 1 diabetes re-exposes an autoimmune recipient to target
islet autoantigens, and therefore represents a potentially useful model to study recurrent autoreactivity. We
have found that islet transplantation via the Edmonton protocol is associated with a mild to moderate reduction
in lymphocyte numbers. This results in increased circulating concentrations of the homeostatic cytokines IL-7
and IL-15 and a chronic increase in circulating proliferating Ki67 positive CD45RO memory CD3 cells, suggestive of homeostatic proliferation. Autoreactive T cells were found to be markedly enriched in the Ki67 positive
cells, and autoreactive T cells would be expanded in vitro in the presence of antigen, IL-7. Proliferation could
be prevented by MMF, but not by rapamycin, FK506 or Cyclosporin. These findings suggest that the islet
transplantation by the Edmonton protocol can result in expansion of autoreactive T cells.
Defective DC-induced regulatory pathways in autoimmune Type 1 Diabetes
F. Facciotti, M. Verga, P. Monti, E. Hauben, M. Falcone
Genetic susceptibility to autoimmune diseases such as Type 1 Diabetes (T1D) results from defective immunoregulation. Certain subtypes of dendritic cells (DC) play a key role in inducing and modulating T cell subsets that exert regulatory function. For example, CD1d-expressing DC are crucial for activation of NKT cells.
We demonstrated that bone marrow-derived DC of NOD mice, either immature or matured with different
stimuli (LPS, IL-1β or TNF-α), expressed lower levels of CD1d compared to DC of normal mice and failed to
activate IL-4-secreting regulatory NKT cells. We also found that DC from NOD mice secrete lower levels of
regulatory cytokines such as IL-10. The presence of IL-10 in the pancreatic microenvironment could critically
affect generation of IL-10-secreting Tr1 cells. Taken together, our preliminary data suggest that lack of immunoregulation in T1D-prone individuals could be related to defective DC that fail to stimulate regulatory T
cells (NKT, Tr1 and CD25+CD4+ cells) while maintaining ability to prime autoreactive T cells. Future goal is
to characterize the phenotype and functional features of tolerogenic DC and restore their function in diabetic
Amino acid metabolism and human pathology
A. Battezzati, L. Sereni Piceni, L. Luzi
In the last years we developed an expertise on tracing the kinetics of proteins and specific amino acids. The
purpose is double: on one side it is to investigate the pathophysiology of well-defined clinical conditions such as
diabetes, hypoglycemia, liver and kidney diseases. On the other side, it is to investigate the impact that genotypes related to amino acid metabolism (such as those predisposing to hyperhomocysteinemia) have on human
disease. Part of the work has been focused on the kinetics of the amino acids glutamine and alanine because of
their central role in gluconeogenesis, ureagenesis and protein metabolism. We elucidated the role of these
amino acids in response to hypoglycemia in healthy subjects, in hypoglycemic syndromes, in diabetic patients
prior and after pancreas and islet transplantation. Despite the known role of the liver in glucose production and
in amino acid metabolism, we also found that in the absence of the liver (during the anhepatic phase of liver
transplantation), other organs can equally produce glucose from glutamine and alanine. Much interest is currently devoted to the study of methionine and homocysteine kinetics in healthy subjects and in carriers of mutations causing hyperhomocysteinemia, a condition strongly related to atherothrombosis. The aims of this study
are to identify the metabolic pathways which mediate the pathogenic effects of hyperhomocysteinemia and to
provide a sensitive and specific marker of thrombotic disease. More recently, a new line of research was implemented, focused on the nutritional regulation of human metabolic pathways. This line includes analysis of food
and of their functional properties in order to fight chronic degenerative diseases.
Fat-induced insulin resistance and the metabolic syndrome
G. Perseghin, P. Scifo, F. De Cobelli, A. Esposito, A. Del Maschio, L. Luzi
This project was undertaken to accurately dissect out the relative contribution of fat-induced insulin resistance at the levels of different organs and tissues to the whole body features of the metabolic syndrome. We developed MR Spectroscopy (MRS) techniques to non-invasively measure intracellular substrate concentrations
and disposal in alternative to the biopsy technique. MRS of the skeletal muscle: we set up a 1H MRS protocol
to assess intramyocellular triglyceride (IMCL) content and we are applying this technique to the study of fatty
acids-induced insulin resistance at the levels of the skeletal muscle in pre-diabetic states, diabetes, obesity, liver
diseases, myotonic dystrophy and infectious disease. MRS of the heart: we developed 31P MRS techniques to
non-invasively assess High Energy Phosphates (HEP) metabolism of the human heart. This technique in vivo
and in association with the MRI determination of morphological and haemodynamic parameters constitutes a
unique tool to study the functional and metabolic heart performance in healthy and disease conditions. In collaboration with the Transplant Unit directed by Prof Secchi we are studying the early metabolic and functional
heart alterations in type 1 diabetes and we are going to extend the study to individuals with metabolic syndrome.
Modulation of cell signal transduction and human pathophysiology
I. Terruzzi, L. Luzi
We have previously demonstrated that lipids and amino acids, by similar molecular mechanisms, induce insulin- resistance in the rat myocardium. We hypothesized that nutritional (amino acids) and hormonal (insulin)
signals could modulate protein anabolism in human vascular endothelial (HUVEC) and smooth muscle
(HVSMC) cells. In order to investigate the pathogenic mechanisms involved in the formation of the atheromasic lesions, we compared in HUVEC and in HVSMC the independent and combined effects of insulin and
amino acids on the phosphorylation of p70 S6 kinase and 4E-BP1 and other key kinases regulating cell growth
and survival. The results show that anabolic stimuli modulate p70S6k and 4E-BP1 activity differently in the two
vascular cell types, and suggest that insulin stimulates protein synthesis for a longer time in HUSMC than in
HUVEC. We are presently investigating the effect of nutrients and hormones, proliferative stimuli and energy
substrates, in the modulation of the molecular mechanisms at the basis of development and proliferation involved in pathogenic processes.
Early identification of patients at risk to develop diabetic nephropathy
G. Zerbini, A. Maestroni, D. Gabellini, D. Ruggieri, L. Luzi
An inherited predisposition to essential hypertension is presently considered the most important cause of susceptibility to Diabetic Nephropathy (DN) in diabetic patients. This is confirmed by the evidence that DN is
characterized by an increased activity of erythrocyte Na-Li countertransport (SLC), the most consistent intermediate phenotype of essential hypertension. During the last year we have demonstrated that SLC is mediated
by an alternative splicing of the first isoform of Na-H exchange (NHE). As a result of our study, the first isoform of NHE is now a candidate gene to explain the pathogenesis of DN. To clarify whether a polymorphism of
the first isoform of NHE might explain the elevated activity of SLC found in DN, we have recently started to
investigate the possible association of the polymorphisms of the first isoform of NHE with SLC activity and
with the expression of the alternative splicing of the first isoform of NHE in patients with or without DN. The
identification of the molecular basis of DN would be of great importance for both the early detection of patients at risk to develop DN and the identification of more effective therapy protocols.
Leptin expression in cartilage and bone cells
A. Rubinacci, I. Villa, S. Cinti, G. Marotti
Aim of the present study was to immunohistochemically evaluate in vivo leptin expression in cartilage and
bone cells of the osteogenic lineage in growing rats and adult humans. In growing rats bone leptin is expressed
in chondrocytes and stromal cells, but not in osteoblasts. In adult human bone leptin is expressed in chondrocytes, stromal cells and bone lining cells; osteocytes, both in rats and humans, were found to be leptin positive
only occasionally. Thus leptin appears to be expressed in the cells of the osteogenic lineage, which are permanent and inactive in respect to osteoblasts. Our results suggest that leptin could act as a paracrine factor in bone
by modulating the activity of these cells, which are involved in the triggering of bone formation and/or bone resorption.
Effects of growth hormone on osteoprotegerin production in human osteoblast-like cells
I. Villa, E. Mrak, R. Lanzi, A. Rubinacci
In a previous study we showed that 6 months of Growth Hormone (GH) replacement therapy in GH deficient patients were able to induce a significant increase of plasma OPG, which is an endogenous inhibitor of osteoclastogenesis. Since the OPG increment was inversely correlated to the changes in plasma bone metabolism
markers, it could reflect an enhanced OPG production from bone. In order to verify this hypothesis, we studied the effect of GH on human osteoblast-like cells. GH treatment was able to stimulate OPG secretion in a
dose-dependent manner. We also showed by RT-PCR that GH induced an increase in OPG mRNA levels, indicating that the hormone has a stimulatory effect on OPG gene expression. The stimulatory effect on OPG production was prevented by pretreating the cells with Tyrphostin AG490 (10mM), an inhibitor of the Janus kinase 2 which is the intracellular pathway activated by GH. Similar results were obtained with a receptor antagonist of GH, pegvisomant at 5x10-8M. In conclusion, our results support the view that GH is able to modulate
bone remodeling by directly influencing osteoblast function and interaction with osteoclast genesis and differentiation.
Bone as an ion exchange organ: evidence for instantaneous cell-dependent calcium efflux from bone not
due to reabsorption.
A. Rubinacci, M. Marenzana, J. Kunkel, A. Shipley
In this study we tested the hypothesis that basal level and short term correction of plasma Ca2+ by Ca2+ fluxes from and into bone are controlled by an active partition system without activation of the bone remodelling
system. We measured direct Ca2+ fluxes using the Scanning Ion-selective Electrode Technique (SIET) on living
bones immersed in a physiological medium. SIET measurements of the Ca2+ fluxes were performed on
metatarsal bones of weanling mice after drilling a hole through the cortex to expose the internal bone extracellular fluid to the bathing medium, which composition mimicked the extracellular fluid. We measured influxes
of Ca2+ at the centre of the cortical hole that reversed to effluxes when Ca2+ was absent in the bathing medium. Only the efflux was nullified by adding Na+-Cyanide, demonstrating its cell-dependence. The timeframes
of the exchanges and the stability of the Ca2+ fluxes over time suggest the existence of an exchangeable Ca2+
pool in bone. The calcium efflux dependency on viable cells suggests that an active partition system might play
a central role in the short-term error correction of plasma Ca2+ without the contribution of bone remodelling.
Simultaneous determination of densitometric, geometric and histomorphometric parameters of femoral
neck from pQCT scans by a dedicated image analysis program
M. Maranzana, F. Colasante, U. Di Rauso, G. Moro, G. Fraschini, A. Rubinacci
Femoral neck fracture is the critical outcome of osteoporosis. Aim of this study was to develop an automated
image analysis software for a multiplatform public-domain image-processing package (Scion Image/NIH-Image), and to determine the parameters of interest from pQCT scans of whole femoral neck sections obtained
from patients undergoing hip replacement surgery for fractures (F) or for primary degenerative arthritis (NF).
Designed outputs included, besides standard BMDv, standard histomorphometric static parameters, and Connectivity Index (CI). The algorithm had a high reproducibility (CV=2%). Trabecular BMDv was significantly
higher (67.25%, p<0.005) in NF than in F, whereas cortical BMDv did not significantly differ. Independent parameters of trabecular architecture such as Tb.N, CI and BV/TV were significantly higher in NF than in F.
Tb.Sp was higher in F than in NF (191%, p<0.05). The parameters determined by the developed algorithm allowed to discriminate F from NF group. By simultaneously defining material and structural properties of the
femoral neck, the current analysis method might improve the clinical estimation of fracture risk in the clinical
Intestinal flora modulation for the treatment of inflammatory bowel disease
M. Guslandi, M. C. Petrone
The enteric flora is known to play a pathogenetic role in Inflammatory Bowel Disease (IBD). Hence antibiotics are employed as a supportive therapy in both ulcerative colitis and Crohn’s disease. A possible, safer alternative is represented by probiotics, microorganisms which, when taken by oral route, exert beneficial effects on
the human intestine. In previous studies we have shown that some probiotics are useful, when added to
mesalazine, in the treatment of both ulcerative colitis and Crohn’s disease. Further studies are in progress to ascertain the possible role of other probiotic agents as well as of locally-acting antibiotics in the treatment of active and inactive IBD, either alone or in combination with standard medical therapy.
Gabexate prophylaxis in patients at risk of post-ERCP pancreatitis
A. Mariani, S. Curioni
Acute pancreatitis occurs in 3-5% of cases after diagnostic or therapeutic ERCP and in 12-15% of cases in
subjects identified at high risk, on the basis of patient- and technique-related conditions. At present, no data are
available on the efficacy of some pharmacological prophylaxis for prevention of post-ERCP pancreatitis in
high-risk patients, those patients in whom there is a real need for prevention. Routine prophylaxis with Gabexate has been proven effective in preventing post-ERCP pancreatitis in standard-risk patients; however, this
strategy may be not cost-effective, since a large number of patients are treated to prevent few complications. On
the other hand, the “on demand” treatment in selected cases could be preferable, if effective. Aim of the study
is to prospectively assess the efficacy of the “on demand” treatment with Gabexate in cases in which techniquerelated risks for post-ERCP pancreatitis occur, in a controlled, randomised vs placebo, multicenter trial involving 500 patients, over a two-year period.
K-Ras gene mutations in cytological specimens by EUS-guided fine needle aspiration biopsy: prospective
study on differential diagnosis of pancreatic focal lesions and patient’s outcome
P. G. Arcidiacono, S. Carrara, M. Rossi
Imaging techniques have low specificity in differential diagnosis of pancreatic focal lesions. EUS-guided fine
needle biopsy has increased the specificity rates up to 90% with a 100% PPV; however, EUS-guided cytology
has a low NPV that does not exceed 75%. K-ras gene mutations have been found in 90-95% of pancreatic adenocarcinomas. The combination of cytology and K-ras mutations has been reported to markedly improve both
sensibility and specificity. Aim of the study is: a) to assess the diagnostic accuracy for malignancy of the EUSguided fine needle tissue collection plus search for K-ras mutations on cytological specimens obtained from either pancreatic focal lesions or enlarged lymphnodes in a prospective series of patients with pancreatic focal lesions (carcinoma, mass forming chronic pancreatitis, cystic lesions); b) to evaluate the patient’s outcome on the
basis of K-ras status.
Risk factors for post-ERCP pancreatitis
A. Mariani, E. Masci
Reported rates of pancreatitis after diagnostic and operative ERCP are quite variable due to several reasons,
including risk factors. We designed a prospective epidemiological multicenter Italian study on a series of 1,000
consecutive patients undergoing ERCP procedures to establish: a) the frequency of both patient- and ERCP-related risk-factors and their role in the occurrence of post-ERCP pancreatitis; b) the clinical characteristics of
ERCP-induced pancreatitis in the first week after its development; c) a pharmaco-economic evaluation of prevention and management of post-ERCP pancreatitis.
Use of conscious sedation during endoscopic ultrasonography of the esophagus and stomach
L. Fanti, M. Agostoni
Patients undergoing Endoscopic Ultrasonography (EUS) usually receive intravenously administered sedation.
All patients submitted to EUS are randomised to receive standard sedation i.v with midazolam and pethidine
administered by the gastroenterologist (group A) or patient-controlled sedation/analgesia with propofol and
fentanyl (group B). An anaesthesiologist is present throughout the procedure. Patients, gastroenterologist and
nurse were blinded to the randomisation. Patients self-administered boluses as often they require. Patients in
the group A received 2 ml of saline solution when they pressed the button; patients in the group B received 2 ml
of a premixed solution of propofol and fentanyl. Additional medication was given by anaesthesiologist on the
basis of patient’s discomfort. After the procedure patient and gastroenterologist rated their satisfaction with sedation using a Visual Analogue Scale (VAS).
Immunotherapy and local radiotherapy tissue ablation in pancreatic cancer: the role of endoscopic
ultrasonography (EUS) and EUS-guided fine needle aspiration biopsy (FNA)
P. G. Arcidiacono, S. Carrara, M. Rossi
Pancreatic Cancer (PC) is the fifth leading cause of cancer death and is extremely difficult to treat, so it has
become a target for novel therapies, such as immunotherapy, with cancer vaccines, and radiofrequency tissue
ablation. EUS is an accurate technique for PC staging and performing operative procedures; the ability to position the EUS probe in direct proximity to the pancreas through the stomach and duodenum produces high resolution images and guides FNA of pancreatic lesions. The aims of our study are: a) tumoral tissue acquisition
(by EUS-guided FNA) for the development of antigens for the vaccine; b) EUS-guided radiofrequency tissue
ablation of the PC with a 19 G needle. This technique will be first standardized for pancreatic application in
porcine pancreas and the endosonographic assessment of tumor response will be based on EUS-FNA performed at the end of the treatment.
Mucin expression in intraductal papillary mucinous tumors of the pancreas obtained by endoscopic
ultrasonography-guided fine needle aspiration biopsy (EUS-FNA)
P. G. Arcidiacono, S. Carrara, M. Rossi
IPMT of the pancreas is a recently proposed pancreatic tumor entity, with an unclear natural history and a
controversial management. Alterations in the expression of mucins may have a correlation with some neoplastic
changes in gastrointestinal tumors. Few studies have analyzed the mucin immunophenotype of pancreatic adenocarcinoma and IPMT, showing an overexpression of MUC1 and MUC5 in pancreatic ductal carcinoma and
in IPMT with malignant tendency, while IPMTs with benign evolution show MUC2 positivity and MUC1 negativity. The aim of our study is to use EUS-FNA to obtain cytological samples of the pancreatic duct and to examine the phenotypic expression of mucins in order to determine the utility of mucins in the differential diagnosis between benign and malignant forms of IPMT.
Detection of carcinoma of the pancreas, biliary tree and ampulla of Vater at early stage by means of Optical
Coherence Tomography (OCT) during ERCP
P. G. Arcidiacono, E. Masci, B. Mangiavillano
Carcinoma of the pancreas, biliary tree and ampulla of Vater are malignant diseases often detected in an advanced stage, when patients are judged unresectable. Optical Coherence Tomography (OCT) is, instead, an
emerging new medical technology capable of generating high-resolution cross-sectional imaging of tissue microstructure in situ and in real time by a micro-probe optical-fibre (diameter 250 mm) inserted into endoscope
operative channel with a resolution of approximately 10 mm and a penetration-depth of 2-3 mm. There are only few studies in literature about the use of the OCT in the biliary tree, during endoscopic retrograde cholangiopancreatography (ERCP), while no data are available for pancreas and ampulla of Vater. The aim of the
study is to evaluate, during ERCP, the possibility to detect carcinoma of the pancreas, biliary tree and ampulla
of Vater at very early stage. Correlation between histology and OCT images will be done, to guide interpretation of the in vivo images.
Conscious sedation in gastrointestinal endoscopic procedures
L. Fanti, M. Agostoni
It is a standard practice in patients undergoing complex endoscopic procedures to receive intravenously
sedative medication. Patient-controlled sedation is becoming increasingly popular and has a potential usefulness during endoscopy. Patients submitted to colonoscopy and to endoscopic ultrasonography (EUS) are randomised to receive conscious sedation by an infusion pump containing different doses of propofol and have the
operation of PCS pump explained to them before the start of the procedure. Patients self-administer boluses as
often as they require. An anaesthesiologist is present throughout the procedures. Pain and sedation score, procedure duration and recovery score are recorded during and after the procedure. Monitoring includes electrocardiography, heart rate, peripheral oxygen saturation and blood pressure (every 15 minutes).
Polymorphisms of calcium-sensing receptor and primary hypercalciuria
G. Vezzoli, T. Arcidiacono, V. Paloschi, M. L. Syren, D. Coviello, G. Bianchi
We found that polymorphism Arg990Gly of Calcium-Sensing Receptor (CaSR) gene is related to calcium excretion in osteoporotic or stone forming patients, with allele 990Gly associated to primary hypercalciuria. We
studied the functional effect caused by allele 990Gly or 990Arg in human embryonic renal cells HEK293
(which do not express CaSR) transfected with one of these two alleles. To test their effect we measured the variations of intracellular calcium (iCa) by Fura-2 fluorescent dye, after exposition of cellular suspension to extracellular calcium. An earlier and greater increase of iCa was detected in the presence of the variant 990Gly, compared to what occurring in HEK293 cells, transfected with allele 990Arg. EC50 (extracellular Ca concentration
developing the 50% of the maximal effect) was 2.95±0.16 mmol/l in cells transfected for 990Gly and 4.79±0.28
mmol/l in cells transfected for 990Arg (n=8, p=0.0001, Mann-Whitney U test). No variations of iCa were observed in non-transfected cells. These findings suggest that variant 990Gly causes a gain of function for CaSR
and thus contributes to hypercalciuria inhibiting Ca reabsorption in ascending limb of Henle loop.
Polymorphisms of calcium-sensing receptor and hyperparathyroidism in uremic patients
R. Quartagno, D. Spotti, G. Slaviero, M. Melandri, P. Stella, C. Lanzani, P. Cravedi, M. L. Syren, G. Vezzoli
A relationship between polymorphisms of Calcium-Sensing Receptor gene (CaSR) and the development of
hyperparathyroidism was hypothesized in uremic patients. To test this hypothesis, we enrolled 116 haemodialyzed patients due to different renal disorders. These patients were genotyped for CaSR exon 7 SNPs
(Ala986Ser, Arg990Gly, Glu1011Gln). Preliminary findings showed no differences in iPTH plasma concentrations related to alleles. Plasma levels of Alkaline Phosphatase were higher in patients bearing the variant 990Gly
(173±38 [n=11] vs. 113±8 [n=105], p=0.024, Mann-Whitney U test) and were lower in patients bearing the
variant 986Ser (93±8 [n=34] vs. 129±11 [n=82], p=0.009). Calcium x Phosphate product was lower in patients
990Gly (51±3 vs. 64±5; p=0.048). The enrolment of uremic patients is ongoing.
Genetic and nutritional determinants of kidney stone disease
G. Vezzoli, T. Arcidiacono, V. Paloschi, G. Casari, A. Trinchieri, L. Borghi, G. Gambaro, G. Bianchi
The present project is aimed to test the association between genotype at different loci and calcium kidney
stone disease. We have genotyped 581 patients and 358 healthy controls for SNPs marking different candidategenes for kidney stone disease or marking blocks of SNPs in linkage disequilibrium on candidate-gene. Calcium and citrate were measured in 24-hour urine of these subjects. The frequency of genotypes was different in
cases and controls for rs1501899 (Stone formers: AA 42.1%, AG 46.2%, GG and 11.7%. Controls: AA 53.1%,
AG 35.9%, GG 7.4%. Chi-square=11.8, DF=2, p=0.003). A border-line difference was found with SNP
rs6783556 (Stone formers: AA 59.7%, AG/GG 40.3%. Controls: AA 65.8%, AG/GG 34.2%. Chi-square=3.4,
DF=1, p=0.06). Both SNPs were on the untranslated 5’ region of the Calcium-sensing receptor gene. This suggests that CaSR or some other genes in linkage with SNP rs1501899 on chr3 increases the risk for kidney
stones. Furthermore, we have also enrolled 20 monozygotic twin pairs and 24 dizygotic twin pairs to evaluate
heritability of citrate excretion, which resulted 0.577 (57% of variance of citrate excretion is due to genetic
Interaction between Nedd4l and alfa-adducin polymorphisms on blood pressure
C. Lanzani, J.-M. Lalouel, C. Tantardini, R. Weiss, S. Fattori, M. T. Sciarrone Alibrandi, L. Citterio, G. Bianchi,
P. Manunta
Genes coding for proteins participating in Na re-absorption are important candidates since their sequence
variation may contribute to the inherited tendency for increased arterial blood pressure. The transepithelial Na
transport consists of two steps: the Na transport across the luminal membrane mediated by Na channels and
the driving Na transport across the basolateral membrane carried out by the Na-K pump. In this study we investigated the interaction between NEDD4L polymorphism affecting the former type of transport and the alpha-adducin (ADD1) polymorphism affecting the latter type. We studied 545 never treated hypertensive patients referred to our outpatient clinic for ambulatory blood pressure monitoring (ABPM) and 252 patients
with the acute salt loading protocol (NaCl 0.9% 2L/2hr e.v.). An epistatic interaction between ADD1*ADD3
was found: patients carrying the ADD1 Trp+NEDD4L A/G genotype showed higher both ABPM blood pressure (127.9 ± 1.50 vs 117.4 ± 2.89 mmHg, p = 0.005) and the largest diastolic BP variation (+6.85 ± 0.95 vs 1.9
± 0.71 mmHg, p = 0.006) after saline infusion. Therefore this may be considered a major genetic quantitative
effect on BP.
Interaction between alpha-beta-gamma adducing and ACE polymorphisms in renal failure
P. Manunta, G. Zerbini, D. Spotti, S. Valentini, C. Lanzani, L. Persichini, E. Messaggio, D. Cusi, G. Bianchi
Recent studies provided evidence for specific role of ADD2 in the progression of renal damage. In these studies we investigated whether ADD polymorphisms effect the rate of progression of renal failure in patients with
Type I (T1DN 156 patients followed for 18 years (range 1-20) and Type II (T2DN 243 patients followed for 21
years (range 13-23) diabetic nephropathy. Patient T1DN carriers of both the ADD2T and the ACE DD genotypes showed a faster kidney function deterioration over time: ADD2T&ACE DD, reached dialysis 2,1 yrs after
the diagnosis of T1DN, while those carrying the more protective genotype, ADD2T&ACE II, started dialytic
treatment after 6,3 yrs (p<0.001). In T2DN, ADD1 and ADD2 genotypes showed, when tested alone, no association with progression rate. However, T2DN patients carriers of both the mutated ADD1 460Trp allele and
mutated ADD2 T allele showed the faster kidney function deterioration over time: ADD1Trp+ADD2T 1.85±0.02 vs ADD1Gly+ADD2C/C –0.51±0.02 mg/ml/year, p=0.01 after correction for possible confounding
variables. ADD1interaction with ADD2 or ACE polymorphisms is important for the glomerular function deterioration in diabetic nephropathy.
Moderate hyposodic diet: effect on alpha-adducin, ACE genes and Endogenous Ouabain (EO) on blood
pressure fall
C. Lanzani, S. Fattori, S. Tedoldi, M. Jankaricova, E. Messaggio, L. Zagato, M. T. Sciarrone Alibrandi,
G. Bianchi, P. Manunta
Genetic and humoral influences modulate the enormous heterogeneity of blood pressure (BP) response to
low Na intake. We studied the association between ADD1 and ACE polymorphisms to BP and EO changes after reduction in Na intake in 61 never treated hypertensive patients (baseline characteristics age 48±1, BP
143±1/92±1 mmHg, urinary Na excretion 181±8 mEq/24 h). Results: ACE DD compared to ACE II carriers
display a greater fall in BP (DBP: -6.32±1.47 mmHg vs. -1.49±1.22 mmHg ANOVA p<0.05) and a rise in EO
(?EO 20.8±10.5% vs. 4.8±8.2%). However, the largest increase in EO (+58±17%) was detected in ACE DD
and ADD1 Trp carriers that also display a less decrease in BP (-1.2±2.45 mmHg). The interaction between
ACE and ADD1 was significant (p<0.05). These findings suggest that the fall in BP in ACE DD patients may
be limited by the Trp allele through an enhancement (or increase) in EO levels.
Searching for ADD3 intronic SNP meaning
L. Citterio, F. D’Avila, C. Lanzani, L. Zagato, P. Manunta, G. Bianchi
The heterodimeric structure of adducin inside the cell consists of ADD1+ADD2 or ADD1+ADD3 combination. In the kidney, ADD1 expression coexists with ADD3. The epistatic interaction of ADD1-460Trp and
ADD3-IVS11+386G (SNP5) polymorphisms is associated with mean BP (p=0.03) in our ABP-monitored population (n=500) and with pressure-natriuresis relationship in 300 Na-loaded patients (p=0.007). We investigated whether the ADD3 intronic SNP5 is a causal mutation by SNP and haplotype analysis. SNP5 is present at
40% of frequency in Caucasians, at 4% in Africans and is absent in Asians. We screened and validated six further intronic and coding SNPs; only intronic SNP0, SNP3, SNP5 and SNP6 were genotyped in TDT families
and ABP. SNP3 may represent the “tag SNP” of the single haplotype block and SNP5 creates the haplotype
variation. This peculiarity may help to assess the clinical relevance of this SNP and to exclude the presence of
haplotype admixture population. Ongoing analysis: updating of SNP map and inclusion of ADD3 HapMap
Project data; haplotype phase calculation on TDT; distribution of SNP5 and SNP6 according to ADD3 full
length and splicing variant messengers.
ANP gene polymorphisms and cardiac hypertrophy in essential hypertension
P. Stella, C. Lanzani, L. Zagato, L. Citterio, G. Bigatti, P. Manunta, S. Rubattu, S. Valentini, G. Bianchi
Low levels of Atrial Natriuretic Factor (ANP) are associated to cardiac hypertrophy (LVH) in animal models;
no evidences are known in human LVH. Cardiac dimensions (by echocardiography), 3 ANP gene polymorphism, a promoter variant (ANP1) and two coding mutations were studied in 228 moderate essential hypertensive patients. Compared to wild homozygotes, heterozygotes for ANP1 showed increase of cardiac mass (g/m2)
(110.5±39 vs 96±23 p=0.004), left posterior wall (PW, mm) (10.4±2.1 vs 9.7±1.5 p=0.03), and septal wall (SS;
mm) (11.2±3.4 vs 10.5±1.9 p=0.05). No mutated homozygotes were found. In contrast, subjects carrying exon
3 coding mutation had a decrease of MVS (81.8±27 vs 103.2±31 vs 94.6±22, p=0.033), PW (8.5±1.5 vs,
10.1±1.8 vs, 9.7±1.4, p=0.016), and SS (9.7±2, vs 11.1±2.2, vs 10.4±1.8, p=0.047). These relationships were independent from anthropometric and clinical factors. Heterozygotes for ANP1 showed lower level of plasma
proANP compared to homozygous: 1395±228 and 3110±547 and fmol/L respectively (P<0.01). Thus, ANP
seems to play a role in the development of LVH in human hypertension.
Bone density and bone metabolism in childhood diseases
S. Mora, M. Sciannamblo, G. Barera, G. Weber, G. Russo, G. Chiumello
Reduced bone mineral density is a common finding in untreated celiac disease. We have documented remarkable improvements of bone mass in children and adolescents after short-term gluten-free diet. We also recently
reported imbalances of bone metabolism in celiac youth. Increased bone formation and bone reabsorption
markers have been found in untreated patients. A longitudinal follow-up demonstrated that bone metabolism
alterations are not completely corrected by a strict gluten-free diet. In collaboration with Alessandro Rubinacci
and Isabella Villa at HSR we are exploring the hypothesis of autoimmunity involvement in the genesis of bone
mass impairment in celiac patients. Controversies exist on the presence of bone metabolism and bone density
impairments in patients with Congenital Adrenal Hyperplasia (CAH). Two factors may affect bone physiological mechanisms in these patients: the use of corticosteroids (drugs that lead to severe osteoporosis if used
chronically), and disturbances of sexual hormones. For this reason, we promoted a study on bone density and
bone metabolism in a large group of pre- and post-pubertal CAH patients.
Metabolic, body composition, and bone mineral alterations in HIV-vertically infected children and
S. Mora, M. Sciannamblo, A. Viganò, J. H. Lin, V. Gilsanz
The use of Highly Active Antiretroviral Therapy (HAART) in HIV-infected children and adults has drastically reduced the progression of the infection and the mortality rate. However, important side effects of combination treatment are now evident. We are currently investigating the role of HAART on lipid metabolism, body
composition, and bone mass in a large group of HIV vertically infected children, in collaboration with Alessandra Viganò at Sacco Hospital, Milan. We found low bone mineral density values in HAART-treated children,
and we documented important alterations of bone metabolism in such patients. However, the role of antiretroviral treatment is not clear. We started a collaboration with Joann M Linn at UCLA and Vicente Gilsanz at Childrens Hospital Los Angeles to study the bone density and bone metabolism on a large group of HIV-infected
children and adolescents on different antiretroviral regimens.
Calcium sensing receptor and calcium homeostasis
S. Mora, I. Zamproni, M. C. Proverbio, G. Chiumello, J. Hu, A. M. Spiegel
The calcium-sensing receptor (CaR) plays a critical role in calcium homeostasis. The CaR is activated by elevations in extracellular calcium concentration, leading to an inhibition of PTH secretion and renal calcium reabsorption. Mutations of the CaR gene lead to constitutively active or inactive forms of the receptor. Activated
forms of the receptor are responsible for the hypocalcemia-hypercalciuria syndrome. We are currently investigating the function of CaR in patients carrying activating mutations, in collaboration with Allen M. Spiegel and
Jianxin Hu at NIDDK. We recently reported two novel activating mutations of the CaR, which are responsible
for severe forms of autosomal dominant hypocalcemia (ADH, OMIM #146200).
Bone mineral physiology
S. Mora, I. Zamproni, M. Sciannamblo
Although hereditary factors play a major role in determining bone density, environmental factors may account for up to 20% of the variability of bone mass. Since the greater changes in bone mass occur during
growth, we investigated the role of diet and physical exercise in healthy children and young adults. The study
represents a collaborative effort, which involves several investigators affiliated to the Universities of Milan,
Pavia and Pisa. Physiological changes of bone density and bone maturation, along with the respective relationships with bone metabolism have been assessed in healthy children of different ethnic backgrounds, in collaboration with Vicente Gilsanz, at Childrens Hospital Los Angeles.
Molecular studies on the susceptibility to type I diabetes
F. Meschi, R. Bonfanti, G. Chiumello, I. Zamproni, M. C. Proverbio, S. Mora
Various studies attempted to identify type I diabetes genetic susceptibility loci. Several candidate genes have
been tested in case-control studies or combined linkage and association-based studies. Few systemic total
genome searches have also been undertaken. The lack of conclusive results prompted us to carefully select a
large cohort of diabetic patients characterized by an early onset of the disease, and to study them and their families using different approaches. We are currently testing two candidate genes (CTLA4 and VDR), in collaboration with the Dipartimento di Scienze e Tecnologie Biomediche, University of Milan. Up to now we studied 159
patients for FokI VDR polymorphism. We found differences in allele frequency between diabetic patients with
early onset of the disease and control subjects.
Genetic defects in the etiology of congenital hypothyroidism
G. Weber, M. C. Vigone, G. Chiumello, I. Zamproni, S. Mora
Congenital hypothyroidism affects about 1:3000 to 1:4000 infants and may be caused by defects in thyroidal
ontogeny of hormone synthesis. The impressive advances in molecular genetics led to the characterization of
numerous genes that are essential for normal development and hormone production of the hypothalamic-pituitary-thyroid axis. Mutations in many of these genes now provide a molecular explanation for a subset of the
sporadic and familiar forms of congenital hypothyroidism. We are currently involved in studies that are devoted
to the search of molecular defects in several genes (PAX8, TTF1, TTF2, TSHR, THOX2), in collaboration with
Luca Persani at the University of Milan.
D.1. Alberti, L; Proverbio, MC; Costagliola, S; Romoli, R;
Boldrighini, B; Vigone, MC; Weber, G; Chiumello, G; BeckPeccoz, P; Persani, L. Germline mutations of TSH receptor
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Perforin mutations in the pathogenesis of human lymphomas and other malignancies
M. Bregni, R. Clementi, F. Caligaris-Cappio, F. Ciceri, M. Bernardi, J. Peccatori, A. Pescarollo
Perforin is a cytolytic protein, mainly expressed by activated Cytotoxic T Lymphocytes (CTLs) and Natural
Killer (NK) cells, which plays a critical role in immune surveillance, being essential for cell killing via non–fasmediated mechanisms. Mutations in the perforin gene have a causative role in hemophagocytic lymphohistiocytosis, as demonstrated by Stepp et al. in 1999. In view of the experimental findings in perforin knock-out mice,
which spontaneously develop late-onset lymphoma, we analysed 29 patients with lymphoma by sequencing
analysis of the perforin gene. We found that four patients, with either Hodgkin or non-Hodgkin lymphoma,
had bi-allelic mutations of the perforin gene. We hypothesize that the impairment of immunosurveillance, due
to perforin mutations, could be responsible for the development of lymphoma. This mechanism could also be
responsible for the development of other lympho- and myeloproliferative diseases or of selected solid tumors,
such as melanoma, in which immunosurveillance plays a critical role. To confirm our hypothesis, mutation
analysis will be extended to a larger cohort of patients affected by the above mentioned diseases.
High-dose sequential chemotherapy plus rituximab (R-HDS) followed by Autologous Stem Cell
Transplantation (ASCT) in Non-Hodgkin’s Lymphomas (NHL)
A. Pescarollo, F. Ciceri, M. Bernardi, J. Peccatori, P. Servida, M. Bregni
High-dose therapy followed by ASCT for NHL is a therapeutic option initially confined to relapsed or progressive disease. ASCT toxicity has decreased by improving supportive care and by the use of haemopoietic
growth factors and mobilized peripheral stem cells.The use of Rituximab, to harvest lymphoma-free circulating
progenitor stem cells after high-dose cyclofosfamide or cytarabine represents a way to potentiate the “in vivo”
purging effect in addiction to HDS. Since 1999 our Division participated in 2 multicentric clinical trials for
treatment of high risk diffuse large cell and follicular lymphomas at diagnosis (age-adjusted International Prognostic Index >/=2). High-risk follicular lymphomas are randomized to receive R-HDS or R-CHOP. Primary
objective: to compare CR achievement, remission duration, OS and EFS. Secondary objective: molecular remission and evaluation of minimal residual disease (GITMO protocol: Gruppo Italiano Trapianto Midollo Osseo).
Diffuse large B-cell lymphomas received R-HDS at diagnosis if unfavorable clinical presentation to increase the
probability of CR and EFS (GITIL protocol: Gruppo Italiano Terapie Innovative nei Linfomi).
Selective exploit of graft versus leukaemia activity in allogeneic Haematological Stem Cell Transplantation
K. Fleischhauer, F. Ciceri, C. Bonini, E. Zino, S. Di Terlizzi, B. Mazzi, S. Rossini, C. Bordignon
The therapeutic efficacy of allogeneic transplantation of Haematological Stem Cells (HSCT) is largely due to
an anti-tumour effect mediated by the donor’s lymphocytes (“Graft versus Leukaemia”; GvL). On the other
hand, the donor’s alloreactive T lymphocytes can also cause Graft versus Host Disease (GvHD), one of the
main clinical complications after allogeneic HSCT. GvL is mediated by T cells specific for minor histocompatibility antigens (mHags), and by Natural Killer (NK) cells with patient-specific alloreactivity on the basis of
HLA-B and C genotyping. Two main strategic approaches are being used for the design of clinical immunotherapy trials in high risk haematologic patients: 1) molecular and immunological follow-up of reconstitution of T cells specific for the haematopoietic cell-specific mHags HA-1 and HA-2 in transplanted patients. 2)
elucidation of the molecular mechanisms underlying a selective GvL effect mediated by alloreactive NK clones
after HLA-B or C mismatched HSCT, by correlation of clinical outcome of HSCT with KIR genotyping, with
kinetics of NK reconstitution, and with functional characteristics of alloreactive NK clones.
HSV-TK engineered donor lymphocytes after haplo-identical hemopoietic stem cell transplantation
F. Ciceri, C. Bonini, M. Bernardi, J. Peccatori, M. T. Stanghellini Lupo, A. Pescarollo, L. Callegaro, M. Bregni,
C. Bordignon
Haplo-identical Stem Cell Transplantation (haplo-SCT) is a promising therapeutic option for patients with
high risk hematologic malignancies lacking an HLA matched donor. This therapeutic strategy is limited by the
delayed immune recovery secondary to T cell depletion, resulting in prolonged risk of post-transplant infections and high mortality. In this setting, the infusion of small numbers of unmanipulated donor T cells results in
severe and often lethal forms of GvHD. We previously showed that the infusion of Donor Lymphocytes (DLI)
expressing the herpes simplex virus Thymidine Kinase (Tk) is an efficient tool for controlling GvHD while preserving anti-tumor activity. We recently designed a dose-finding clinical protocol of Tk-DLI for immune reconstitution and relapse prevention after haplo-SCT. Eight pts with high risk hematologic malignancies who underwent haplo-SCT received escalating doses of Tk-DLI starting from 42 days after SCT: three pts received
1x106/kg and five pts received 1x107/kg CD3+ cells. At the time of infusion, no circulating CD3+ cells could
be detected. Circulating transduced cells were documented in 2/3 and 5/5 of pts receiving the low and high
Reduced intensity conditioning followed by allografting of hematopoietic cells from related and unrelated
donor for patients with poor-risk hematologic malignancies
F. Lunghi, F. Ciceri, J. Peccatori, M. Bernardi, A. Pescarollo, C. Corti, E. Guggiari, P. Servida, M. Bregni
High-dose chemoradiotherapy followed by allogeneic Haematopoietic Stem Cell Transplantation (HSCT) is
often limited to young patients in good medical conditions because of the increased risk of regimen-related toxicity, and Graft-Versus-Host Disease (GVHD) which occurs with increasing age and poor performance status.
On the other hand, the median age for hematologic malignancies is over 50 years, and therefore only a minority
of patients can benefit from HSCT. In an effort to reduce Transplant-Related Mortality (TRM), in patients over
45 years of age or heavily pretreated, or affected by medical comorbidities, nonmyeloablative conditioning regimens have been developed. The rationale for reducing the chemoradiotherapy dosage relies on the concept
that the curative potential of HSCT is due to the Graft-Versus-Tumor (GVT) effect. The GVT effect is active in
patients with chronic myeloid leukemia, acute leukemia, myelodysplastic syndromes, myeloma, and lymphoma.
These programs of nonmyeloablative conditioning are currently available for allotransplant from related and
unrelated donor, in cooperation with the Gruppo Italiano di Trapianto di Midollo Osseo (GITMO).
Intensive treatment of high risk Myelodysplastic Syndromes (MDS) according to patient age and
performance status
M. Bernardi, F. Ciceri, E. Guggiari, M. Ponzoni, F. Lunghi, L. Camba, M. Bregni
High-risk MDS (HR-MDS) patients (pts) have 75% probability to develop secondary Acute Myeloid
Leukemia (sAML) and have a median survival of 4-14 months. The median age of MDS pts is 65 years. Allogeneic Stem Cells Transplantation (AlloSCT) is the therapy of choice for younger patients. High dose
chemotherapy with autologous SCT (ASCT) rescue is advisable in patients without a donor, provided the Complete Remission (CR) of the disease is obtained before stem cells’ collection. Since 1999, we have been treating
HR-MDS and sAML pts up to 70 years old and with good Performance Status (PS), with an aggressive therapeutic program comprising an induction cycle, the FLAG-IDA regimen, ± a second cycle and a final stem cell
transplantation. Up to now 40 pts have been treated. The FLAG-IDA regimen has proved to be effective (CR >
70%). According to our data, a transplant procedure after optimal disease debulking is feasible in the majority
of HR-MDS and sAML patients up to 70 years. We propose to pts > 70 years old, with a good PS, a reduced intensity outpatient chemotherapeutic program based on the rationale of the FLAG-IDA regimen.
Non-ablative chemotherapy with allogeneic blood cell transplantation for the treatment of
relapsed/refractory solid tumors (breast carcinoma, ovarian cancer, renal cell cancer, prostate cancer)
M. Bregni, J. Peccatori, F. Ciceri, I. Sassi, D. Niederwieser
Preclinical and clinical reports suggest that a Graft-Versus-Tumor (GVT) effect exists after allogeneic
hematopoietic cell transplantation in some solid tumors: breast cancer, renal carcinoma and ovarian cancer.
This immunologic effect has been observed after full donor chimerism, and it is associated with occurrence of
Graft-Vs-Host (GVH) disease. Submyeloablative, fludarabine-containing regimens, have been recently developed in patients with hematological and non-hematological malignancies with the goal of reducing transplant
related mortality, and induction of graft-versus-leukemia/tumor effect. In cooperation with the European
Blood and Marrow Transplantation Group (EBMT), we have developed a Phase I-II protocol for patients with
solid tumors (breast, ovarian, renal cancer) in advanced disease of HLA-matched, familial allogeneic stem cell
transplant as adoptive immunotherapy. Hormono-refractory prostate cancer is being treated with the same program in a monoinstitutional study. A prognostic model for survival of patients with renal cancer undergoing allograft has been developed within the EBMT. We have also initiated a cooperative program of matched unrelated donor allotransplant.
New therapeutic strategies in multiple myeloma
J. Peccatori, M. Bernardi, A. Pescarollo, M. Bregni, F. Ciceri
Multiple myeloma has a median overall survival of less than 3 years. High dose chemotherapy with autologous bone marrow transplant was found superior to conventional therapy. Recent studies indicate that double
transplants are superior to a single transplant. Allogeneic transplant is another option, however, earlier attempts
were hampered by high Transplant Related Mortality (TRM). Allogeneic transplant results have improved during the last 5 years, and TRM has been reduced with the use of a non-myeloablative conditioning regimen. In
2003 our unit joined in an EBMT phase-II study comparing non-myeloablative allogeneic stem cell transplant
following autologous transplantation to double autologous transplant. The aim is to demonstrate a difference in
progression free survival, TRM, relapse rate and survival. From a biological point of view we are testing the feasibility of an in vitro purging on leukapheretic product by a gene-transduction protocol. We are also investigating the role of radio-pharmaceuticals which accumulate in areas of active bone turnover: in collaboration with
HSR Nuclear Medicine we are developing a radioconiugated-phosphonate and soon a phase I study will start.
Molecular modification of idiotypes from B cell lymphomas for expression in mature dendritic cells as a
strategy to induce tumor-reactive CD4+ and CD8+ T cell responses
S. Muraro, A. Bondanza, M. Bellone, P. D. Greenberg, C. Bonini
The majority of non-Hodgkin’s B cell lymphomas (NHL) are characterized by the clonal expansion of a single
cell expressing a unique rearranged immunoglobulin gene. This idiotype (Id) is a tumor-specific antigen that
can be immunologically targeted. The therapeutic efficacy of Id-based vaccines correlates better with detection
of cellular immune responses, although these have not been as well characterized as the humoral responses.
This study exploited a molecular approach to modify the Id of 38C13 lymphoma for processing via Class I and
II antigen processing pathways, and evaluated protein expression in dendritic cells (DCs) to simultaneously
stimulate tumor reactive CD8+ and CD4+ lymphocytes. Recombinant vaccinia viruses (rVV) were constructed
coding for Id fused with the targeting signal of the lysosomal-associated membrane protein1 (Id-LAMP1) to
promote antigen presentation in the context of MHC class II. Mature DCs infected with rVV/Id-LAMP1 elicited both CD4+ and CD8+ Id-specific T cells, and protected animals from tumor challenge. Id-specific CD8+
cells were required to mediate the effector phase of a therapeutic response, and CD4+ cells were beneficial in
the induction phase.
Dissecting the natural history of Chronic Lymphocyti Leukemia (CLL)
P. Ghia, C. Scielzo, A. Camporeale, R. M. Carletti, M. Alessio, G. Casorati, M. Ponzoni, C. Doglioni,
F. Caligaris-Cappio
The early transforming events leading to CLL are unknown. The identification of monoclonal B-lymphocytes
with a phenotype closely reminiscent of CLL in the blood of otherwise healthy adults may provide a model to
study these events. CLL cells have the phenotypic profile of memory B cells activated and anergized by antigen
and display a preferential immunoglobulin variable region (IgVH) gene usage, indicating the possibility of antigen involvement. The molecular and functional features of CLL are heterogeneous (interclonal diversity) and
often translate into remarkable clinical differences. In addition, each clone may carry some degree of intraclonal heterogeneity, based on the expression of intracellular or membrane molecules and depending on the
anatomical site. Furthermore, CLL cell is dependent on external stimuli originating from the microenviron-
ment (e.g. antigen stimulation, T cell-help). We are dissecting the natural history of CLL using proteomic technology and functional assays to elucidate the biological mechanisms to underline the different steps of the disease and to biologically define categories of patients with a specific clinical risk.
Vgamma9/Vdelta2 T lymphocytes in the immune response against Multiple Myeloma (MM)
S. Girlanda, C. Fortis, D. Belloni, E. Ferrero, P. Ticozzi, M. Tresoldi, A. Vicari, F. Caligaris-Cappio, M. Ferrarini
Amino-Biphosphonates (AB) activate human Vgamma9/Vdelta2 T cells and promote their cytotoxicity
against multiple myeloma (MM) cells. TCR-mediated effector functions of gamma-delta cells are enhanced upon triggering of the activating receptor NKG2D by MICA, a stress-inducible antigen expressed by epithelial
and some hematopoietic tumors. We found that the myeloma cell lines U266 and LP1, as well as primary bone
marrow (BM) plasmacells obtained from 6/10 untreated MM patients and from 5/5 patients with monoclonal
gammopathy of undetermined significance (MGUS), expressed surface MICA. Preliminary data indicate that
MICA expressed by U266 and LP1 cell lines is not per se sufficient to elicit in vitro killing. However, treatment
of MM cell lines with AB strongly up-regulates cytotoxicity and cytokine production; both functions also depend on MICA engagement, as demonstrated by inhibition experiments. We plan to evaluate whether MICA
expressed by monoclonal plasma cells is functional and correlates with disease stages and to explore the possibility that pamidronate-activated Vgamma9/Vdelta2 lymphocytes may be exploited in the immune therapy of
early stages MM and possibly also of MGUS.
Hypoxia and proteasome inhibitor: implications for Multiple Myeloma (MM) therapy
P. Ticozzi, C. Foglieni, S. Scabini, L. Veschini, D. Belloni, M. Ferrarini, F. Caligaris-Cappio, E. Ferrero
Angiogenesis is crucial for growth and spreading of tumor cells. This is true for solid tumors but not fully
demonstrated for hematological malignancies, including Multiple Myeloma (MM) where neoangiogenesis is
prominent. So far, stimuli triggering angiogenesis are poorly identified. Hypoxia, frequently associated with actively growing solid tumors, is an emerging candidate. We found that hypoxia turns on the angiogenic program
of HUVEC in vitro, through the activation of the hypoxic mediators HIF1alfa, EPAS1 and Glut1. Proteasome
inhibition is a promising new investigational avenue for MM therapy, and proteasome inhibitors (PI) have entered numerous clinical trials. We plan to investigate whether PI exerts their pro-apoptotic activity also against
HUVEC, whether hypoxia enhances the sensitivity of HUVEC to PI-induced apoptosis and how these features
relate to MM plasma cells growth in MM. The overall aim is to explore the possibility that interfering with permissive environmental conditions, such as hypoxia-induced mediators, may be a strategy in the treatment of
Molecular and structural analysis of the Immunoglobulin genes in B cell chronic lymphocytic leukemia
P. Ghia, C. Scielzo, A. Camporeale, M. Frenquelli, R. M. Carletti, I. Bruno, M. Degano, F. Caligaris-Cappio
There is increasing evidence for a role of antigenic stimulation in the pathogenesis of Chronic Lymphocytic
Leukemia (CLL). The nonrandom composition of the B-cell receptor (BCR) expressed by the leukemic cells
suggests that CLL B cells recognize distinct antigens. The nature of the cognate antigen is still unknown and
may be responsible for the typical heterogeneity in the clinical course of CLL. We have previously demonstrated that among CLL patients expressing the IGHV3-21 gene on their leukemic cells, some carry a very similar if
not identical antigen binding site (in particular CDR3 regions), though originating from distinct geographical
regions. We plan to extend this analysis on all IGHV genes expressed in our European cohort of patients, in
order to highlight distinct and repetitive use of particular sequences involved in the antigen binding site. The
analysis at the nucleotide and amino acid levels of the most relevant cases, together with the determination of
the crystal structure of the associated Igs, may help to gain insights in the nature and origin of the antigenic
stimuli implicated in the pathogenesis of the disease.
Natural immunity and hematological malignancies: gammadelta t cells in the surveillance against chronic b
cell leukemias
M. R. Zocchi, S. Catellani, P. Ghia, S. Stella, F. Caligaris-Cappio, A. Poggi
To define whether gammdelta T cells are involved in limiting the onset and spreading of CLL, we have
analysed 32 untreated patients (23 low risk and 9 intermediate risk stage, according to Rai). We found that in 15
patients circulating Vdelta1 T were significantly increased (100-300cells/ml) compared to 15 healthy donors
(50-100 cells/ml). Despite their functional cytolytic machinery, these lymphocytes do not kill the autologous tumor cells, which lack the MHC-related MIC-A antigen and the UL16 binding proteins (ULBPs), both recognized by Vdelta1 T cells via NKG2D. The anti-tumor activity could be reconstituted by transfecting leukemic
cells with MIC-A or by inducing expression of MIC-A or ULBP3, on the cell surface upon exposure to transretinoic acid. Interestingly, in 6/18 patients who did not have an increased number of circulating Vdelta1 T
cells the disease progressed in the last year. These results point to a role for Vdelta1 T lymphocyte in the defence against CLL and highlight a potential therapeutic use of retinoic acid in enhancing the activation of the
gammadelta T cell subset which preferentially responds to CLL tumor cells.
The Cancer Immunotherapy and Gene Therapy Program (CIGTP) is part of the Dept. of Oncology, and has
been constituted in 1998 to co-ordinate the activity of 7 Research Units, which were already studying different
aspects of tumor immunology with many active collaborations and complementary expertise. The co-ordination of the research is instrumental to define our strategic targets for cancer immunotherapy, to rationalize the
management of both human and technical resources, and to optimize the translation of basic and pre-clinical
studies into their clinical applications.
The CIGTP studies three strategies to attack tumors. The first two strategies aim at inducing tumor-specific T
cell responses by: 1. Active vaccination; and 2. Adoptive immunotherapy. The third strategy aims at inducing
tumor debulking by targeting tumor associated vascular endothelium with cytotoxic biological compounds.
This approach, contrary to conventional cytotoxic antineoplastic therapies, should lead to tumor cell death
without causing concomitant immunosuppression. In our view, therefore, it may become possible to combine
active or adoptive immunotherapy with tumor targeting with cytotoxic biological compounds, to obtain a more
radical elimination of cancer cells from the patient.
The processing of apoptotic tumor cells reveals naturally generated autoantigens
S. Frontini, S. Antonacci, A. Capobianco, M. Alessio, M. G. Sabbadini, A. A. Manfredi, P. Rovere Querini
Animal models bearing a characterized defect of the phagocytic clearance of apoptotic cells provide an excellent in vivo system to reveal novel antigen specificities, after immunization with apoptotic cells. We are studying
in particular mice deficient in the tissue transglutaminase (in collaboration with M. Piacentini, Rome), for
which spontaneous syngeneic neoplasms are available. We are identifying antigens preferentially recognized into apoptotic cell lysates using antibodies from immunized mice, in collaboration with M. Alessio in the Proteomic unit of this Institution. As a probe, we rely on high affinity autoantibodies collected at different times
upon immunization. Of interest, the preliminary identification of relevant antigens at least partially corresponds
to molecules with a demonstrated role as auto-antigens in human subjects. We are also assessing whether the
immunogenicity of tumors is biased in clearance defective mice. Strategies aimed at interfering with the normal
immunosuppressive clearance of dying cells could prove valuable in cancer treatment.
Identification of cryptic and naturally processed epitopes within a carcinoembryonic antigen sequence
recognized by CD4+ T cells
M. Crosti, G. Consogno, R. Longhi, M. P. Protti
The expression profile, its role in tumor progression and its immunogenicity make the carcinoembryonic
antigen (CEA) an attractive target for immunotherapeutic purposes. We previously identified an immunodominant naturally processed CEA epitope (CEA177-189/355-367). To study the existence of subdominant epitopes, CD4+ T cells were stimulated with a pool of synthetic peptides, corresponding to CEA sequences predicted to bind MHC class II molecules, in the absence of the immunodominant epitope. We obtained several
DR*07 and DR*14 restricted CEA97-111 specific CD4+ T cells clones. Since not all the clones recognized the
naturally processed epitope, we analyzed their recognition of truncated peptides, to verify whether they recognized different epitopes. We showed that sequence CEA97-111 contains three different epitopes, among which
two are naturally processed and one is cryptic. Studies are in progress to verify which epitopes are preferentially recognized by CD4+ T cells from neoplastic patients bearing CEA positive tumors.
Identification of shared tumor associated antigen(s) within allogenic pancreatic adenocarcinomas
P. Filipazzi, A. Zerbi, P. Dellabona, V. Di Carlo, M. P. Protti
The identification of shared tumor associated antigens in Pancreatic Adenocarcinomas (PA) is a fundamental
prerequisite for the development of strategies of specific active vaccination with autologous Dendritic Cells
(DC) pulsed with allogenic Natural Tumor Peptides (NTP). To this aim we obtained NTP from three different
PA cells lines (A8184, PaCa 44 and DM), among which two commercially available and one established in our
laboratory (DM). CD4+ and CD8+ T cells from two healthy donors and one PA patient (DM) were stimulated
with allogenic NTP-pulsed autologous DC. We showed that NTP from PA are immunugenic and activate
MHC restricted responses, they are cross-presented by the autologous DC and they contain shared tumor associated antigens. Indeed, i) CD4+ T cells from the same donor stimulated with NTP from two tumor cell lines
recognized the same PA cell line; ii) CD8+ and CD4+ T cells from two different donors, stimulated with the
same NTP, recognize different PA cell lines; and most importantly, iii) CD8+ T cells from the patient, activated
with allogenic NTP, recognized the autologous tumor.
Immuno-proteomics of cancer: Serological Proteome Analysis (SERPA) of colorectal carcinoma
L. De Monte, L. Cannizzaro, M. Frasson, M. Braga, A. Bachi, F. Sanvito, C. Doglioni. M. P. Protti, M. Alessio
The immune recognition of self proteins in cancer reflects the attempts of the host immune system to eliminate cells expressing qualitatively or quantitatively aberrant proteins, the so called Tumor Associated Antigens
(TAAs). In order to identify specific colon TAAs we used an approach which combines serology with proteomics technologies. TAAs are identified by comparing the reactivity of proteins resolved by 2D-electrophoresis (2DE) with patients’ sera. Sera from 52 colon carcinoma patients and 39 age-matched healthy controls have
been used to screen 2-DE gels displaying the proteoma derived from LS180 and HT29 colon carcinoma cell
lines. Immunostaining image analysis revealed tumor-related immunoreactive spots recognized with a frequency ranging between 19 to 40% of the tumor patients sera. The overall immunoreactivity for these protein spots
covers 84.6% of the colon carcinoma population tested. Tumor specific protein spots have been characterized
by mass spectrometry analysis. Immunohistochemistry and Western blot analyses revealed that protein expression was either specific for tumoral cells or increased in tumor.
Discovery of tumour associated antigens expressed by Non Small Cell Lung Carcinoma (NSCLC)
P. Gruarin, G. Canderan, D. Montagna, C. Traversari, V. Russo, P. Dellabona, G. Casorati
Discovery of novel Tumor Associated Antigens (TAA) is a critical prerequisite for immunotherapy in Non
Small Cell Lung Carcinoma (NSCLC), owing to its poorly characterized antigenic repertoire. We attempt an
expression cloning strategy, whereby tumor-specific CD8+ T cells are used to screen a transfected cDNA library derived from the autologous tumor. We have obtained 52 cytolytic CD8+ T cell clones specific for the autologous NSCLC cell line CaPo13, and selected 5 of them to identify the putative TAA(s). To improve their use
in the expression cloning approach, we have grafted their TCR into the mouse T cell lymphoma 58ab, which
does not express an endogenous TCR, nevertheless can express functional exogenous ones. This cell line is
characterised by an unlimited growth, and produces substantial amounts of IL-2 upon TCR-dependent activation, allowing to detect its activation by antigen. In the meanwhile, a sixth T cell clone, which recognises
CaPo13 cells in a HLA-Cw0201 restricted manner, is being utilised as a cellular probe to screen COS-7 cells,
transfected with HLA- Cw0201 and pools of a cDNA library produced from CaPo13 cells, for the expression
of the putative TAA.
Improving dendritic cell based vaccination strategies in pre-clinical animal models
M. T. S. Bertilaccio, M. Grioni, E. Degl’Innocenti, A. Corti, M. Bellone
In pre-clinical mouse models of transplantable and spontaneous cancer development we investigated the
therapeutic potential of Dendritic Cell (DC)-based cancer vaccines. We found that DCs are highly efficient in
controlling the growth of immunogenic tumors and small tumor burdens. However, DCs alone are not able to
significantly impact on the clinical course of large tumor burdens. Hence we designed a novel therapeutic approach which combines the tumor debulking potency of chemotherapy with DC vaccines. To improve the therapeutic index of chemotherapeutic agents without increasing their toxicity we pre-treated the tumor bearing
animals with NGR-TNF, an engineered cytokine that selectively targets the tumor vasculature and temporary
modifies its permeability. We verified that the selected doses of chemotherapeutic agents administered in association with NGR-TNF do not impair the immunogenic potential of DC-based vaccines. The therapeutic potential of the combined strategy is being tested in a model of spontaneous prostate cancer development.
Early dendritic cell-based vaccination prevents T cell tolerance and controls tumor development in a
transgenic model of prostate cancer
E. Degl’Innocenti, M. Grioni, M. T. S. Bertilaccio, A. Boni, M. Freschi, A. Monno, C. Arcelloni,
N. M. Greenberg, M. Bellone
Dendritic cell-based vaccines are promising therapeutic strategies for cancer. We investigated the impact of
bone-marrow derived Dendritic Cells (DC) pulsed with the immunodominat cytotoxic T Lymphocyte (CTL)
epitope Tag-IV in the tumor-prone Transgenic Adenocarcinoma Mouse Prostate (TRAMP) model. We found
that a single injection of peptide-pulsed DC in six weeks old TRAMP mice, bearing mouse prostate intraepithelial neoplasia, elicited a strong and long lasting Tag-specific CTL response. The DC-induced CTL response indeed, was still detectable in TRAMP mice of 24 wks of age, and was associated with reduced disease progression. Our data support a relevant role for exogenous DCs in controlling the induction of peripheral tolerance to
tumor associated antigens and disease progression.
The nuclear HMGB1 factor released by dendritic cells controls the clonal expansion of naïve T
I. Dumitriu, P. Baruah, A. Capobianco, M. Giazzon, S. Antonacci, A. A. Manfredi, M. E. Bianchi,
P. Rovere Querini
Dying cells release high mobility group proteins. In the extracellular environment, these moieties behave as
inflammatory mediators. We found that the High Mobility Group Box 1 (HMGB1) protein is sufficient to
transform poorly immunogenic apoptotic lymphoma cells in efficient vaccines, capable of eliciting a sustained
and effective protection upon re-challenge with living tumors. Moreover both myeloid and plasmacytoid maturing dendritic cells actively secrete nuclear HMGB1, and that this event, via an autocrine- paracrine circuit
involving the Receptor for Advanced Glycation End-products (RAGE) is required for the clonal expansion and
functional differentiation of naïve CD4 T cells. The results suggest that dendritic cells elicit and sustain their
own activation reconstituting the environment associated to excessive/deregulated cell death, a primordial
event shaping the metazoan immune system.
Characterization of the CD4+ T cell response against renal cell carcinoma natural tumor peptides
E. Tassi, S. Seresini, A. Bachi, M. P. Protti
Renal cell carcinomas (RCC) are largely infiltrated with CD4+ and CD8+ T cells and RCC patients show 1520% response after IL-2 immunotherapy, thus suggesting potential immunogenicity for this tumor but only few
renal tumor antigens have been described so far. To verify the existence of RCC shared antigens, natural tumor
peptides were obtained from a cell line established in our laboratory from a primary RCC. Autologous dendritic cells were pulsed with the natural tumor peptides and used to stimulate CD4+ T cells from healthy donors
and RCC patients. From two healthy donors and one patient, CD4+ T cell clones with several HLA-DR restrictions were obtained. The cells strongly proliferated and released IFN-g in the presence of HLA-DR matched
RCC, EBV-transformed lymphoblastoid cells lines, melanomas, cervical carcinomas, colon carcinomas, pancreatic adenocarcinomas but not autologous T cell blasts, thus showing a broad range of reactivity. We excluded
the recognition of known RCC tumor associated antigens. Studies using a biochemical and genetic approach
are in progress to identify the antigen(s) recognized by the clones.
Impact of a dendritic cell based vaccine on the polarization of T lymphocytes
G. Iezzi, A. Boni, E. Degl’innocenti, M. Grioni, M. T. S. Bertilaccio, M. Bellone
Activated CD8+ T cells can differentiate into Tc1 cells, producing high levels of IFN-g, and Tc2 cells producing mostly IL-4, IL-5, and IL-10. Whereas Tc1 cells are potent cytotoxic T lymphocytes mainly involved in the
defence against intracellular pathogens and cancer cells, the role of Tc2 cells, which have been associated in
cancer with disease severity and progression, is largely unknown. We found that fully activated dendritic cells
(DC) pulsed with low amounts of antigen and in the presence of IL-4 favoured the induction of Tc2 cells. Tc2
and not Tc1 cells altered, through a cell-to-cell contact mechanism, the antigen presenting cell function of DC.
Indeed, Tc2-conditioned DC displayed a reduced expression of MHC class II and co-stimulatory molecules,
produced IL-10 instead of IL-12, and favoured the differentiation of both naïve CD4+ and CD8+ T cells towards type 2 cells. We propose that IL-10 producing Tc2 cells play a role as modulatory cells by skewing the
differentiation of naive T cells towards type 2 phenotypes during the late phase of an ongoing immune response.
Clonal dynamics of antigen-specific cytotoxic T lymphocytes in a transgenic model of prostate cancer
E. Degl’Innocenti, M. Grioni, M. T. S. Bertilaccio, C. Arcelloni, M. Bellone
Whether recognition of Tumor Associated Antigens (TAA) by the immune system leads to activation or tolerance is a central question in cancer immunology. In the tumor-prone transgenic adenocarcinoma mouse
prostate (TRAMP) model we have been following the fate of the immune response against a non-mutated tumor TAA selectively expressed in the prostate epithelium during the endogenous transformation from normal
cells to tumors. We found that young [up to six-seven weeks (wk) old] male TRAMP mice, despite a dim and
patchy expression of the SV40 large T antigen (Tag) overlapping foci of prostate intraepithelial neoplasia, displayed a strong Tag-specific Cytotoxic T Lymphocyte (CTL) response. This response however was weaker than
the one found in wild type littermates, and was characterized by a reduced frequency of TAA-specific CTLs. In
TRAMP mice the Tag-specific cytolytic activity started to decrease after wk 7 and disappeared by wk 11, when
Tag was already widely expressed in pathologic prostate lobes. These results suggest that the induction of peripheral tolerance is a rather rapid event, and correlates with disease progression and antigen expression in the
Soluble factors that bind to dying tumor cells control the cross-presentation of tumor, self or viral antigen
to T lymphocytes
P. Baruah, I. Dumitriu, V. Russo, P. Rovere Querini, A. A. Manfredi
We are investigating the role of soluble factors belonging to the innate immune response, with attention to
those produced in tissues under the control of primary pro-inflammatory signals and to factors regulating the
systemic response to tissue damage. We found that the soluble pattern recognition receptor PTX3 behaves as a
flexible adaptor of the function of the most potent antigen presenting cells, the dendritic cells. This effect becomes critical when dendritic cells are simultaneously challenged with apoptotic tumor cells. In collaboration
with the groups of V. Barnaba (Roma) and A. Mantovani (Milano), we have also found in the presence of PTX3
the cross-presentation of viral, self and tumor antigens expressed by dying cells abates, limiting the activation of
antigen-specific T lymphocytes; in contrast, PTX3 does not influence the ability of dendritic cells to activate T
cells specific for soluble antigens. The generation of factors that edits cross-presentation at the site of tumour
cell death possibly contributes to limit tumour immunogenicity and to the success of anti-neoplastic therapies.
Chromogranin A in tumor biology
A. Gasparri, B. Colombo, F. Curnis, E. Ferrero, A. Corti
Chromogranin A (CgA), a protein secreted by many neuroendocrine cells, is abnormally expressed in neuroendocrine tumors. Detection of CgA in biological fluids and tumor tissues has proven useful for tumor diag-
nosis and prognosis. However, little is known on the effect of excessive production of CgA on the tumor behavior and response to therapy. In the attempt to gain information on the pathophysiological role of CgA we have
investigated the effect of CgA on tumor growth and response to chemotherapy in animal models. Chronic production of CgA by neoplastic cells inhibited tumor growth, indirectly, by affecting the tumor cell microenvironment. In vitro studies showed that fibroblasts and smooth muscle cells adhesion to extracellular matrix proteins
can be modulated by CgA. Moreover, we have found that CgA can inhibit TNF-induced endothelial permeability and vascular leakage and inhibits the response of tumors to chemotherapy. These findings suggest that abnormal secretion of CgA by neuroendocrine tumors is not simply an epiphenomenon of cell secretory activity,
but it could play important functions in tumor physiology and response to therapy.
Study of the naturally occurring CD4+ T cell response against the HPV-18 E6 protein in healthy donors
and patients bearing neoplastic cervical lesions
S. Seresini, R. Longhi, M. P. Protti
HPV infection is strongly associated with the development of neoplastic cervical lesions. The E6 and E7 proteins are important in the induction and maintenance of cellular transformation, therefore are good candidates
for cancer vaccines development. HPV-18 is responsible for a very aggressive form of cancer and poor survival.
As for other HPV types, immune surveillance has probably a role in the control of the infection. However, very
little is known on HPV-18 immunogenicity. We previously found that HPV-18 E652-66 and E697-111 are recognized by CD4+ T cells from healthy donors in association with several HLA-DR alleles and contain naturally
processed epitopes. We next verified whether CD4+ T cells from healthy donors reacted ex-vivo with the synthetic peptides corresponding to the identified sequences, and to the recombinant E6 protein. We found three
donors out of 16 tested with CD4+ T cells that specifically proliferated in the presence of HPV-18 E6 antigens
and produced IFN-g in the presence of the E6 protein, thus strongly suggesting the potential for this protein to
elicit in the host a natural productive immune response.
Investigating the pro and anti-inflammatory activities of chemotactic soluble uPAR fragments
F. Furlan, V. Basso, M. Resnati, F. Blasi, A. Mondino
Chymotripsin-cleaved soluble uPAR (D2D388-274) elicits migration of monocytic cells through the interaction with FPRL-1, and modulates the ability of monocytes to migrate in response to MCP-1, Rantes and fMLP.
The ability of D2D388-274 to prevent chemokine-induced cell migration is mimicked by MMK, a synthetic ligand for FPRL-1, and correlates with the inhibition of chemokine-induced integrin-dependent cell adhesion. We
are currently investigating the intracellular events induced by FPRL-1 occupancy leading to the inhibition of integrin activation. Since the levels of soluble uPAR fragments are found elevated in cancer patients, their local
concentration could either favour inflammatory processes (favouring the recruitment of monocytic cells) or
prevent inflammation (preventing monocyte-derived antigen presenting cells to migrate to peripheral lymphoid
tissues). Thus, understanding the mechanisms of action for these chemokines and the signalling pathways involved might lead to innovative therapeutic strategies.
Investigating the mechanisms for TCR-MHC class II tetramer interaction to characterize CD4+ t cell
M. Moro, V. Cecconi, M. P. Protti, P. Dellabona, G. Casorati
We have developed fluorochrome-labeled, soluble HLA-DR-peptide tetramers to characterize, by flow cytometry at the single cell level, CD4+ T cells specific for tumour antigens. We have produced HLA-DR1101
tetramers, a frequent HLA-DR allele in the Caucasian population, in insect cells in a versatile empty form,
which can be loaded after purification with different peptides of interest. To set up the staining conditions,
HLA-DR1101 tetramers are loaded with the tetanus toxoid TT830-844 (p2) peptide, a well characterised epitope which binds HLA-DR1101 with high affinity and allows to define the optimal condition for the ex-vivo visualization of antigen specific CD4+ T cells. We find that a stable TCR/HLA-DR1101-p2 tetramers interaction
depends critically on the reorganization of the TCR on the plasma membrane upon T cell activation. Furthermore, either an active TCR clustering process or enhanced membrane fluidity are required for HLA-DR
tetramer staining of CD4+ T cells. We are currently investigating the possible mechanisms facilitating the TCR
clustering in the tetramer contact area, namely: plasma membrane fluidity, TCR topography, physiological and
polarized TCR recycling.
Functional and phenotypic maturation of CD1d-dependent human Va24iNKT cells
C. De Lalla, N. Festuccia, G. Andolfi, F. Ficara, A. Aiuti, P. Dellabona, G. Casorati
Human CD1d-dependent invariant Va24(Va24i)NKT cells are a subset of autoreactive T lymphocytes implicated in regulating various autoimmune, infectious and tumour conditions. In adults, fully mature Va24iNKT
cells homogeneously exhibit an innate effector-memory phenotype in the absence of exogenous immunization,
and produce IFN-g at high frequency upon ex-vivo activation. Little is known about the maturation process
followed by human Va24iNKT cells during development, whether their phenotype is really innately acquired,
and what are the possible mechanisms regulating their maturation. We have addressed the issues, and studied
Va24iNKT cells in foetal thymi, cord-blood, post-natal thymi and adult peripheral blood. We found that human Va24iNKT cells are generated as semi-mature cells and begin a phenotypic, though not functional, maturation already during foetal life. The process involves pre-natal proliferation, and may require sustained signalling, possibly antigen- and/or homeostatic cytokine-driven, which must proceed into post-natal life to reach
full maturation
Independent roles for CD4+ CD8+ thymocytes and thymic antigen-presenting cells in positive and
negative selection of mouse invariant Va14i NKT cells
P. Pittoni, J. Schumann, E. Tonti, H. R. MacDonald, P. Dellabona, G. Casorati
CD1d-dependent NKT cells are T lymphocytes expressing a conserved semi-invariant TCR consisting, in
mice, of the invariant Va14-Ja18(i) TCR paired mostly with Vb 8.2 and Vb7. The cellular requirements for their
thymic positive and negative selection are only partially understood. We generated transgenic mice expressing
human CD1d (hCD1d) either on the cells implicated in the selection of Va14i NKT cells: CD4+CD8+ double
positive (DP) thymocytes or antigen-presenting cells (APCs). In the absence of mouse CD1d (mCD1d), the expression of hCD1d on DP thymocytes, but not on APCs, is sufficient to select Va14i NKT cells that, however,
attain lower numbers than controls and expressed essentially Vb8.2. The low number of Vb8.2+ Va14i NKT
cells selected by hCD1d on DP thymocytes is not reverted by the concomitant expression of mCD1d, which instead restores the development of Vb7+ Va14i NKT cells. Vb8.2+, but not Vb7+, NKT cell development is impaired in mice expressing hCD1d on APCs and mCD1d. Taken together, our data reveal that CD1d expression
by DP thymocytes suffices for positive selection of Va14i NKT cells and that both DP thymocytes and APCs independently mediate negative selection.
Dendritic cells licence the anti-neoplastic function of natural killer lymphocytes at the tumor site
A. Capobianco, A. A. Manfredi, C. Rugarli, P. Rovere Querini
Upon the synchronized death of tumor cells, antigens became available that are valuable for immunizing patients against their own neoplasms. However, immune responses in cancer patients treated with cytotoxic
agents usually either do not initiate or abort. We speculate that the lack of immunization ensuing tumor cell
death derives from the lack of efficient antigen presenting cells. We therefore relied on the bidirectional activation between NK cells and dendritic cells, injecting them simultaneously into exponentially growing, highly tumorigenic B16 murine melanomas. Both subsets were required for melanoma rejection and immune protection
of treated animals. We are actively investigating the role of soluble factors, including in particular IL-18 and
gamma-IFN, in this cross-talk, relying on specifically engineered mouse models.
Ameliorating natural and vaccine-induced anti-tumor immune responses by combined chemotherapy,
immunotherapy and bone marrow transplant
V. Zimmermann, A. Casati, R. Hess, M. Bellone, A. Mondino
We have found that effector CD4+ T lymphocytes naturally differentiate in tumor-bearing mice. By contrast,
central memory T lymphocytes accumulate only after tumor resection. We have also shown that dendritic cellbased vaccines elicit CD4+ T cell memory only when injected into naïve and not in tumor-bearing mice. This
indicates that both natural and vaccine-induced T cell responses might be limited by the presence of the tumor.
To ameliorate current vaccination strategies, we have combined DC-based vaccination with tumour debulking
by doxorubicin and melphalan-based chemotherapy, showing that this combination does not hamper the vaccination efficacy. We are currently investigating the therapeutic efficacy of the combined therapy in mice sponta-
neously developing a prostate cancer (TRAMP mice). In these mice, we will also investigate whether DC-dependent vaccination could be combined with bone marrow transplant (BMT). BMT ought to restore the immunocompetence in tumor-bearing mice, improving the therapeutic efficacy of vaccination.
Developing new strategies for adoptive immunotherapy
S. Caserta, A. Mondino
The aim of these studies is the development of new strategies to improve the detection of tumor-specific CD4
T lymphocytes and to expand these cells in vitro for adoptive immunotherapy purposes. We previously developed artificial antigen presenting (aAPC) prepared by immobilising purified peptide-linked class II multimers
together with recombinant anti-CD28 monoclonal antibodies, able to expand peptide-specific CD4 T cells in
vitro and in vivo. We have now investigated the possibility to combine the aAPC with recombinant cytokines
able to favour the proliferation, and survival of T lymphocytes. Our results indicate that recombinant cytokines
such as IL-2, IL-7, IL-4, IL-12 can be used with aAPC to favour the expansion and differentiation of antigenspecific lymphocytes. Moreover, we have also found that the ex vivo culture with recombinant cytokines such as
IL-2, IL-7, and IL-15 favours the accumulation of in vivo primed tumor-specific CD4 T cells. Ongoing experiments are addressing the ability of the ex vivo expanded T cells to confer protection upon transfer into tumorbearing mice.
The mammalian target of Rapamycin control T cell proliferation, differentiation and antigen
S. Colombetti, V. Basso, S. Innocentin, A. Mondino
We have demonstrated that the T Cell Receptor, the CD28 coreceptor, and the IL-2 receptor independently
regulate T cell proliferation and T cell differentiation through only partially overlapping intracellular signalling
pathways. One of the intracellular signalling molecules shared by these surface receptors is the mammalian target for Rapamycin, an immunosuppressive agent used in many clinical conditions. This is a serine/threonine kinase critical for the cell metabolism and for the growth factor response. We have demonstrated that mTor-signaling is required for TCR/CD28-dependent, IL-2 independent T cell proliferation, while it is dispensable for
IL-2-dependent T cell expansion. Moreover, mTor-signalling is also required for cell polarization and acquisition of effector functions. Ongoing studies are defining the targets for mTor signalling, and the proteomic profile of Rapamycin-resistant lymphocytes to better define the immunomodulatory capacity of this drug.
Tumor vascular targeting and chemotherapy
F. Curnis, A. Gasparri, A. Sacchi, A. Corti
Drug delivery and penetration into neoplastic cells distant from tumor vessels are critical for the effectiveness
of solid-tumor chemotherapy. We have developed two strategies for increasing the local concentration of
chemotherapeutic drugs in tumors and their therapeutic index, based on tumor vascular targeting. Firstly, we
have found that vascular targeting with TNF, a cytokine able to increase vascular permeability, alters tumor barriers and increases the penetration of chemotherapeutic drugs in subcutaneous tumors in mouse models. Targeted delivery of TNF to tumor vessels was achieved by coupling this cytokine with peptides containing the
NGR or the RGD motives that target the tumor neovasculature. Secondly, in collaboration with M. Ponzoni of
the Gaslini Institute of Genoa we have observed that encapsulation of doxorubicin into liposomes coupled to
the NGR-peptide markedly improves drug uptake by tumors and its therapeutic index. Vascular targeting, either indirectly with NGR-TNF or directly with NGR-targeted liposomes, can be a novel strategy for increasing
the therapeutic index of chemotherapeutic drugs. A phase I study on NGR-TNF has been started in 2004.
Uncoupling IFN-gamma anti-tumor effects from counter-regulatory mechanisms by vascular targeting
F. Curnis, A. Gasparri, A. Sacchi, A. Cattaneo, A. Corti
Because of its immuno-modulatory and anti-cancer activities IFN-gamma has been used as an anti-cancer
drug in several clinical studies, unfortunately with modest results. Attempts to increase the response by increasing the dose or by repeated continuous injection often resulted in lower efficacy, likely due to counter-regulato-
ry effects. We have found that targeted delivery of low doses of IFN-gamma to CD13, a marker of angiogenic
vessels, can overcome major counter-regulatory mechanisms and delay tumor growth in murine models which
poorly respond to IFN-gamma. Tumor vascular targeting was achieved by coupling IFN-gamma to GCNGRC,
a CD13 ligand, by genetic engineering technology. Anti-tumor effects were induced with a dose about 500-fold
lower than the dose used in patients. Unlike IFN-gamma, targeted-IFN-gamma activated TNF-dependent antitumor mechanisms without inducing soluble TNF-receptor shedding in circulation, suggesting that anti-tumor
and counter-regulatory mechanisms were uncoupled by this approach. Vascular targeting with low doses of
IFN-gamma seems promising as a novel strategy for cancer treatment.
Vessel inflammation and leukocytoclasia in systemic small vasculitis
M. G. Sabbadini, F. Fazzini, G. Dell’Antonio, E. Dal Cin, C. Doglioni, A. A. Manfredi, P. Rovere Querini
Inflammatory damage within or through the vessel wall, and widespread inflammation, are the hallmark of
systemic small vessel vasculitis. The long pentraxin PTX3, which is generated in extra-hepatic sites under the
control of primary pro-inflammatory signals, increases during active phases of vasculitis (with A. Mantovani,
Milano). Endothelial cells produce PTX3 in involved sites. We are verifying whether PTX3 predicts disease relapses, revealing early endothelial involvement. In collaboration with A. Mantovani and C. Kallenberg
(Groeningen), we confirmed that PTX3 plays an anti-opsonic role, preventing the internalisation of dying neutrophils by macrophages and antigen presenting dendritic cells. We also found that mononuclear phagocytes,
but not immature dendritic cells, recognising dying cells actively release PTX3. In turn, ANCA auto-antibodies
influence the production and the effect of PTX3. Moreover, PTX3 accumulates at site of tissue damage, including the kidney parenchyma. Our results suggest that tissue generated pattern recognition receptors and ANCA
are both required for the maintenance of tissue inflammation and possibly for tissue damage in vasculitis patients.
Innate immune factors are recruited at the site of tissue damage during lupus nephritis
M. G. Sabbadini, P. Rovere Querini, G. Bonavida, E. Bozzolo, G. Dell’Antonio, E. Dal Cin, C. Doglioni,
A. A. Manfredi
Renal involvement is characteristic of Systemic Lupus Erythematosus. Although acquired immune responses
play a pivotal role, the factors that determine the individual susceptibility are not understood. Soluble pattern
recognition receptors belonging to the pentraxin family are defective in SLE patients, possibly contributing to
the natural history of the disease; we recently found that the prototypical long pentraxin PTX3, which is generated in peripheral tissues under the control of primary pro-inflammatory stimuli, is also defective and fail to increase during active phases of the disease. We therefore verified by immuno-histochemistry whether this factor
was recruited at the inflammatory site in patients with active nephritis. We studied in parallel whether the bestcharacterized ligand for PTX3, the first component of the classical pathway of complement activation, C1q correlates with PTX3 and whether uncleared apoptotic cells, which also selectively bind to PTX3 association, also
correlate with the clinical involvement. Our results suggest that innate factors are directly involved in tissue
damage and represent a target for molecular intervention.
Anti-β2 glycoprotein I antibodies in the natural history of experimental systemic lupus erythematosus
M. G. Sabbadini, P. Rovere Querini, A. Bondanza, A. A. Manfredi
The offspring of NZB/NZW F1 mice develop a syndrome resembling Systemic Lupus Erythematosus. We
followed the outcome of the processing of apoptotic cells both in this model and in non autoimmune prone
control mice. NZB/NZW F1 mice develop an accelerated disease (weight loss, proteinuria, lupus anti-coagulant activity, kidney involvement) only when challenged with dying cells in the presence of adjuvants. In contrast, normal mice repeatedly injected with apoptotic cells failed to develop any autoimmune feature. In contrast, we observed a transient autoimmune response in normal mice, which did not spontaneously lead to clinical disease or antigen spreading. We are exploiting this model to directly assess the role of anti-β2 glycoprotein
I antibodies in the hierarchy of autoimmune response elicited by apoptotic cells, with attention to the differen-
tial processing of the soluble molecule associated when derived from mouse, bovine or human serum. The results indicate that minimal differences possibly play an important role in the generation of this autoimmune response, which is directly implicated in the associated clinical patterns (abortion, thrombocytopenia, thrombosis).
Rheumatoid arthritis as a model for chronic inflammation and tissue damage
M. G. Sabbadini, A. Corti, A. Marinosci, E. Baldissera, F. D’Auria, P. Di Matteo, P. Rovere Querini,
A. A. Manfredi
Unknown triggers induce synovial inflammation in patients with Rheumatoid Arthritis (RA). Synovitis leads
to pain, soft tissue swelling, stiffness, joint erosions and destruction. RA therefore is an ideal model to identify
factors causing chronic tissue damage. RA moreover represents one of the few conditions in which pathogenetic therapies targeting single molecules have been demonstrated to influence the natural history of a disease. In
collaboration with the group of A. Corti in this Institution, we are actively pursuing the characterisation in patients undergoing different phases of the disease (in terms of systemic inflammation and tissue involvement) of
factors involved in the TNF regulation pathway, with attention to molecules linking neuroendocrine and inflammatory cells, like chromogranin A (CgA). Preliminary results indicate that CgA represents a candidate
marker to identify RA patients with systemic involvement. The predictive value of CgA to identify patients at
risk for extra-articular disease is being investigated.
Hereditary periodic-fever disorders as natural models to study homeostatic mechanisms in vivo
M. Tresoldi, S. Franchini, L. Ferri, M. G. Sabbadini
The hereditary periodic-fever disorders, characterized by bouts of fever, rash and joint pain, share several features with rheumatologic diseases. The TNF-receptor associated periodic syndrome (TRAPS) is caused by mutations of TNFRSF1A gene. We studied, in collaboration with the group of L. Obici (Pavia) a TRAPS patient
with a previously non characterized mutation con in codon 30, resulting in the substitution of a cysteine with
tyrosine (CYS30TYR). Analysis of the maternal and paternal DNA showed no polymorphism of TNFRSF1A,
demonstrating that the CYS30TYR mutation had appeared “de novo”. Serum soluble TNF-R1 levels in plasma
were normal, while they were much reduced in the supernatant of patient’s monocytes after PMA stimulation.
The results suggest that an impaired shedding of TNF-R1 was associated with the CYS30TYR mutation. The
effects of this event on actual signalling and induction of apoptosis upon treatment with different death receptors agonists are being actively investigated.
The Viral Immunology Program (VIP) has been running since 2002 and coordinates the activities of different
Units involved in either basic research – mostly located at DIBIT – or clinical research, located at the Centro
San Luigi hosting the Division of Infectious Disease. Although the major focus of this program remains HIV infection and AIDS, other viral diseases, namely hepatitis B and C, and SARS, have been object of recent studies.
In addition, research on tuberculosis and on virus-related oncogenesis as a consequence of infection by human
papilloma virus, human herpes virus-8, and HBV/HCV have become focus of attention. Concerning
HIV/AIDS research, in addition to the continuation of studies on its pathogenesis, particular attention has
been devolved to the immunologic reconstitution consequent to antiretroviral and immune-based therapies. In
addition, significant efforts are being devoted to the research of an effective vaccine capable of either preventing HIV infection or curtailing its pathogenicity.
Validation of an ELISPOT-IFNgamma assay for the diagnosis of TB infection and disease
P. Mantegani, P. Scarpellini, L. Codecasa, L. Galli, A. Lazzarin, C. Fortis
The tuberculin skin test (TST) is largely utilized for both diagnosis and screening of TB infection. However,
its broad cross-reactivity results in poor specificity. We have recently selected and pooled 5 highly immunogenic
peptides derived from ESAT-6 and CFP-10 proteins secreted by M. tuberculosis, but not by BCG strains and
by the majority of environmental isolates, and tested in an ELISPOT-IFNgamma assay in TB-infected and uninfected individuals both HIV+ and HIV-. The test resulted highly specific and sensitive for diagnosis of TB infection (Scarpellini P. et al., J Clin Microbiol 2004). To further validate the test a group of Contact of subjects
with active TB will be studied. The results of the ELISPOT assay will be correlated with the results of TST.
Moreover, a group of HIV-infected and severely immuno-compromized patients will also be studied for detection of TB infection.
Specific Biologic and Molecular Signatures of the SARS-Coronavirus Strain HSR1
E. Vicenzi, F. Canducci, D. Pinna, N. Mancini, S. Carletti, A. Lazzarin, C. Bordignon, G. Poli, M. Clementi
The etiological agent of the recent epidemic of severe acute respiratory syndrome (SARS) has been identified
in a new coronavirus (CoV), designated as SARS-CoV. We have isolated a SARS-CoV strain by inoculating
VERO cells with a saliva specimen of an Italian patient affected by a severe form of pneumonia of unknown etiology with a history of travel from Vietnam to Italy in March 2003. Ultrastructural analysis of infected VERO
cells showed the presence of many virions within cell vesicles and around the cell membrane. The full length viral genome sequence was highly homologous to those derived from the Hong-Kong Hotel M isolate. By optimizing both a real-time reverse transcription-polymerase chain reaction (RT-PCR) TaqMan assay and an infectivity plaque assay we could define that approximately 360 viral genomes were required to generate a plaque
forming unit (pfu). In addition, heparin (100 µg/mL) inhibited infection of VERO cells by 50%, as estimated
by this plaque assay. Overall the molecular and biological characteristics of the strain HSR1 support the evidence that SARS-CoV form a new fourth genetic coronavirus group with distinct genomic and biological features.
NK and GammaDelta T Cells in Anti-Viral Surveillance: Role in the Control of HIV-1 Infection
M. R. Zocchi, C. Fortis, M. Alfano, A. Poggi
We have shown that the release of perforin, granzymes and IFN-gamma, occurring upon contact of NK cells
with HIV-1 infected cells, is dependent on the activation of PI-3K and CAMKII which are inhibited by HIV-1
Tat. Recently, we found a similar impairment in NK cell function, in patients with early HIV-1 infection. Moreover, in HIV-1 infected patients, an increase in circulating Vdelta1 T cells was observed; this was likely due to
the interference of the cystein-rich domain of HIV-1 Tat, present in the serum of these patients and containing
CXC and CC chemokine-like sequences, with the chemotactic activity of CXCL10 and CXCL12. The effect of
these chemokines is dependent on the activation of PI-3K and CAMKII triggered by their specific receptors
(Blood 103:2205-2213, 2004). As Vdelta1 T cells are mainly localized in the mucosal tissue and are involved in
the surveillance against viral infections, the above mentioned mechanism might account for the reported decrease of this subset in intestinal mucosa in HIV-1 infection. As Vdelta2 T cells are decreased in a group of
HIV-1 patients, we are currently verifying the hypothesis that apoptotic mechanisms are operating in this case.
Phenotypic analysis of NK cell subsets during primary HIV infection and follow-up
P. Mantegani, L. Galli, A. Moretta, C. Tassandin, A. Lazzarin, G. Tambussi, C. Fortis
NK cells represent the first line of defence against viral infections. Most (>90%) circulating NK cells belong
to the CD16+CD56dim cytotoxic subset while a minor subset (<10%) is represented by the non-cytolytic
CD16-CD56bright subset. The function of both subsets depends on the balance between several inhibitory and
activating receptors differently expressed on their surface. Our objectives are to analyse the expression of some
activating and inhibitory receptors on circulating NK cell subsets during the acute phase of HIV infection and
during 1 year of follow-up, and to correlate it with immunological and virological parameters and with therapy.
PBMC from individuals with primary HIV infection will be analysed by flow cytometry for surface expression
of CD3 and CD56 molecules and several NK receptors, either inhibitory or activating, by a triple fluorescence
detection method gating on CD3+ and CD3- lymphocytes.
Mucosal humoral immunity involved in HIV resistance
C. Pastori, G. Taskaris, C. Alberti, D. Ferrari, C. Uberti Foppa, A. Lazzarin, L. Lopalco
Since HIV-1 is sexually transmitted, the genital mucosa represents the main site for initial host-virus contact.
As the risk of adults becoming infected through the oral route seems to be relatively low, identifying specific
immuno-repertoires at the level of different mucosal tissues could elucidate the mechanisms of preventing HIV
infection. We studied the humoral immune responses at both genital and systemic levels of HIV-exposed but
uninfected individuals and we detected antibodies with HIV blocking capacity including serum and mucosal
IgA to either CCR5 (one of the main HIV coreceptor) or gp41. We are focusing on immuno and virologic characterization of such HIV neutralizing antibodies. Moreover, we will also identify a reliable standardized assay to
detect and quantify the antibodies at different immuno-compartments. The identification of specific immunorepertoires at mucosal level could help in designing new therapeutical interventions and vaccinal strategies.
Determinants of disease progression in HIV-1 infected children
G. Scarlatti, C. Ripamonti, M. Cavarelli, A. Pastore, I. Karlsson, E. M. Fenyo
Patterns of HIV-1 disease progression differ among infected children: some have a Fast Progression (FP),
while others, the Slow Progressors (SP), stay healthy for prolonged periods. This may reflect the interaction of
genetic, viral and immunologic factors. 50% of HIV-infected individuals who develop AIDS carry an R5 virus.
We have previously shown that the genetic mutation of the chemokine receptor 5 correlates with disease progression, and that R5 viruses from SP children are more sensitive to b-chemokines and grow less easily in tonsil
tissue culture as those form FP. However, Karlsson et al recently showed that R5 viruses have a wide arrow of
receptor specificity, as demonstrated by the use of cell lines transfected with different CCR5/CXCR4 chimera.
Our preliminary results show a close correlation between broad chimeric CCR5/CXCR4 usage of the viral isolates obtained in early age and fast disease progression. Indeed, these results may have relevant implication for
interventions with CCR5 inhibiting drugs and formation of escape variants. We intend to further investigate the
aspect of receptor specificity in relation to antibody and chemokine neutralization escape.
Molecular and biological features of mother-to-child HIV-1 transmission
G. Scarlatti, M. Cavarelli, I. Karlsson, M. Rescigno
The WHO estimated that during year 2004, despite effective antiretroviral therapy (ARV), there have been
approximately 800.000 newly infected children in the world. HIV-1 Mother-To-Child Transmission (MTCT)
occurs during pregnancy, delivery and breast-feeding. Mother-to-child HIV-1 transmission is a multifactorial
event, to which viral, immunological and genetic factors contribute. We have shown that viruses with CXCR4
usage may be favoured during transplacental passage. Furthermore, our recent results suggest that mothers
transmit broad chimeric CCR5/CXCR4 viruses when the mother carries such viruses, which may have relevant
implications for the use of CCR5 inhibiting drugs. A large portal of entry for HIV is certainly the mucosal surface; the baby swallowing large amounts of contaminated fluids. Intestinal enterocytes and M cells were shown
to trancytose selectively virus through binding with GalCer. Recently, however, it was shown that Dendritic
Cells (DC) can favour passage of microbes. As HIV uses DC-SIGN expressed on DCs, as receptor to infect
trans-receptive cells, we want to apply this model to MTCT of HIV.
Pertussis toxin B-oligomer inhibits hiv infection and replication in hu-PBL SCID mice
M. Alfano, C. Lapenta, M. Spada, S. M. Santini, S. Racca, F. Dorigatti, G. Poli, F. Belardelli
Bordetella pertussis toxin B-oligomer (PTX-B) has been shown to inhibit HIV infection and replication in
vitro. The potential antiviral effect of PTX-B was here tested in an in vivo surrogate model of HIV infection, i.e.
severe combined immunodeficiency mice reconstituted with human PBL (hu-PBL-SCID) and infected with a
CCR5-dependent (R5) HIV-1 strain. SCID mice inoculated intra-peritoneal (i.p.) with PTX-B and then infected with the R5 strain SF-162 were sacrificed 7 days later and analyzed for human PBL lymphoid tissue reconstitution, infection of human PBL, plasma viremia and viral rescue from ex vivo cultivated intra-peritoneal human PBL. Mice treated with 500 ng/animal of PTX-B showed no evidence of viral inhibition. Daily administration of PTX-B (50 ng/mouse) strongly inhibited virus infection and replication, as determined by undetectable
viremia, absence of infected hu-PBL, and lack of rescue of infectious HIV in most animals. Furthermore, PTXB injection 2 h before and twice after infection prevented HIV-1 infection and replication in all (10/10) tested
animals. Thus, PTX-B potently inhibited virus infection and replication in hu-PBL-SCID mice supporting the
Pertussis toxin B-oligomer inhibits intra- and extracellular Tat function and HIV expression
C. Rizzi, M. Alfano, A. Bugatti, M. Camozzi, G. Poli, M. Rusnati
By targeting different infected and uninfected cell types, the endogenous HIV-1 transactivating protein Tat
significantly contributes to both virus replication and to the onset of AIDS-associated pathologies. We have
previously demonstrated that the B-oligomer of pertussis toxin (PTX-B) inhibits Tat-dependent HIV-1 long
terminal repeat (LTR) transactivation in T lymphoid Jurkat cells (M. Alfano et al., J. Virol. 74:8767-8770, 2000).
Here we demonstrate that PTX-B inhibits both Tat-dependent virus expression and interleukin-8 secretion in
chronically infected U1 promonocytic cells and Tat-dependent HIV-1 LTR transactivation in non-permissive
epithelial HL3T1 cells. PTX-B-mediated inhibition occurred both when Tat was produced endogenously by
transfected cells and when cells were incubated with extracellular Tat. In this later case, PTX-B did not interfere with extracellular Tat uptake. Thus, the general anti-HIV-1 effect exerted by PTX-B in different cell types
may be accounted for in part by its ability to interfere with either intracellular or extracellular Tat.
Immunomodulation as a strategy of viral pathogenesis
A. Smith, F. Santoro, C. Paolucci, G. Di Lullo, S. Burastero, P. Lusso
HHV-6 is a potentially immunosuppressive agent. We have investigated the phenotypic and functional alterations induced by HHV-6 on blood-derived human Dendritic Cells (DC). We found that, despite the non-productive nature of DC infection by HHV-6, pre-exposure to either HHV-6 A or B impairs the maturation of DC
driven by IFN-g and LPS. Moreover, HHV-6, but not the closely related b-herpesvirus HHV-7, dramatically
and selectively suppresses the secretion of IL-12 by DC. Functionally, DC previously treated with HHV-6 are
impaired in their ability to stimulate allogeneic T-cell proliferation. These data identify interference with the
functional maturation of DC as a primary mechanism of virus-induced immunomodulation, which may facilitate the viral spread and persistence in vivo.
B cell repertoire and anti-HIV humoral immunity in primary HIV infected patients under HAART
C. Pastori, F. Chiodi, A. De Milito, C. Tassandin, K. Titanji, G. Tambussi, A. Lazzarin, L. Lopalco
Highly Aggressive Antiretroviral Therapy (HAART) induces a decline of anti-HIV abnormal immunity in established HIV infection; nevertheless, the impact of early HAART on memory B cells and on specific anti-HIV
humoral immunity during primary HIV infection is not yet clarified. We observed that the early introduction of
four combinations drugs induces HIV specific IgG able to block the infectivity of different clades of HIV-1.
Moreover, in collaboration with F. Chiodi (Karolinska Institutet) we are investigating on activation, differentiation and survival of B cells including analyses of phenotype and apoptosis of naive and memory B cells. Preliminary results indicate that many of the B cell dysfunctions appear early during infectious process and they are
partially recovered in patients under HAART treatment. The effect of different therapy regimens in acutely infected subjects on the development of neutralizing antibodies and on the activation/differentiation of both
naive and memory B and T cells will be relevant for shedding light on the pathogenetic mechanisms involved in
HIV infection.
Selective increase of serum IgE following enfuvirtide treatment
H. Hasson, P. Biswas, L. Galli, S. Burastero, A. Danise, A. Bigoloni, E. Carini, A. Lazzarin, A. Castagna
Enfuvirtide (ENF) is the first entry inhibitor in HIV therapy. It is a synthetic peptide which inhibits HIV-1 by
binding to a gp41 region that is involved in the fusion of the virus. ENF is formulated for twice-daily administration by the subcutaneous route. We evaluated the effect of ENF treatment on humoral parameters possibly
related to ENF-associated injection site reactions (ISRs) by a 24 week prospective study in failing multidrug resistant patients with CD4 counts <100 cells/µl receiving ENF for at least 4 weeks. A significant and selective increase of IgE was observed in all patients, independently of their altered or normal IgE at baseline (BL). The increase was not associated to hypereosinophilia. No cases of systemic hypersensitivity were observed. ISRs were
more frequent in patients with altered BL IgE. IgE increased significantly in all patients, regardless the different
stratifications of their BL CD4 counts. Of note, the ENF-induced increase of CD4 occurred significantly in all
patients, independently of their BL IgE levels. Thus, the positive immunological and virological response associated to ENF treatment is accompanied by a selective increase of IgE levels.
Persistence of CCR5 usage among primary HIV-1 isolates of individuals receiving intermittent IL-2
S. Ghezzi, F. Pacciarini, S. Nozza, S. Racca, F. Dorigatti, A. Lazzarin, F. Veglia, G. Tambussi, G. Poli
Intermittent Administration of Antiretroviral Therapy (ART) and IL-2 to HIV-1 infected individuals determines a sustained increase of circulating CD4+ T lymphocytes. Here we report that the frequency of HIV isolation from PBMC of infected individuals receiving intermittent IL-2 administration together with ART is similar
to that of control individuals treated only with ART. Positive HIV isolation from PBMC was correlated to higher levels of viremia, but not to the number of circulating CD4+ or CD8+ T cells both at entry and after 12 mo.
Negative HIV isolation from PBMC independently predicted increased numbers of CD4+ T lymphocytes following 12 mo of ART+IL-2, but not of ART alone. Fourteen out of 21 (66.6%) individuals with a primary
CCR5-using (R5) HIV-1 isolate at entry and receiving ART+IL-2 maintained viruses with this co-receptor use,
in contrast to 3 out of 7 (42.8%) of those only on ART. Conversely, only 1 out of 21 (4.7%) individuals harboring an R5 virus at entry showed HIV with concomitant usage to CXCR4 after 12 mo of ART+IL-2 in contrast to
2 out of 7 (28.5%) patients treated with ART alone.
Structure-based rational design of HIV-1 entry inhibitors
P. Lusso, F. Sironi, V. Pavone, R. Longhi
Our discovery of the HIV-suppressive activity of chemokines has opened new perspectives for the therapy
and prevention of HIV infection. However, chemokines possess inherent pro-inflammatory activities and cannot be used in their natural form. We have previously identified the major structural determinants of the antiviral and pro-inflammatory functions of RANTES, the most effective antiviral chemokine that binds CCR5.
Based on a prototype biologically active RANTES-derived linear peptide, R11-29, characterized by the presence of 2 aromatic clusters at both extremities connected by a hydrophilic linker, we have generated a series of
mutated peptides with the aim of improving their specific antiviral activity and pharmacological profile. We
have derived shorter linear peptides displaying a more rigid structure, while maintaining a satisfactory solubility, which block various primary HIV-1 strains at nanomolar levels. The fine structural analysis of the antiviral
determinants of RANTES may be instrumental in the rational design of more effective HIV entry inhibitors.
Generation of antigen presenting systems in inducing HIV blocking murine humoral immunity as anti
HIV strategy
C. Pastori, C. Alberti, E. Soprana, R. Longhi, G. Taskaris, A. G. Siccardi, L. Lopalco
In spite of repeated exposures to HIV by sexual route, some individuals remain seronegative (ESN). A subset
of ESN produces HIV blocking antibodies specific to CCR5 (one of the main HIV coreceptors) and mucosal
IgA directed to a small conserved region of env-gp41. In order to induce and reproduce protecting anti-CCR5
and anti-gp41 antibodies in individuals at high risk of HIV infection, we generated chimeric immunogens containing the relevant CCR5 and gp41 peptides in the context of the viral antigen such as hepatitis B virus (HBV)
surface Ag and Flock House Virus (FHV) capsid protein. Administered in mice via systemic or mucosal route,
the immunogens elicited anti-CCR5 IgG and IgA (in serum and vaginal fluids) shared the same specificity of
the previously identified human anti-CCR5 antibodies. We will also test “in vivo” the HIV protective role of antibodies by using Hu-PBL-SCID-mouse. If one or more immunogens (or DNA vaccines) prove to elicit “in vivo” anti-CCR5/gp41 antibodies with the expected properties, and with no toxicity, we could then experiment
the same vaccines in monkeys and eventually in humans.
Innovative approaches to the design of new drugs and vaccines to prevent and cure HIV infection
S. Burastero, A. Lazzarin, C. Paolucci, D. Breda, F. Sironi, P. Lusso
We set up an immunization protocol using recombinant vaccinia viruses in an HIV-free model to infect
murine cells and obtain the membrane expression of Env proteins. These cells were reacted with soluble CD4
in order to obtain complex specific epitopes. We isolated several monoclonal antibodies which bound preferentially to the gp120 molecule engaged with the CD4 receptor or, viceversa, with the CD4 receptor engaged in
gp120 binding. One of such complex specific monoclonal (DB-81) was more extensively studied. We found
that DB-81 is an IgG1, k, anti-CD4 monoclonal antibody specific for the first two domains of the molecule,
which recognizes an epitope distinct from Leu3a, Okt4a, Okt4, SIM-2 and SIM-3. DB81 displays a wide spectrum of neutralization of primary HIV isolates, has poor complement fixing ability and does not significantly
down-regulate membrane CD4 expression. These are promising characteristics in the perspective of validating
this reagent as a novel HIV entry inhibitor.
CCR5 usage as an antibody-protection strategy for HIV-1
P. Lusso, E. Berger, F. Sironi, F. Santoro, C. Ripamonti, G. Scarlatti, R. Longhi, P. Earl, S. Burastero
Primary HIV-1 isolates maintain their external gp120 envelope glycoprotein in a protected conformation in
order to avoid neutralization by host antibodies. We have identified a novel conserved gp120 neutralization epitope (D19), which provides the first evidence of a correlation between HIV-1 biological phenotype (i.e., CCR5
vs. CXCR4 usage) and neutralization sensitivity. The epitope is contained within the V3 domain of gp120 and is
conserved in the majority (23/29, 79.3%) of subtype-B strains, as well as in selected strains from other clades.
Strikingly, in CCR5-restricted (R5) isolates, the D19 epitope is invariably cryptic, albeit inducible by sCD4; by
contrast, it is constitutively accessible in CXCR4-using (X4, R5X4) strains. Accordingly, R5 isolates are resistant
to neutralization by mAb D19, whereas CXCR4-using isolates are neutralized regardless of the presence of
sCD4. These results suggest that the in vivo evolution of HIV-1 coreceptor usage may be influenced by the selective pressure of host antibodies.
Characterization of globally prevalent HIV strains in relation to HIV vaccine development
G. Scarlatti, A. Nordqvist, A. Pastore, R. Volpi, M. L. Kalish, F. Barré-Sinoussi, E. M. Fenyo, A. Careddu
According to UNAIDS the distribution of HIV-1 subtypes worldwide is: A 25, B 16, C 48, D 4, and E 4%.
The apparent predominance of subtype C has corroborated the idea that this subtype could have cell/tissue
specific replication advantages. We study the viral replication, chemokine receptor use and macrophage tropism of 57 HIV isolates of different subtypes from all over the world. The majority of viruses used CCR5, 48 of
these CCR5 alone, 4 with CXCR4 and 3 with minor co-receptors CCR1, CCR3 or CXCR6. Interestingly, all 3
subtype D HIV isolates used CXCR4, while only 1 of 37 subtype C used CXCR4, thus, suggesting that the X4
phenotype is more frequent among subtype D than among other subtypes. The remaining 3 viruses with CXCR4 use were R5X4 and equally distributed among subtypes. Furthermore, all viruses, including those using
CXCR4, could infect and replicate in monocyte-derived-macrophage (MDM) cultures. However, isolates of the
same genetic subtype or similar co-receptor use varied in their capacity to infect MDM. We conclude that
MDM tropism is a general property of HIV of different subtypes and co-receptor use, and does not explain the
large diffusion of subtype C viruses.
Host-Virus interaction in the pathogenesis of viral hepatitis
L. G. Guidotti
The hepatitis B virus and hepatitis C virus are noncytopathic DNA and RNA viruses that cause acute and
chronic hepatitis and hepatocellular carcinoma. Over 500 million people are chronically infected worldwide
and over 2 million die from these infections every year. The focus of our research is to discover the mechanisms
responsible for viral clearance and disease pathogenesis in order to devise new therapeutic approaches to prevent and cure these infections.
Neutrophils contribute to the pathogenesis of CTL-induced liver disease
G. Sitia, M. Iannacone, L. G. Guidotti
We previously showed that following transfer of HBV-specific CTL into HBV transgenic mice many host-derived antigen non-specific inflammatory cells are recruited into the liver and this process amplifies the severity
of CTL-induced liver disease. The severity of liver disease is also ameliorated by the depletion of polymorphonuclear neutrophils (PMNs) which abolishes the recruitment of all antigen non-specific mononuclear cells
into the liver. This effect occurs in the presence of high intrahepatic levels of chemokine gene expression, suggesting that PMN-dependent functions, in addition to chemokine production, are necessary for the recruitment
process to occur. In keeping with this, we recently showed that metalloproteinases (MMPs) known to be pro-
duced by PMNs (i.e. MMP-8 and MMP-9) are rapidly induced in the liver of CTL-injected mice and they facilitate the trafficking of antigen-nonspecific mononuclear cells throughout the liver and the amplification of
pathogenetic events initiated by antigen-specific CTLs.
Platelets contribute to the pathogenesis of CTL-induced liver disease
M. Iannacone, G. Sitia, L. G. Guidotti
Ongoing studies show that platelets play an important role in CTL-induced liver disease. Indeed, using HBV
transgenic mice as recipients of HBV-specific CTLs and mice acutely infected with adenovirus, we showed that
platelets accumulate within necroinflammatory foci of the liver and their depletion profoundly reduces liver
disease severity. Importantly, the inhibition of liver injury occurs under conditions where priming and effector
functions of virus-specific CTLs are normal, although CTL capacity to home to the liver is reduced. In keeping
with this finding, analysis of CTL-platelets interactions under flow conditions in vitro suggests that virus-specific CTLs strongly adhere to activated platelets. Based on these results, it appears that platelets may exacerbate
CTL-induced immunopathology by at least three different mechanisms: first, they facilitate the accumulation of
these cells at the site of inflammation; second, they may directly cause hepatocyte damage and, third, they could
enhance cytopathic functions of other liver-infiltrating inflammatory cells. These unprecedented observations
suggest that platelets play a previously unsuspected role in CTL-induced immunopathology.
A role for platelets in the control of Lymphocytic Choriomeningitis Virus (LCMV) infection
M. Iannacone, G. Sitia, L. G. Guidotti
The studies aforementioned indicated an unexpected role for platelets in the pathogenesis of the immunemediated liver disease. Unlike natural infections, HBV is not infectious for mice and, therefore, those studies
could not unveil a contribution of platelets in the control of an acute viral infection. To investigate this process,
adult immune-competent mice were depleted of platelets and infected with a noncytopathic strain of LCMV.
Surprisingly, a high percentage of platelet-depleted mice died within few days of infection. In addition, the animals that survived failed to clear LCMV from the liver and other organs despite the fact that they mounted a
functional adaptive immune response. Studies are currently undergoing to understand the mechanisms whereby platelets contribute to the control of LCMV infection.
Identification of a novel disease entity caused by HHV-8
L. Dagna, M. G. Viganò, A. Lazzarin, G. Tambussi, M. G. Sabbadini, P. Lusso, M. Malnati
HHV-8 is the causative agent of Kaposi sarcoma. We have developed a highly sensitive quantitative-calibrated real-time PCR assay for the accurate measurement of HHV-8 plasma viremia. This method was employed to
establish an etiologic link between HHV-8 and a unique relapsing inflammatory syndrome in a 61-years-old
HTLV/HIV-seronegative woman who presented with recurrent episodes of fever, lymphadenopathy,
splenomegaly, edema, arthrosynovitis, and skin rash. Atypical Kaposi sarcoma developed 1 year after the initial
clinical presentation. A close correlation between these clinical manifestations and the titers of HHV-8 DNA in
plasma and PBMC was documented. Histological studies on an enlarged lymph node heavily infected with
HHV-8 demonstrated an atypical lymphoproliferative disorder. These results underscore the importance of
quantitative molecular diagnostic methods for the identification of viral pathogens as causative agents of seemingly “idiopathic” disorders.
Study of the HPV related parameters useful in the prevention, diagnosis and monitoring of anogenital
F. Lillo, D. Ferrari, C. Uberti Foppa, M. Origoni, G. Taccagni, M. Cusini, A. Lazzarin
HPV related virological markers useful in the set up of appropriate preventive programs have been studied,
particularly in the HIV positive population. We confirmed that viral load is a helpful parameter in the selection
of women with increased risk of developing cervical high grade lesions and cancer. Analogue studies are ongoing in the male HIV positive population for the surveillance of anogenital tumours, also increasing in this population. Another aspect of the study is the epidemiological observation of the more frequent HPV types and
variants responsible for an increased risk of lesion, with the aim to provide information useful for the set up of
specific vaccines. An analogue epidemiological study is ongoing in an age-selected population in Mombasa
(Kenya). To verify whether the detection of HPV genomic sequences in extralesional sites could be a useful
marker of metastatic micro-dissemination of invasive carcinoma, samples of primary cervical cancer and dissected lymph nodes have been selected and, although very preliminarily, our data suggest the possibility of using HPV typing to trace extralesional expansion of the primary tumour in hystologically negative nodes.
Viral mechanisms of liver carcinogenesis
G. Morsica, S. Bagaglio, L. Aldrighetti, R. Finazzi, L. Albarello, S. De Mitri, M. Bernardi, A. Lazzarin
We have previously shown an association between HCV infection and liver carcinogenesis in patients with
HCV-related HCC. In particular two regions, the non structural NS5A domain and the PKR -phosphorylation
homology domain, PePHD may interact with the host protein PKR controlling protein synthesis and cellular
growth. Further studies are currently in progress on the natural evolution of HCV viral strains in cirrhotic patients who developed or not HCC. Furthermore, with the aim to explore the effect of HCV mutant on the expression of PKR, we developed a quantitative PCR assay to quantify m-RNA PKR in tumoral and non-tumoral
liver tissues of HCC patients infected with HCV-PePHD mutants.
Molecular mapping of resistance conferring mutation in M Tuberculosis
P. Cavallerio, F. Piana, P. Scarpellini, L. Codecasa, G. B. Migliori, P. Cichero, D. M. Cirillo
Tuberculosis is one of the leading causes of death worldwide and Multi Drug Resistant (MDR) TB is associated to high case-fatality rate. Rapid identification of resistance is crucial for prompt administration of appropriate therapy, for preventing development of further resistance and spreading of MDR strains. Reagents are commercially available for rapid detection of resistance. Routine large-scale use of these tests will increase the cost
of TB diagnosis. The aim of the project is to identify a selected population in which these tests are cost-effective
for patient management and disease control. We are mapping resistance associated mutations in strains collected for the TB Resistance Surveillance Project and we are comparing the specific mutations frequency to epidemiological information based on personal data and molecular typing of strains. On 80 MDR strains already
typed we showed that all had a mutation in the rpoB “hot spot”region and 74% had the same point mutation. 3
clusters of transmission were recognized. We are now mapping isoniazid resistance associated mutations. Future development will include detection of mutations associated with resistance to second-line drugs.
Operational research on Tuberculosis in Resources-Limited Countries
F. Piana, G. B. Migliori, D. M. Cirillo
We perform field-visits, supported by WHO and in close collaboration with National Health Authorities, to
countries for assessment of TB programs, laboratory networks, training in TB control and preparation of Global Fund proposals. Several projects started in 2004: in Mozambique, in collaboration with the National TB Program, we started a nationwide Drug Resistance (DR) Surveillance project aimed to detect the mono and multiresistance rate and to assess the efficacy of the local strategy of TB control. Results are expected by late ‘05. In
Kosovo we performed an assessment study on cost-effectiveness of rebuilding a National TB Program. Reviewing data from ’99 to present, we showed that success on TB control was obtained post-war with international
help and without inducing donors’ dependence. We are performing molecular typing and genetic determination of resistance on samples collected in the assessed countries using stained diagnostic slides as study material. Extraction of DNA from fixed slides allows to collect typing and resistance data in absence of culture. Slides
handling does not require a BSL3 facility, they are stable and international shipping is easy and inexpensive.
Clinical evaluation of Gamma Interferon Assays for the detection of active Tuberculosis and latent
F. Piana, P. Cavallerio, L. Codecasa, D. M. Cirillo
Early diagnosis of Latent TB Infection (LTBI) is crucial for tuberculosis control. Current LTBI diagnosis,
based on Tuberculin Skin Test (TST), is affected by BCG vaccination or exposure to non-tuberculous my-
cobacteria. 2 tests, based on gamma interferon (IFN) production upon stimulation with TB specific antigens,
are now commercially available for LTBI diagnosis. The aim of the project is to evaluate sensitivity and specificity of the 2 tests and to assess cost-effectiveness of a large scale use. With the Regional Reference Centre we
recruited 550 patients and we divided them in 3 groups: active TB high risk, infectious case close contacts and
low risk. Contact groups, currently under screening, include 150 hospital contacts, with haematological malignancies, of the same index case. For active TB high risk patients we will compare results of the tests with TST,
culture and X-rays examination. For contacts, stratified according to the time spent with the index case, and for
low TB risk group we will evaluate the gamma IFN test and TST. Preliminary results show a diagnostic usefulness of the gamma IFN tests for detection of LTBI patients in the close contact group, compared to TST.
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The cerebrovascular patient: from bedside to bench and viceversa
M. Sessa, M. Comola, F. Corea, S. Mammi, A. Poggi, L. Roveri, S. Dylgieri, S. Ferrari, F. Fumagalli,
C. Ghidinelli, G. M. Nuzzaco, M. C. Spinelli
Stroke is the first cause of disability and the third cause of death in Western countries. It is then evident the
need of an optimized management of the cerebrovascular patient, the relevance of prevention, and the importance of identifying causes and mechanisms of ischemic damage. To achieve these goals: a) we have set a Stroke
Unit for the management of cerebrovascular patients; b) we are participating in 3 international clinical trials
evaluating new therapeutic approaches in primary and secondary prevention (SITS-MOST, AbESST II, and
PRoFESS); c) we have started two projects aimed at the identification of risk factors, other than carotid stenosis
grade, in patients affected by atherosclerotic carotid disease. In collaboration with the Neuroradiology Unit, we
are performing an MR study of the carotid plaque aimed to characterize the different components of the vulnerable plaque and to define potential correlation with inflammatory markers and clinical outcome. In collaboration with the Unit of Interventional Cardiology, we are evaluating in the acute cerebrovascular patients plasma levels of plaque vulnerability biomarkers, such as the metalloproteinase PAPP-A.
Dementia and cognitive impairment
G. Magnani, R. Mossini, E. Schiatti, M. Franceschi, M. Falautano, A. Barbieri, E. Altamura, G. Comi
Aim of our Unit is to identify early markers of disease to achieve the diagnosis in the early stage of
Alzheimer’s disease (AD). The Dementia Unit has identified, as part of its research program, the AD Research
Project of Health Department on advanced MR techniques for diagnosis and prognosis in AD and Mild Cognitive Impairment (MCI). Moreover we have analysed the prevalence of behavioural and psychological symptoms
of dementia (BPSD) and the relationships between BPSD and cognitive functions in 324 patients with AD. Patients with AD present BPSD both in the early and advanced course of the disease and the prevalence of BPSD
approaches 80%. Our study suggested that in AD patients the presence of BPSD is frequent at the time of diagnosis of dementia and the BPSD are correlated with older age and a worse performance of IADL. Much emphasis has been given to projects with the Clinical Neurogenetic Unit to identify genetic factors in AD and with
the Functional Exploration of the CNS Unit in studies on programming of voluntary movement and motor
control in normal aged subjects, MCI and AD patients. We are also much interested in the future therapeutic
interventions of cognitive impairment.
Headache research unit activity
B. Colombo
Our Unit is deeply involved in the evaluation of mechanisms underlying migraine with aura and the understanding of the role of brain lesions associated with this pathology. We are studying the genetic components
linked to migraine with aura, particularly the ion channels involved in families affected by this disease. We are
investigating the brain activity with neurophysiological approaches (high resolution EEG) to evaluate the different modalities of signal composition in migraine with aura patients vs normal subjects. With MRI-spectroscopy we are studying the composition of brain lesions associated with migraine to understand the tissue
damage due to the dysfunction caused by the vascular event. With f-MRI we are studying the neuronal functions in patients affected by migraine and brain lesions to better understand the plasticity of brain activity. With
Otovestibular techniques we are investigating the correlations between migraine and vertigo. We are involved
in clinical trials, particularly in migraine prophylaxis, both with Topiramate (in high frequencies headaches)
and with Frovatriptan in menstrual migraine. We are involved in epidemiological studies regarding migraine
and epilepsy.
Clinical trials in movement disorders
M. A. Volontè, S. Lalli, E. Molinari
The Movement Disorders Unit has the main aim of providing correct clinical evaluation and diagnosis to patients with Idiopathic Parkinson’s disease, Parkinsonism (Supranucler Palsy, Multisistemic Atrophy, Corticobasal Degeneration), Chorea, Dystonia, Myoclonus and Tremors. Most of them are neurodegenerative progressive diseases. We are involved in clinical pharmacological trials to provide our patients with innovative
drugs; we are also able to support our patients in the complicated phases of the disease with a multispecialistic
team including the surgical approach with Deep Brain Stimulation of Subtalamic Nucleus. Our ongoing research project concern the role of iron and ferritin in the pathophysiology of neurodegeration. Recently a single
mutation of the ferritin L-chain was linked to a neurodegenerative disease, called neuroferritinopathy, in which
iron and ferritin deposits are formed in the basal ganglia of the brain, indicating, in this region, an abnormal
regulation of iron homeostasis. In collaboration with the Functional genomics - Protein engineering Unit we
examine the biological sample of our phenotypically defined patients with movement disorders.
Clinical trials in Multiple Sclerosis (MS)
V. Martinelli, M. Rodegher, L. Moiola, B. Colombo, P. Rossi, F. Martinelli-Boneschi, G. Comi
Our Clinical Unit is involved in many International clinical trials (phase II,III or IV) to validate new therapeutic strategies on MS patients in different phases of the disease. In particular we are coordinating a multicentre trial on the effect of a combined therapy with Beta-Interferon and Mitoxantrone on patients with bad prognostic factors in the early phase of the disease. Moreover we completed the enrollment of patients in different
phase-II studies to evaluate preliminary safety and efficacy data on MRI-measures of newly-developed immunosuppressive drugs: BMS-188667,CCI-779,CFTY720D2201, and of Xaliproden, a neuroprotective and anti-inflammatory drug. Two different multicentre clinical trials have been planned to directly compare beta Interferon 1a or 1b versus Copaxone, the 3 approved drugs for the treatment of MS. We carried out a clinical and MRI
study comparing the efficacy and safety of the oral versus the iv administration of a high dose of steroids during
the recovery from acute relapses. Finally we are the coordinating Centre of a phase III study aimed at evaluating the efficacy and tolerability of Copaxone on patients with a first demyelinating event suggestive of MS.
Epidemiology and characterization of specific aspects of multiple sclerosis
V. Martinelli, B. Colombo, M. Rodegher, L. Moiola, P. Rossi, F. Possa, M. Falautano, F. Martinelli-Boneschi,
G. Comi
By using a computerized data collection we collected and analized, clinical and laboratory data on more than
1200 MS patients who have been treated with Beta Interferon or Copaxone in our MS Centre since 1996, to
identify possible predictors of clinical efficacy or development of side effects. A rehabilitative approach, aimed
to improve meta-memory knowledge and acquire memory techniques, was undertaken on MS patients with
mild memory impairment. We also carried out a neuropsychological evaluation on Primary and Secondary Progressive MS patients with and without cognitive impairment, and compared clinical, brain MRI and elettroencephalographic (EEG) data. Moreover, we started an observational study to monitor the possible effects of different immunomodulant drugs on the cognitive performances, emotional status, fatigue and quality of life in
patients with RR MS.
We assessed the prevalence of pain-related symptoms in a cohort of 424 consecutively recruited MS outpatients during a 3-month observational period. More than half of the MS patients reported the occurrence of at
least one kind of pain. The frequency of neuropathic and somatic pain was increased in cases vs controls, while
no difference was observed regarding headache and migraine. A primary progressive course of disease, a longer
disease duration, an earlier onset of disease and the use of immunosuppressant drugs were predictors of a
greater burden of neuropathic and somatic pain symptoms. We began to collect clinical and genetic data on an
homogeneous cohort of MS patients affected by a primary progressive course, aimed to identify eventual susceptibility and disease-modifying genes. The study included the preparation of a questionnaire of data collection, in which specific questions will be asked on the recurrence of the disease in families.
Molecular genetics of Charcot-Marie-Tooth neuropathies
A. Bolino, S. Benedetti, S. C. Previtali, M. Sessa, P. Carrera, R. Fazio, A. Quattrini, M. Ferrari, G. Comi
Patients with different clinical forms of inherited neuropathies are coming at the San Raffaele Institute for
clinical evaluation. We designed a flow-chart for genetic analysis of 10 genes involved in demyelinating, axonal,
and intermediate forms of Charcot-Marie-Tooth disease. The molecular screening on these patients will allow
us to identify new mutations in known CMT genes and to perform further genotype-phenotype correlations.
Data from clinical evaluation and laboratory tests are included in a database we created to perform genotypephenotype correlations and to follow the progression of the disease.
Monitoring treatment effects in multicenter multiple sclerosis trials using magnetic resonance techniques
M. Filippi, M. A. Rocca, M. Rovaris, F. Agosta, B. Benedetti, P. Tortorella, G. Comi
From the assessment of treatment efficacy in several clinical trials in MS, we found that: in patients with clinically isolated syndromes suggestive of MS treatment with interferon beta-1a is effective in reducing conversion
to clinically definite MS and slowing progressive loss of brain tissue; in patients with Secondary Progressive
(SP) MS, treatment with intravenous immunoglobulin showed no benefit on clinical and conventional MRI
metrics of disease progression over a two-year follow up. Conversely, in a subgroup of these patients, magnetization transfer MRI metrics of normal-appearing brain tissue showed reduced worsening of tissue damage over
time (even if not statistically significant) in treated vs. placebo patients; in patients with SPMS, progressive tissue loss occurs despite the suppression of MRI-visible inflammation, following autologous stem cell transplantation; using the data from a clinical trial of glatiramer acetate, we quantified measurement errors associated
with two techniques for assessment of brain atrophy in MS and demonstrated that a fully automated, normalized technique increases the study power to detect treatment effect on brain volume changes in MS.
Functional MRI correlates of reparative mechanisms in multiple sclerosis and other cns diseases
M. Filippi, M. A. Rocca, F. Agosta, P. Valsasina, A. Falini, P. Tortorella, G. Scotti, G. Comi
Using fMRI, we showed that: a) in patients with clinically isolated syndromes suggestive of MS, movement-related cortical changes occur during the performance of simple and complex tasks and the extent of these
changes might be one of the factors predicting the subsequent evolution to definite MS; b) during a simple motor task, relapsing-remitting MS patients activate brain regions that are recruited by controls during more complex tasks, involving object manipulation. During these latter tasks, MS patients have a widespread, bilateral activation of several regions located in the frontal, parietal and temporal lobes; c) patients with MS, following injury of the motor pathways, show recruitment of a widespread sensorimotor network, which might help to limit the appearance of overt clinical deficits; d) in patients with Devic’s neuromyelitis optica (DNO) movementassociated cortical changes extend beyond the ‘classical’ sensorimotor network and involve visual areas devoted
to motion processing. The correlation found between fMRI changes and the extent of cord damage suggests
that such functional cortical changes might have an adaptive role limiting the clinical outcome of DNO.
In vivo assessment of multiple sclerosis and other neurological disorders using quantitative magnetic
resonance techniques
M. Filippi, M. Rovaris, M. A. Rocca, B. Benedetti, A. Falini, E. Pagani, G. Comi
Using quantitative MR techniques, we found that: a) in patients at presentation with clinically isolated syndromes of the CNS suggestive of MS no abnormalities can be detected in the cervical cord using magnetization
transfer (MT) MRI, suggesting the absence of intrinsic structural damage of the cord soon after the onset of the
disease; b) in patients with early-onset MS, average mean diffusivity of the normal-appearing brain tissue is
slightly increased compared with matching healthy volunteers. This modest CNS involvement might explain
why these patients have a more favorable clinical course than adult-onset MS; c) using DT and MT MRI, gray
matter damage can be detected in patients with Devic’s neuromyelitis optica.
Systemically-injected neural precursor cells recapitulate lymphocyte pathways to enter the central nervous
system of mice with experimental autoimmune encephalomyelitis
S. Pluchino, L. Zanotti, E. Brambilla, G. Salani, M. Bacigaluppi, A. Cattalini, R. Furlan, G. Constantin,
G. Martino
We have explored the (immune) mechanisms sustaining the capacity of adult neural precursor/stem cells
(aNPCs) in promoting remyelination and functional recovery following systemic injection in mice affected by
experimental autoimmune encephalomyelitis (EAE). Selective aNPC homing capability was mainly mediated
by the membrane expression of cell adhesion molecules (CAM) and chemokine receptors. aNPCs constitutively expressed VLA-4, CD44, CCR2, CCR5, CXCR3 and CXCR4. RANTES and stromal-derived factor (SDF)1alpha, dose-dependently modulated aNPCs chemoattraction in vitro. Intravital microscopy showed numerous
aNPCs arresting in inflamed brain venules after injection into the carotid artery of lipopolysaccharide (LPS)treated mice. In vivo blockage of VLA-4 significantly reduced CNS homing of aNPCs. Thus, systemically-injected aNPCs home specifically to inflamed brain areas during EAE, owing to the constitutive expression of the
same molecules used by enchephalitogenic lymphocytes to enter the CNS. The expression of functional receptors, might be considered as a prerequisite for delivery of somatic stem cells into inflamed CNS through the
blood compartment.
Persistent therapeutic effect of i.v.-injected neural precursor cells in mice with relapsing-remitting
experimental autoimmune encephalomyelitis
S. Pluchino, L. Zanotti, E. Brambilla, M. Deleidi, M. Bacigaluppi, G. Salani, R. Furlan, G. Comi, G. Martino
We injected intravenously (i.v.) syngenic aNPCs into mice affected by proteolipid protein (PLP)139-151-induced relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE) at either the disease onset or
after the first relapse. aNPCs entering both the brain and the spinal cord and selectively reaching inflamed CNS
areas survived up to three months after transplantation. At sacrifice, R-EAE mice transplanted with aNPCs
showed a significant decrease of disease score, cumulative score and number of relapses. A significant decrease
of the extent of demyelination and axonal loss was also found. The majority of the transplanted aNPCs maintained over time an undifferentiated phenotype within the host CNS. Surviving aNPCs accumulated within
perivascular areas where reactive astrocytes, inflamed endothelial cells, and encephalitogenic T cells produced
neuro- and gliogenic regulators (BMP-4, Noggin). Here, transplanted aNPCs induced apoptosis of CNS-infiltrating T cell. In CNS inflammation, transplanted adult neural precursor/stem cells (aNPCs) are thus capable
to promote neuroprotection even by maintaining undifferentiated features and exerting unexpected immunelike functions.
Intrathecal administration of an IL-4-expressing helper dependent adenoviral vector is not immunogenic
or toxic in the presence of pre-existing anti-adenoviral immunity and induces clinical and functional
recovery in relapsing EAE
E. Butti, A. Bergami, E. Brambilla, A. Cattalini, U. Del Carro, S. Pluchino, F. Mavilio, G. Martino, R. Furlan
We studied the therapeutic potential of adenoviral-mediated IL-4 gene therapy by inducing relapsing EAE in
SJL mice by immunization with PLP139-151. We injected the IL-4-releasing vector intracisternally the day of
disease onset. IL-4 gene therapy was able to decrease the cumulative clinical score and to significantly decrease
the number of clinical recurrences. We also tested motor evoked potentials (MEP) and found improved performance in mice treated with IL-4 gene therapy as compared to control mice. To assess the immunological impact of this treatment, we serially measured albumin content, immunoglobulin content, and anti-adenoviral antibodies in sera and CSF from macaca fasciculata intrathecally injected with the IL-4-releasing vector. Pre-immunized monkeys had anti-adenoviral neutralizing antibody levels in sera comparable to human patients affected by adenoviral infection while non pre-immunized monkeys became very weakly positive, less than 1/10, after
IL-4 gene therapy. Naive macaques were negative for anti-adenoviral antibodies in the CSF, while pre-immunized animals had anti-adenoviral antibody CSF levels compatible with passive transfer from the serum.
Immunological patterns identifying disease course and evolution in multiple sclerosis patients
R. Furlan, M. Rovaris, F. Martinelli-Boneschi, A. Bergami, M. Deleidi, F. Agosta, G. Comi, M. Filippi,
G. Martino
Reliable, and easy to measure, immunological markers able to denote disease characteristics in Multiple Sclerosis (MS) patients are still lacking. We applied a multivariate statistical analysis on results obtained by measuring – by real-time RT-PCR – mRNA levels of 25 immunological relevant molecules in PBMCs from 198 MS patients. The combined measurement of mRNA levels of IL-1, TNF, TGF, CCL20 and CCR3 was able to distinguish MS patients from healthy individuals. CXCR5, CCL5, and CCR3 combined mRNA levels identify primary progressive MS patients while TNF, IL-10, CXCL10 and CCR3 differentiate relapsing MS patients. Our
results indicate that multi-parametric analysis of mRNA levels of immunological relevant molecules in PBMCs
may represent a successful strategy for the identification of putative peripheral markers of disease state and disease activity in MS patients.
New Antigens In Paraneoplastic Neurological Syndromes (PNS)
R. Fazio, V. Lampasona, M. L. Malosio, A. Bachi, G. Martino
Paraneoplastic Neurological Syndromes (PNS) are rare neurological diseases arising as remote effect of cancer. The diagnosis of PNS is made by detection of antibodies against neuronal antigens. Some typical antibodies
are universally accepted as paraneoplastic antibodies. In clinical practice we also find atypical antibodies.
Among these atypical antibodies the best characterized are anti-Tr antibodies, which are associated with development of Hodgkin disease. Anti-Tr antibodies have a typical ICC pattern, but the target antigen is not known
yet. using a preparative tissue isoelectrofocusing we identified two major protein bands sequenced by mass
spectroscopy (MALDI-TOF). Two potential targets were identified: Aldolase C (zebrine) and Rab-GD1. Incubation of Tr-positive sera with Aldolase-C antigen partially removed the ICC staining on cerebellar sections and
data from confocal microscopy on PC12 cells both suggest only a partial identity between the Tr-antigen and aldolase-C. We are currently evaluating the role of Rab-GD1 as a target antigen anti-Tr antibodies as well as identifying and characterizing other atypical onconeural antigens.
Target validation in peripheral nervous system disorders
S. C. Previtali, M. Scarlato, G. Dina, N. Riva, P. Dacci, M. C. Malaguti, G. Comi, A. Quattrini
The Neuropathology Unit is a leading center for the histological diagnosis of neuromuscular disorders, and
provides a bank of about 3,500 biopsy samples including muscle and sural nerves. The pathogenetic mechanisms sustaining most of the neuromuscular disorders are not known or poorly understood. A great opportunity is to bridge results obtained in animal models, where the defective pathway is known and longitudinal and
prospective studies, with informations achieved by human biological samples and clinical data. This strategy
has been successfully applied in collaboration with other groups, to integrate records from transgenic mouse
models and mutated patients to better understand the pathogenesis of a subset of Charcot-Marie Tooth (CMT)
neuropathies, such as CMT1B and CMT4B. Furthermore, the principal goal of this research plan is to identify
new protein markers for peripheral neuropathies. These protein markers and gene represent attractive candidate targets that may have important implication for diagnosis, prognosis and treatment.
Role of basement membrane, adhesive receptors and Schwann cell cytoskeleton on peripheral nerve
D. Triolo, G. Dina, M. C. Malaguti, I. Lorenzetti, S. Amadio, U. Del Carro, A. Messing, A. Quattrini,
S. C. Previtali
Axonal loss is a consistent and undesirable event in many peripheral neuropathies and is responsible for disabling and permanent deficits. In these neuropathies the physiological ability of nerve to regenerate is lost or reduced. Schwann cells play a key role in nerve regeneration and their function is modulated by basement membrane, adhesive receptors and cytoskeleton constituents. We intend to explore the role that these molecules
may have on axonal degeneration-regeneration induced by crush injury of the sciatic nerve. Transgenic mice depleted of molecules that belong to the basement membrane, adhesive receptors or cytoskeleton will be investigated to dissect the role of this pathway. Preliminary results show a delay in nerve regeneration and remyelination in mice lacking the Glial Fibrillary Acidic Protein (GFAP), a Schwann cell specific cytoskeleton constituent. The peripheral nerves of GFAP null mice normally develop and function due to upregulation of the vimentin intermediate filament which is not sufficient to compensate for the absence of GFAP during nerve regeneration.
The role of extracellular matrix (ECM) in human peripheral nerve regeneration
S. C. Previtali, M. C. Malaguti, D. Triolo, G. Dina, M. Scarlato, P. Dacci, I. Lorenzetti, G. Comi, A. Quattrini
Our knowledge of the regulatory mechanisms and signaling cascade underlying the regeneration program in
human peripheral nervous system is still very limited. Furthermore the majority of the molecular players involved in nerve regeneration await identification. Hence, comparative studies to reveal the role of the extracellular matrix in axonal regeneration will be performed in human nerve samples. This is of particular interest in
chronic axonal neuropathies. We selected biopsies from patients with axonopathy of undetermined origin and
we investigated differences in the expression of several extracellular matrix components such as laminins, collagen, fibronectin, and vitronectin between those with axonopathy but efficient axonal regeneration as compared
to those with axonopathy and inefficient regeneration. Primary results could sustain a different assortment of
ECM molecules in regenerating versus non-regenerating nerves. Since inefficient regeneration affect at least
30% of all the axonal neuropathies, independently from the pathogenesis, any information would definitely
contribute to ameliorate their outcome.
Molecular genetics of Charcot-Marie-Tooth neuropathies
A. Bolino, S. Benedetti, S. C. Previtali, M. Sessa, P. Carrera, R. Fazio, A. Quattrini, M. Ferrari, G. Comi
Patients with different clinical forms of inherited neuropathies are coming at the San Raffaele Institute for
clinical evaluation. We designed a flow-chart for genetic analysis of 10 genes involved in demyelinating, axonal,
and intermediate forms of Charcot-Marie-Tooth disease. The molecular screening on these patients will allow
us to identify new mutations in known CMT genes and to perform further genotype-phenotype correlations.
Data from clinical evaluation and laboratory tests are included in a database we created to perform genotypephenotype correlations and to follow the progression of the disease.
Cerebrovascular disease: neurophysiological monitoring of biological parameters
M. Cursi, G. F. Fanelli, A. Tirelli, G. Elia, C. Paleari, M. Sblendido, L. Giacomotti, M. V. Meraviglia,
F. Minicucci
New projects are related to monitoring techniques. In Intensive Care Unit an automatic or semiautomatic
method of neurophysiological data analysis could be the best way to obtain on-line information on cerebral disease modification and related therapy. We have carried out the project for a new method of EEG analysis able
to detect dangerous variation of cerebral blood flow. An easy to understand index of flow modification is given
by this method, allowing also non trained people to use EEG data. We are confident that this method will be
useful not only in critically ill patients but also in other areas of neurological pathology, such as stroke or syncope. Calculation of IMT (Intima Media Thickness) of carotid artery represents an updated tool to evaluate the
clinical outcome after pancreas and kidney transplantation and the restenosis after carotid endoarterectomy.
We are still evaluating the IMT of the carotid artery in normal and diabetic people. Pathogenetic factors of the
slow pathological evolution of the plaques needs a long follow up of patients; a new multi-centric study will
probably start and our present work will offer the background knowledge. We are also involved in a pilot study
on safety and efficacy of Diverter, a new intraluminal device for avoiding cerebral embolic lesion. Examination
of carotid bifurcation anatomy is our first step of work, but a long term monitor of maintained flow and patency of this filter is the real goal of our participation.
Electrophysiological and psychophysiological evaluation of higher cortical function in motor neuron
L. Leocani, R. Fazio, E. Munerati, F. Cerri, M. Cursi, P. Annovazzi, N. Riva, G. Comi
Over the last years subtle cognitive deficits, particularly related to frontal executive functions, have been noted in non-demented patients with Amyotrophic Lateral Sclerosis (ALS), which had been considered in the past
a disease confined to the motor system. In Primary Lateral sclerosis (PLS) the studies concerning cognitive
functions are scarce. We evaluated electrophysiological correlates of executive functions (event-related potentials -ERPs- and reaction times -RTs- to the Stroop frontal test) in ALS and PLS. ALS patients had slower and
RTs were less precise compared to normal subjects, while PLS showed only RT slowing. ALS but no PLS patients showed reduced amplitude of frontal ERPs compared with normal subjects. ERPs and RT findings confirm the involvement of frontal lobe function in ALS and also suggest that this function is relatively spared in
PLS. Event-related potentials and RTs to the Stroop test may be a useful tool in the assessment of cognitive
function in these motor neuron diseases.
Evaluation of cortical function abnormalities in migraine
L. Leocani, P. Annovazzi, B. Colombo, L. Bernasconi, G. Comi
Central neuronal hyperexcitability is proposed to be the putative basis for the physiopathology of migraine.
Studies using Event-Related Potentials (ERPs) as well as transcranial magnetic stimulation studies suggest the
presence of such hyperexcitability, either during attacks or interictal periods, although results have been conflicting so far. Higher order brain functions and electrophysiological correlates of cortical activation were studied in migraine patients using reaction times (RT’s) and Event-Related Potentials (ERP) to the Stroop test. We
found slower RTs in patients if compared to controls, supporting the hypothesis of a mild cortical function impairment even in interictal periods in this group of patients. ERPs to the Stroop test were increased in migraine
patients compared to controls over occipital and parietal areas. The latter finding this result is consistent with
previous findings of lack of habituation and overload of cortical excitatory vs inhibitory processes in migraine
Impaired short-term motor learning in multiple sclerosis: evidence from virtual reality
L. Leocani, E. Comi, M. Cursi, A. Inuggi, V. Martinelli, M. Rovaris, P. Rossi, M. Comola, G. Comi
Virtual reality has been proposed as a rehabilitation tool in Multiple Sclerosis. The aim of our study was to
evaluate the short-term effects of visuo-motor training in multiple sclerosis (MS) using a ‘virtual reality’ system.
MS patients and normal subjects performed, at basal evaluation and after a short series of repetitions, a motortracking task with their right arm, following an object’s trajectory projected on a screen in a virtual reality environment. Performance (distance from ideal trajectory) was worse in depth compared to frontal planes. MS patients showed normal post-training improvement in the frontal plane, but reduced improvement compared
with normals in the depth planes. Our method provided accurate estimation of motor performances, which, as
expected, were significantly impaired in MS patients. Motor learning was reduced in MS patients for task features requiring a more complex integration of visual and sensory-motor information (depth estimation). These
findings stress the need of a careful customization of rehabilitation strategies, which must take into account the
actual patients’ motor, sensory and cognitive limitations.
Neurophysiological assessment of central motor system in experimental mouse models of EAE and MLD
S. Amadio, P. Morana, M. Miola, G. Martino, S. Pluchino, D. Dolcetta, A. Biffi, L. Naldini, U. Del Carro
Motor Evoked Potential (MEP) using transcranial stimulation is widely and currently used in clinical neurophysiology to evaluate function of central and peripheral motor tracts in a non invasive and repeatable manner.
During the last year our work was aimed to validate this method in order to evaluate its ability to detect corticospinal tracts functional impairment in several experimental murine models. In collaboration with colleagues
of Neuroimmunology Department and Stem Cell Research Institute, we had validated transcranial MEP as a
quantitative method to detect the efficacy of therapies in EAE model: our data supported the hypothesis that
injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Now we are studying
a sjl mouse model of relapsing-remitting MS. Our neurophysiological studies, performed on transplanted MLD
mice made in collaboration with colleagues of TIGET Institute (who carried out an ex vivo gene therapy approach for MLD and GLD based on Lentiviral vectors and haematopoietic stem cells), clearly demonstrated
that ARSA over expression prevents deterioration of motor stimuli conduction along central pathways.
Neurophysiological assessment of peripheral motor system in experimental mouse models
S. Amadio, P. Morana, A. Quattrini, S. C. Previtali, A. Bolino, M. Pennuto, L. Feltri, L. Wrabetz, U. Del Carro
Neurophysiological evaluation of peripheral motor system is currently used both in clinical and in experimental study in order to characterize the pathological feature, the natural course of the illness and therapeutic
modification. In MLD mouse model our data indicate that HSC-based ex vivo gene therapy not only prevented
further disease progression, but also corrected already established functional neurological deficits: the neurophysiological assessment performed on 12 months old mice demonstrated complete normalization of motor
stimuli conduction along not only the CNS but also in PNS. In collaboration with colleagues of Myelin Biology
we are studying the peripheral motor nerve conduction in a mouse model of laminin-2 mutation (merosin deficient congenital muscular dystrophy, CMD) where we are discussing that glial specialization at nodes of Ranvier is important for organization of axonal domains, and provide a molecular basis for the reduced nerve conduction velocity of Congenital Muscular Dystrophy patients. We are also studing different mouse models of
congenital demyelinating neuropathy.
Cognitive neuroscience of language
A. Moro, S. Siri, M. Tettamanti, C. Saccuman, J. Abutalebi, S. Brambati, R. Manenti, S. F. Cappa
The activity in this area is based on the cooperation with the Functional Neuroimaging Section, and with the
Centre of Excellence in High Field Magnetic Resonance. During this year we have completed a number of investigations using functional magnetic resonance dealing with several aspects of language processing in normal
subjects. The main lines of research are the processing of verbs and nouns; the representation of semantic
knowledge; the processing of syntax; the representation of different languages in bilinguals. In addition, we
have continued the investigation of written language processing in familial dyslexia, and the neural correlates of
language recovery in aphasia. The introduction of the 3T machine dedicated to research is already showing
beneficial effects, in terms of quality of the data and overall productivity. The next developments are expected
to derive from the integration of fMR data with neurophysiological findings, in order to obtain real-time temporal information. We continue to pursue active collaborations with research groups with similar programs in
Europe, USA and Canada.
Cognitive neuropsychology of dementia
P. Ortelli, V. Ginex, S. Siri, A. Marcone, S. F. Cappa
This area, based on the application of the models of cognitive neuropsychology to the investigation of patients affected by neurological disorders, has resulted in several publications, mostly focusing on the cognitive
investigation of the dementias. We are now focusing in particular on the investigation of language in
tauopathies, with a particular emphasis on morphosyntactic processing. We have applied an extensive and innovative investigation of all relevant aspects of linguistic processing, and we are going to apply it systematically
to a large group of patients with FTD and other tauopathies. The cognitive results are analysed in parallel with
the results of structural and functional imaging, as well as with the findings of biological investigation. A close
cooperation in this area has been established with the Memory and Aging Group of the University of California
at San Francisco. The collaboration with the Sleep Disorders Unit, dealing with the assessment of neuropsychological dysfunction in patients with sleep disorders, in particular REM-behaviour disorder and the sleep-apnoea
syndrome, is also continuing.
Pain in diabetic neuropathy case study: whole patient management
P. Marchettini, L. Teloni, F. Formaglio, M. Lacerenza
The study reports the complex clinical spectrum of symptoms found in different painful diabetic neuropathies. Accurate clinical evaluation allows disentangling of the multiple pathophysiological pain mechanisms. The therapy is tailored according to the pain diagnosis.
The Lindblom Roller
P. Marchettini, C. Marangoni, M. Lacerenza, L. Teloni, F. Formaglio
The art of thermal testing has evolved since the introduction of the thermal roller. The technique allows bedsite identification of dynamic sensory changes caused by transient spinal ischemia. Two such cases are originally reported.
Sleep Disordered Breathing (SDB): diagnostic procedures and long-term complications
L. Ferini-Strambi, M. Zucconi, A. Oldani, V. Castronovo, M. Manconi, M. L. Fantini, D. Bizzozero, A. Massimo,
G. Lecciso
In a cross-sectional survey in 604 children we demonstrated a high prevalence of SDB in this population.
Moreover, the same study showed that ambulatory monitoring with unattended devices is reliable for the diagnosis of SDB not only in adults but also in children. Complications of SDB include daytime sleepiness and neuro-cognitive impairment. The Epworth Sleepiness Scale (ESS) is a self-administered form developed for assessing subjective sleep propensity during real-life situations. We assessed the validity of an Italian language version
of ESS. Moreover, we compared ESS with a newly developed Resistance to Sleepiness Scale. In a group of SDB
patients we evaluated the cognitive functions before and after a treatment with Continuous Positive Airway
Pressure (CPAP). CPAP did not improve executive functions and constructional abilities even after a 4-month
treatment. Ongoing projects include new studies for evaluating: a) the relationship between nocturnal body
motility-autonomic activity and daytime performances in SDB patients of different age groups; b) the cognitive
performances in SDB before and after treatment with a new CPAP (auto-CPAP) that allows a more stabilized
Restless Legs Syndrome (RLS), Periodic Limb Movements (PLM) and other abnormal motor activity
during sleep
L. Ferini-Strambi, M. Zucconi, A. Oldani, V. Castronovo, M. Manconi, M. L. Fantini, D. Bizzozero, A. Massimo,
G. Lecciso
RLS is a sleep disorder characterized by uncomfortable and unpleasant sensations in the legs that appear at
rest. We have found significant evidence of linkage to a locus for RLS on chromosome 14q13-21 region in a 30member, 3-generation Italian family affected by RLS and PLM. This is the second RLS locus identified so far
and the first consistent with an autosomal dominant inheritance pattern. Clinical and genetic studies in other
large Italian families are ongoing. We participated in international epidemiological studies on RLS; these studies showed that RLS is common in European and US populations (3.4 % in individuals who presented to primary care physicians) and that the prevalence increases with age. Dopaminergic Agents (DAs) are the most successful drugs for RLS treatment. We evaluated the efficacy and safety of some DAs in RLS patients, with particular attention to the “augmentation” phenomenon. This is defined as the earlier onset and increased severity of
RLS symptoms, observed after several weeks of treatment. Ongoing projects include new studies for evaluating:
a) long-term follow up of DA treatment in patients with different degree of RLS severity; b) the clinical significance of RLS and PLM in different neurological disorders.
See: Imaging
Imaging of brain development
C. Baldoli, S. Pontesilli, R. Scotti, P. Scifo, G. Scotti
MRI of fetal brain: in the evaluation of the growing brain new MRI acquisition techniques are now available,
in particular diffusion tensor (DTI) and fast T1-weighted sequences. DTI techniques were optimized to evaluate structural characteristics of the growing brain, with particular regard to pre-myelination processes. For diagnostic purposes, these techniques are able to show parenchymal lesions in an acute stage. Fast T1-weighted
sequences have been optimized to better define morphologic cerebral development and to improve diagnostic
purposes. MRI of neonatal brain: DTI techniques are optimized to follow-up myelination processes in particular in the pre-term brain. Functional MRI has been performed to evaluate cortical response to auditory stimula
in term and pre-term brain, in order to determine which brain regions support language processing in the first
months of life and to evaluate cerebral plasticity in early cortical damage.
MRI in metachromatic leukodystrophy: indications on disease natural history
C. Baldoli, S. Gerevini, M. Sessa, A. Biffi, F. Fumagalli, M. G. Roncarolo, G. Scotti
This project is part of a wider project of the Pediatric Clinical Research Unit of the San Raffaele-Telethon Institute of Gene Therapy aimed to increase the knowledge of the natural history of metachromatic leukodystrophy Magnetic Resonance (MR) imaging has an important role in the diagnosis and follow up of metachromatic
leukodystrophy (MLD). MR images are evaluated for the presence of White Matter (WM) signal alterations
that is shown as hyperintensity on T2-weighted and Flair images, for the appearance of focal or global atrophy
and tigroid aspect. All these findings are defined in a scoring system to evaluate the evolution of the disease. We
study the different types of MLD with conventional MRI and diffusion-weighted MR imaging (DWI) and Diffusor Tensor Imaging (DTI) finding various type of previous results. The variability in DWI findings can be related to the histological stage of the disease at the time of imaging, ranging from intracellular metachromatic
material accumulation to breakdown of myelin membranes.
A multiparametric approach to pre- and intra-operative evaluation of patients with cerebral gliomas:
validation and combined application of Diffusion Tensor Imaging (DTI), Fiber Tracking (FT) and fMRI
A. Gambini, A. Falini, V. Blasi, P. Picozzi, A. Franzin, G. Scotti
The aim of the project is to improve the surgical planning and performance in cerebral gliomas by providing
a complete functional mapping of the grey and white matter adjacent to the tumor. The final objective is to increase patients’ survival allowing a more radical surgical intervention. Functional MRI can identify the cortical
activations related to motor or language tasks; DTI can structurally characterize the tumor and adjacent brain
tissue, and with FT technique it can visualize and reconstruct the pathway of white matter bundles surrounding
the tumor. The application of DTI to tumor patients and even more the combination of this technique with fMRI are at the beginning of their validation. This project includes an experimental phase of technical implementation of DTI and FT and its combination with fMRI results. Once the integration of functional mapping of
grey and white matter adjacent to the tumor is obtained, the final goal is its transfer to the neuronavigation system, so that the whole information is available to the neurosurgeon during the surgical intervention and dynamically used to ameliorate the surgical performance and reduce the risk of inducing lesions.
Voxel Based Morphometry (VBM) and Diffusion Tensor Imaging (DTI) in neurodegenerative diseases:
research for markers of early diagnosis and prognosis
A. Falini, A. Gambini, S. Brambati, A. Castellano, S. F. Cappa, G. Scotti
The aim of the study is the research of markers for early diagnosis and prognosis of neurodegenerative diseases, in particular Fronto-Temporal Dementia (FTD) and Progressive Supranuclear Palsy (PSP) through the
application of advanced MR techniques. The protocol consists of: a) volumetric study processed with Voxel
Based Morphometry (VBM) to objectively quantify the degree of grey matter atrophy and verify the presence of
different regional patterns; b) structural study with Diffusion Tensor Imaging (DTI) and Fiber Tracking (FT),
to verify differences in water diffusion properties and eventually the involvement of specific white matter bundles. The volumetric acquisition is used to segment the brain in grey matter, white matter and cerebrospinal fluid, the volume of grey matter is calculated and the statistical analysis is based on group comparison between patients and controls: areas of statistically reduced grey matter volume in patients are highlighted. DTI is
processed to obtain the Mean Diffusivity (MD) and Fractional Anisotropy (FA) of white matter, grey matter
and whole brain.
Diagnostic and prognostic value of advanced MR techniques in Alzheimer Disease
A. Falini, A. Gambini, M. Bozzali, M. Cercignani, M. Franceschi, G. Magnani, M. Filippi
The aim of the project was to define the clinical utility of MR advanced techniques in patients with Alzheimer
Disease (AD) and Mild Cognitive Impairment. Up to this time 26 patients with AD, 24 with MCI and 23 normal volunteers were recruited. Metabolic modification related to neuronal degeneration was investigated with
whole brain MR spectroscopy. Diffusion Tensor Imaging and Magnetization Transfer Imaging were used to calculate the mean diffusivity and magnetization transfer ratio in the whole brain, in the white and grey components, segmented according to different anisotropy, and in the single lobes. Preliminary data analysis suggest
that metabolic (spectroscopy) and structural (volumetry, diffusion and MTR values) modification can be measured from the early phase of the disease.
The predictive role of clinical and functional MRI findings in patients with post-traumatic vegetative coma
V. Blasi, A. Falini, M. Cadioli, N. Anzalone, A. Franzin, L. Beretta, G. Scotti
Purpose of our study is to investigate the role of functional MRI (fMRI) in predicting the outcome of patients
in post-traumatic Vegetative State (VS). Seven VS patients executed an fMRI study during verbal auditory, musical auditory and tactile stimulation. Neuronal activity correlated with the mentioned stimulations has been determined by means of statistical parametric mapping according to the general linear model. Single subjects results showed a great variability in the pattern of cortical activity among patients. Five more patients and 12 age
matched controls will be studied in order to perform comparison between patients and controls. Furthermore,
patients’ neuroimaging data will be studied by means of correlational analisys with clinical parameters to determine the predictive role of fMRI in determining the probability of VS patients to regain consciousness.
Neural substrates of visuospatial abilities deficits in patients affected by Congenital Hypothyroidism
V. Blasi, R. Longaretti, C. Baldoli, G. Weber, M. C. Vigone, C. Giovanettoni, G. Chiumello, A. Falini, G. Scotti
It is well established that Thyroid Hormone (TH) is essential for early brain development being involved in
neurogenesis, neuronal migration, axon and dendrite formation. Previous studies demonstrated that patients
with Congenital Hypothyroidism (CH) show selective deficits in specific visuospatial abilities such as the ability of locazing objects in space.Purpose of the study is to investigate the neural substrates of the visuospatial
deficits described in CH children by means of fMRI. Fifteen CH patients and 15 age matched controls have
been studied by means of fMRI. The fMRI consisted of an event-related paradigm during which subjects where
asked to fixate a screen where pictures representing two children each with a ball on one hand appeared. Preliminary analyses of fMRI data demonstrated that CH subjects activate more than controls prefrontal cortex
(BA 44-9), while controls activate more than CH an occipital-temporal-parietal network on the right side including inferior parietal lobule (BA 40), precuneus (BA 7), cuneus (BA 18) and fusiform gyrus.
Clinical research in the field of pituitary diseases
M. Giovanelli, M. Losa, P. Mortini, P. Picozzi, L. R. Barzaghi, M. Valle, A. Franzin, C. Ferrari da Passano
The main focus of research of our Unit regards pituitary diseases. Last year, in particular, we explored the
contribution of pretreatment with somatostatin analogs on surgical outcome in acromegalic patients and found
that both efficacy and safety of surgery were not significantly affected by pretreatment with somatostatin
analogs. Another area of active clinical research involved the effects of gamma-knife radiosurgery on tumor
growth in patients with residual nonfunctioning pituitary adenomas after surgical debulking. The results have
shown that in the mid term stereotactic radiosurgery is very effective in preventing tumor regrowth without
causing important side-effects. This is particularly true for the occurrence of hypopituitarism, which seems
much less frequent after radiosurgery than after external frationated radiotherapy. Lastly, we concluded the investigation on the prognostic value of the proliferation index, as measured by Ki-67 immunostaining, in patients with craniopharyngioma. The main result of this study has shown that the risk of tumor recurrence or regrowth is independent of the proliferation index measured in tumor cells. Therefore, the Ki-67 labeling index
cannot be used to assess the risk of recurrence in patient with craniopharyngioma and cannot guide the decision whether perform radiation therapy or not in individual patients. We have active collaboration with other
groups to investigate the regulation of tumor cells obtained from pituitary tumors “in vitro”.
Effectiveness of peritonsillar infiltration with Naropine 0.75% before tonsillectomy on pediatric
postoperative pain
L. Beretta, M. Gemma, L. Gioia, A. De Vitis
Postoperative pain is a major concern after tonsillectomy. This issue is particularly important in children,
since pain is often difficult to evaluate and administration of analgesia is problematic. Intraoperative infiltration
of the tonsillary pillars with local anaesthetics has been tried by different authors with inconsistent results due
to the variety of drugs, administration schemes and pain evaluation methods employed. In a prospective, randomized, double-blind, placebo-controlled study, we evaluated postoperative pain in 3/7- year-old children
submitted to adeno-tonsillectomy and whose tonsillary pillars have been infiltrated with either saline or
Naropine 0.75%. Presently 50 patients have been enrolled in the study (72 patients foreseen). A preliminary
analysis of these first 50 patients shows no difference between groups.
Prone versus knee-chest position for microdiscectomy: a prospective randomized study of intra-abdominal
pressure and intraoperative bleeding
L. Beretta, M. Gemma, A. Rigamonti, M. Messina
Bleeding during microdiscectomy is one of the main concerns for the neurosurgeon, as also minor bleeding
can impair the field of vision of the surgeon. Besides arterial blood pressure, the position of the patient on the
operating table affects the rate of bleeding, since abdominal compression may lead to a rise in the perivertebral
venous system pressure through caval compression. As a matter of fact various studies addressed the topic of
the best positioning of the patient during microdiscectomy, but the majority of them are affected by methodological drawbacks. In a prospective, randomized study, we compare two of the most popular patient positions
for microdiscectomy (the knee-chest and the prone position) by measuring the urinary bladder pressure, which
is presently considered the gold standard for assessing intra-abdominal pressure, and by evaluating the entity of
bleeding. The study has been completed after the enrollment of the foreseen 30 patients. No difference between groups has been evidenced. The present work is in press in the international journal “Spine”.
Keeping and analyzing a multicenter database of severe head injury
L. Beretta, C. Mattioli, E. Grandi
Since 1997 we collaborate with the multicenter Neurolink project, which yielded a database of 1000 severe
head injuries up to now. Besides registering our head injured patients in the database, we perform regularly the
quality control analysis of the entire database. Presently we are performing a review of errors and pitfalls in the
maintainance of such database.
CSF pharmacokynetics of Remifentanil
L. Beretta, M. Gemma, A. Albertin, D. Poli
The modern anesthesia and sedation employs a large variety of drugs, whose effector sites are in the central
nervous system. On the other hand only few studies addressed the CSF (Cerebrospinal Fluid) pharmacokinetics of these drugs. In our neuroanaesthesia practice we routinely place a lumbar CSF drainage in patients undergoing transsphoenoidal neurosurgery for the ablation of pituitary adenomas. This practice warrants CSF
drainage at the end of surgery for the prevention of liquoral fistulas. We performed a clinical pharmacological
study by comparing the plasmatic and liquoral pharmacokynetics of Remifentanil (an anesthetic routinely used
in our operating theater) in patients undergoing trans-sphoenoidal pituitary surgery. The clinical effect of
Remifentanil was assessed by determination of the thermal perception and pain thresholds evaluated in collaboration with the Neurophysilogy Dep. of our Hospital. Presently the work is in progress: 6 patients have been
Evaluation of sedation for MRI in a series of children and babies
L. Beretta, C. Mattioli, S. Piccoli, M. Gemma
Sedation is mandatory in children and babies to maintain immobility during magnetic resonance imaging. We
implemented a database of the activity of sedation in Neuroradiology during the last 3 years. A high degree of
homogeneity is caracteristic of our series: e.v. sodium thiopental for babies younger than 2 ys and propofol for
older patients. The study of particular subpopulations in our series (e.g. metachromatic leukodistrophy or
Prader-Willi syndrome) yields a unique report of sedation in patients with uncommon diseases.
Psychobiology of panic anxiety spectrum disorders: beyond respiration
G. Perna, D. Caldirola, A. Bertani, R. Bussi, M. Cucchi, S. Cammino, L. Bellodi
It is well established that respiration has a central role in panic disorder and we have recently demonstrated
that irregular breathing is the physiological abnormality that undelies panic disorder. The role of respiration appears crucial also examining the “language” of dyspnea in panic patients: we have shown that some specific descriptors of respiratory discomfort seem to be characteristic of panic patients and might be used to better characterize and define this disorder. A recent study from our team has also shown that citalopram, a well established anti-panic agent, is able to improve respiratory function and quality of life in oxygen dependent patients
with chronic obstructive pulmonary diseases. Overall these studies support the view of a strict relationship between panic disorder and medical diseases specifically foucusing on the respiratory system. We are currently
examining the role of a complex respiratory-cardiovascular and balance system dysfunction in panic disorder.
Psychobiology of the panic anxiety spectrum disorders: fear conditioning
G. Perna, E. Favaron, C. Namia, G. Vanni, R. Mellone, S. Biffi, L. Liperi, L. Bellodi
Fear conditioning is one of the main brain mechanisms related to the development of reactive behaviors to
adversive stimuli. We have recently shown that patients with panic disorder have a reduced reactivity of the
pavlovian fear conditioning mechanism and this finding has been confirmed in an independent sample. This result supports the idea that panic is qualitatively different from fear. We are currently testing this mechanism in
first degree relatives of patients with panic disorder with the aim of understanding if this abnormality might be
related to a familial genetic vulnerability. The development of a fear conditioning model in humans might also
allow to test the anti-phobic effects of novel drug treatments.
Chronobiology of mood disorders
C. Colombo, F. Benedetti, B. Barbini, A. Bernasconi, E. Campori, M. Cigala Fulgosi, A. Pontiggia, M. Florita,
E. Smeraldi
Since 1995 we studied the possibility to treat mood disordered patients by acting on their biological rhythms.
We successfully concluded and published studies on the combination of chronobiological interventions (total
sleep deprivation, sleep phase advance, bright light therapy) with lithium salts or serotonergic treatments, and
on the role of both clinical and genomic predictors of antidepressant response (gene polymorphisms pertaining
to the molecular clock and to neurotransmitter systems). As a whole, our results provided new insights of the
pathophysiology of mood disorders, and showed the possibility to obtain complete and sustained antidepressant responses with a one-week treatment, thus reducing disability and social costs of major depression. The reliable, rapid and sound effects obtained in depressed patients led to a good media coverage of our results, and
is leading to the international diffusion of our treatment protocols in other psychiatric clinics.
Genetics of affective disorders and schizophrenia
A. Serretti, C. Lorenzi, R. Cavallaro, P. Artioli, L. Mandelli, A. Pirovano, M. Catalano, E. Smeraldi
Major psychoses are serious disorders that affect about 7% of the population. During the last years we identified several gene polymorphisms influencing features of both mood disorders and schizophrenia. With regard
to pharmacogenetic research, we identified that polymorphisms in the genes encoding for the promoter region
of the Serotonin Transporter (SERTPR), the Tryptophan Hydroxylase Enzyme (TPH), the Serotonin Receptor
1a (5HT1A), the G-protein beta3- subunit and the Circadian Locomotor Output Cycles Kaput (CLOCK) independently predict antidepressant efficacy, while SERTPR variants were associated also with lithium response
and sleep deprivation efficacy. A neural network pharmacogenetic approach was developed for the analysis of
complex interaction between genetic and environmental factors and demonstrated superiority versus traditional techniques. We also observed an association between CLOCK variants and clozapine induced sleepiness. We
investigated the time course of mood disorder searching for both clinical and genetic predictors. Moreover, we
performed a wide-genome scan analysis (by microsatellite repeats) on sib-ships affected by mood disorders; the
aim of the study is to identify if that there are one or more chromosome regions linked to affective disorders.
Neuropsychological assessment and rehabilitation of schizophrenia
R. Cavallaro, S. Anselmetti, M. Bechi, E. Ermoli, F. Cocchi, S. Angelone, E. Smeraldi
Research group activity maintained the focus on the assessment and treatment of the neuropsychological
deficits known to be chronically present in schizophrenia and responsible of the personal, social and relational
functional impairment with innovative protocols. We concluded and presented to the international scientific
community a placebo-controlled study of computer assisted cognitive remediation of neuropsychological
deficits, known to be limiting factors of classical cognitive-behavioural rehabilitation, as a potentiating strategy
the latter. Treatment with individual, deficit-driven, exercise on personal computers showed efficacy on the
main deficitary neuropsychological functions and effectiveness on the personal, social and relational functioning. A new study on the effect of the COMT gene polymorphysm on the efficacy of cognitive remediation in
schizophrenia has been completed and research on the neuropsychological effects of antipsychotic treatment
was continued. New research on language in schizophrenia, according to the models of universal grammar, was
started in collaboration with Prof. Andrea Moro of the Faculty of Psychology of Vita-Salute University.
Neuropsychological aspects of obsessive-compulsive spectrum disorders
P. Cavedini, T. Bassi, C. Zorzi, A. Gorini, E. Marocco, L. Bellodi
Executive functioning deficits have been found by our group in patients with Obsessive-Compulsive Spectrum Disorders (OCSD), including eating disorders and pathological gamblers. Neuropsychological analysis of
decisional processes can explain phenomenological heterogeneity and deepen the understanding of the pathophysiology of these disorders. The results of our studies have highligted the role played by the neuropsychological profile in predicting the response to treatment, thus permitting to plan in advance an augmentation strategy proved to be effective in anticipating response.
Emotional processes in obsessive-compulsive spectrum disorders
P. Cavedini, C. Zorzi, T. Bassi, M. Piccinni, A. D’Annucci, L. Bellodi
Fear conditioning is one of the central mechanisms, on which behavioural therapy of anxiety disorders is
based. We have investigated pavlovian fear conditioning in a sample of patients with Obsessive-Compulsive
Spectrum Disorders (OCSD) and the results have shown that this basic fear mechanism is abnormal compared
to healthy controls. The investigation of fear conditioning mechanisms might help to understand biological
mechanisms underlying avoidance in anxiety disorders and possibly it will help to understand and predict the
response to behavioral therapies. These preliminary results encourage further studies in this field. We are also
studing neurophysiological parameters during decision-making and during the presentation of emotional stimuli, to provide evidences for an abnormal activation of the somatic state in OCSD.
Clinical psychopharmacology and psychopathology in eating disorders
S. Erzegovesi, M. C. Cavallini, G. Diaferia, A. Casolari, T. Bassi, F. Mapelli, B. Negri, L. Bellodi
We have been investigating two main areas: 1) Clinical psychopharmacology. 1a) Drug treatments. OBJECTIVES AND METHODS: Drug therapies are scarcely effective in underweight Anorexia Nervosa (AN). New
treatment strategies in AN are important and needed. We have been trying add-on therapies (e.g. SSRIs plus
olanzapine) in addition to Cognitive Behavioral Therapy (CBT) in AN inpatients. RESULTS: add-on drug
treatments seem to improve outcome in underweight AN inpatients, in comparison with CBT only. 1b) Predictors of drug response. OBJECTIVES AND METHODS We have been trying to identify clinical, genetic and
neuropsychological predictors of response (2,3). RESULTS: The presence of “poor insight” in AN inpatients
predicted a poorer outcome. On the other hand, the “long-long” variant of the serotonin transporter promoter
region predicted a better outcome. 2) Psychopathology: Clinical characteristics, Family History and Comorbidity. OBJECTIVES AND METHODS: The clinical phenotype in ED seems to be etherogeneous. We’ve been
studying ED symptom subtypes, OC symptoms, genetic features, and personality, temperament and character
in Anorexia and Bulimia Nervosa (1, 4-6). RESULTS: EDs can be subdivided in discrete clinical phenotypes
(e.g. poor insight AN, Bulimia Nervosa with Borderline Personality Disorder). Each discrete phenotype seems
to have different outcome and different treatment needs.
Psychometrics of personality disorders
A. Fossati, S. Borroni, I. Carretta, F. Grazioli, M. Donini, M. Battaglia, T. Beauchaine, J. Feeney, E. Barratt,
C. Maffei
Taxometry of personality disorders. This research project was carried out to investigate if the latent structure
of schizotypal, narcissistic, avoidant, dependent, and obsessive-compulsive personality disorders should be better described in terms of dimensions or categories. Both multivariate mixture models and taxometric techniques – namely, mean above minus mean below a cut (MAMBAC) and maximum covariance (MAXCOVHITMAX) analyses – were used in order to identify the existence of latent discontinuities in the distribution of
the characteristics of these personality disorders. Monte Carlo simulations were used to assess the significance
of these findings taking into account also the risk of biasing effects due to variable skewness, kurtosis, etc. on
the results. Impulsivity,aggression, and personality disorders. This research project aimed at investigating the
relevance and specificity of the associations between personality disorders, and dimensions of behavioral
dyscontrol, namely,impulsivity and aggression, using a convergent-discriminant validity design. Both an analytic factor and a multiple linear regression design were used to identify specific relationships bewteen selected
personality disorder and action personality traits, and controlling for the possible confounding role played by
demographic and clinical variables, such as gender and presence of any axis I diagnosis. Moreover, we also tried
to evaluate how sub-components of these action personality traits were effective in discriminating the individual personality disorders loading on the same latent dimension.
Developmental and experimental psychopathology
M. Battaglia, A. Ogliari, A. Zanoni, C. Maffei, A. Citterio, L. Falzone, F. Crevani
Developmental Behavioral Genetics: ongoing research projects include children and adolescent twin studies
of manifestations of anxiety through biometrical fitting models and covariation with endophenotypes. Other
protocols are focused on the influence of functional polymorphisms of the serotonergic system in childhood depression, and on the influence of functional polymorphisms of the cholinergic system in influencing the response to adverse environmental events. -Emotions and recognition of peers’ face expressions in children with
and without Behavioral Inhibition (BI) two independent cohorts of children between the age of 7 and 10, carefully characterized according to their degree of BI are being followed up for: degree of BI at follow up, and the
ability to classify a set of standardized pictures of facial affects in peers.
Biological significance of the cross-talk between the nitric oxide and sphingolipid signalling pathways
C. Sciorati, C. Perrotta, C. De Palma, S. Bulotta, N. Borgese, E. Clementi
Endothelial NO synthase (eNOS) is activated through various pathways involving increases in cytosolic Ca2+
concentration, activation of Akt, and regulation by other kinases and by protein-protein interactions. This enzyme plays a crucial role in mediating biological functions in endothelial cells, including those generated by Tu-
mour Necrosis Factor alpha (TNF). In human endothelial cells we found that eNOS is activated by TNF in a
pathway involving activation of a neutral sphingomyelinase, followed by generation of the sphingolipid ceramide. Ceramide is then converted to Sphingosine 1 Phosphate (S1P) by the sequential activation of neutral
ceramidase and sphingosine kinase 1. S1P eventually activates Akt through phosphorylation at Ser 473 and
eNOS itself through phosphorylation at Ser 1179. We also found that NO inhibits, through generation of
cGMP, the activation of both the acid and neutral sphingomyelinases, suggesting the existence of a cross-talk
between the sphingolipid and NO signalling pathways. We are now exploring the role of this cross-talk in modulating the effects of TNF on endothelial cells, including migration and adhesion of macrophages and dendritic
Nitric oxide-dependent mitochondrial biogenesis and its role in energy metabolism
E. Nisoli, S. Falcone, M. Carruba, O. Cantoni, C. Sciorati, C. De Palma, A. Pisconti, S. Moncada, E. Clementi
Nitric Oxide (NO) regulates acutely mitochondrial function, acting as an oxygen sensing system, through its
interaction with complex IV in the mitochondrial respiratory chain. We have studied whether NO acts as a
long-term modulator of mitochondrial function. We found that NO triggers mitochondrial biogenesis independently of the cell linage and species, since the process was observed in myoblasts, neurons, macrophages
and adipocytes of human, rat and mouse origin. The effect of NO was cyclic GMP-dependent and mediated by
induction of peroxisome proliferator-activated receptor gamma coactivator 1alpha, a master regulator of mitochondrial biogenesis, followed by increased activity of the mitochondrial transcription factor A and of the nuclear respiratory factor. The newly generated mitochondria are functional and generate ATP. The NO-dependent mitochondrial biogenesis appears to play a role in the regulation of energy metabolism, since its impairment
is linked to obesity, metabolic syndrome and defects in cell proliferation and differentiation.
Identification, origin and functioning of neurosecretory granules
J. Meldolesi, P. Podini, G. Racchetti, A. Lorusso, E. Chieregatti, A. Laslop, T. Giordano, E. Cocucci,
M. L. Malosio
Identification, origin, and functioning of neurosecretory granules. This field was debated in the last few years
because a few prestigious labs had proposed minimalistic hypotheses. Our work (Malosio et al. J. Cell Sci.
117,743-749, 2004) has demonstrated the inconsistency of those hypotheses. The problem has also been discussed and further clarified in overview (Meldolesi, Chieregatti and Malosio, Trends in Cell Biol. 14,13-19,
2004). The most demanding commitment in this area is for the identification of master genes of neurosecretion,
to be investigated in wild type and defective clones of PC12 cells. So far we have localized the gene and now we
intend to map, sequence and express it. Finally, synaptic vesicle fusion occurs via the establishment of a complex of proteins, the SNAREs, in collaboration with a Ca2+ sensor. In this field we have published a note in Nature Cell Biology (Meldolesi and Chieregatti 6,476-478, 2004).
Nature and function of enlargeosomes
J. Meldolesi, P. Podini, G. Racchetti, M. Rupnik, T. Kirchhausen, J. Cerny, B. Borgonovo, E. Cocucci,
E. Chieregatti
The most original project of the lab concerns the nature and function of enlargeosome, a new organelle, competent for regulated exocytosis that we discovered in 2002. The continuation of the work has revealed new
properties of the organelle, demonstrating its endocytosis by non-conventional pathway(s) (Cocucci et al., Mol.
Biol. Cell 15,5356-5368, 2004). Moreover, in collaboration with a group at Harvard, we have demonstrated the
role of enlargeosomes in the repair of plasma membrane wounds occurring especially in some cell types (Cerny
et al., EMBO Rep. 5,883-888, 2004).
PLOSL, the Nasu-Hakola disease
J. Meldolesi, P. Podini, M. Mariani, P. Panina, A. Meroni, R. Spreafico, F. Sinigaglia, M. Colonna, G. Sessa
The project supported by a Telethon grant investigates PLOSL, a genetic disease due to mutations of a receptor, TREM-2, or its coupling protein DAP12. Working on the human and mouse brain we demonstrated that
the two molecules are expressed both by microglia and neurons, not at the cell surface, as expected, but especially in an intracellular pool which is redistributed to the surface after stimulation. The published work (Sessa
et al., Eur. J. Neurosci. 20,2617-2628, 2004) is now being pursued to clarify details and pathogenetic mechanisms.
S Molecular study of neurodegeneration
J. Meldolesi, P. Podini, E. Chieregatti, G. Racchetti, A. Lorusso, A. Klajn, R. D’Alessandro, E. Cocucci,
G. Naum-Ongania
The laboratory has initiated a molecular study of neurodegeneration in the framework of a European project
(APOPIS). We focus especially on the role of synapses analyzed functionally by in vitro reconstitution and by
cellular and biochemical approaches. The present results concern synuclein, a protein mutated in some early
forms of Parkinson disease; the work on beta amiloyd is now being developed.
Mechanisms of Protein Kinase C activation in neurons
F. Codazzi, A. Di Cesare, N. Chiulli, A. Albanese, D. Zacchetti, F. Grohovaz
Protein Kinase C (PKC) plays an important role in neurotransmitter release, synaptic plasticity, neurodegeneration, and represents a point of convergence of signal transduction at the post-synaptic level. These functions
appear to be mainly sustained by the neuronal specific isoform, PKC-gamma, a member of the conventional
PKC family, whose activation requires Ca2+ and diacylglycerol. We focused our research on the characterization of the signaling steps leading the activation, of PKC-gamma in hippocampal pyramidal neurons by using
the Total Internal Reflection Microscopy, combined with Ca2+ imaging. We showed that the diacylglycerol necessary to sustain PKC-gamma activation can be produced by stimulation of PLC-delta via a strong calcium influx, through NMDA receptors or voltage-operated Ca2+ channels, independently of stimulation of
metabotropic glutamate receptors. On the other hand, moderate or slower Ca2+ influxes appear to be equally
effective in promoting PKC-gamma activation only when coupled to stimulation of metabotropic receptors. On
the whole we propose PKC-gamma as a powerful coincidence detector of multiple signals, both synaptic and
Multiple activation pathways in astrocytes: role in neurodegeneration
A. Di Cesare, F. Codazzi, N. Chiulli, D. Zacchetti, F. Grohovaz
Changes in the phenotype of astrocytes (a process known as activation) frequently occur in the central nervous system in response to physiological and pathological stimuli. Under pathological conditions, this state of activation is maintained in glial cells and may amplify tissue damage. We are characterizing how various stimuli
can activate astrocytes, by investigating changes in morphology, proliferation rate, secretion of biologically active molecules affecting neuronal activity and survival. Rat cortical astrocytes in culture are exposed to cytokines (IL-1b, TNFa, INFg), trophic factors (FGF2), or neurotoxic metabolites (sphingosylphosphocholine,
beta-amyloid), and then analyzed for both the expression of markers of activation (e.g. COX-2, iNOS) and the
ensuing secretion of neuromodulatory molecules (e.g. PGE2, IL-6, NO, glutamate, ATP). Our results show
that different kinds of activation can be distinguished based on the phenotype, and that specific signal transduction pathways sustain them. We have specifically investigated the role PGE2 plays on astrocytes and the
transduction pathways activated upon binding of PGE2 to its cognate receptor.
Translational control of BACE-1 expression: a possible role in Alzheimer’s disease
M. Mihailovich, F. Grohovaz, D. Zacchetti
BACE-1 is the membrane-bound aspartic protease (beta-secretase) that initiates the production of the neurotoxic beta-amyloid peptide, which accumulates in the brain of patients affected by Alzheimer’s disease (AD).
Therefore, the control of BACE-1 expression appears to be crucial to avoid an excess of beta-amyloid production. Indeed, it has been shown that BACE-1 protein/activity is elevated in a subpopulation of sporadic AD patients, even though the higher BACE-1 levels are not paralleled by an increase in the corresponding transcript.
Within this framework, we are investigating the molecular mechanisms that regulate BACE-1 translation. We
have recently demonstrated that modulation of BACE-1 translation can occur via a tissue-specific alternative
splicing in the transcript leader. The occurrence of this splicing in brain, as well as in neuronal cultures is under
investigation. On the other hand, we have also evidence that mRNA binding factors can control BACE-1 trans-
lation. Their identification might pave the way to novel pharmacological strategies for the prevention and cure
of the disease.
Role of sphingosylphosphocholine in neuronopathic Niemann-Pick type A disease
N. Chiulli, F. Codazzi, A. Di Cesare, D. Zacchetti, F. Grohovaz
Niemann-Pick type A is a genetic disease characterized by the absence of a functional ASM (acidic sphingomyelinase) gene and an abnormal accumulation of sphingomyelin. In this disease, also sphingosylphosphocholine (SPC, a sphingomyelin metabolite) accumulates in various tissues, including the brain, where it might
act as a toxic stimulus. We have studied the effects of SPC on astrocytes and neurons in culture. Our results
show that SPC acts on astrocytes and produces a Ca2+-dependent release of glutamate that, in turn, leads to
[Ca2+]i elevation in the neurons. In addition, we found that chronic SPC treatment is able to drive astrocytes
to a state of ‘activation’ characterized by increased proliferation and release of proinflammatory molecules. In
conclusion, we propose that astrocytes can be the main target for some of the toxic agents that accumulate abnormally in neurodegenerative diseases, and that molecules released by astrocytes can generate signals, leading
neurons to die or to become more susceptible to other toxic stimuli.
Cerebellar malformations and disfunction in Ebf2 null mice
L. Croci, E. Motti, R. Tonini, R. Hawkes, F. Rossi, G. G. Consalez
Our group has developed and partially characterized mice carrying a null mutation of the Ebf2 gene, encoding an helix-loop-helix transcription factor involved in primary neurogenesis and neuronal differentiation. Ebf2
null mice feature a severe defect in motor learning as well as remarkable abnormalities in cerebellar corticogenesis, with defective foliation and a sharp reduction in the total number of cerebellar Purkinje Cells (PC), caused
by impaired migration and a selective loss of specific PC subpopulations. This leads to significant alterations in
the cerebellar map, accompanied by defects in PC axon guidance, maturation, pruning and myelination. Additionally, electrophysiology studies conducted on postnatal and adult tissue slices reveal a significant decrease in
the spontaneous firing rate as well as an abnormally depolarized threshold for the action potential in mutant
PC. A search for Ebf2 target genes has been conducted in various wildtype and mutant tissues, and its analysis
is currently in progress.
The synapsins: regulators of neurotransmitter release and of synaptogenesis involved in epilepsy and
mental retardation
F. Valtorta, A. Menegon, D. Bonanomi, C. Albertinazzi, G. Maila
The synapsins are Synaptic Vesicle (SV) proteins whose deficiency in mice causes increased seizure susceptibility. The recent identification of a SYN1 mutation in a family with X-linked epilepsy and/or mental retardation indicates a role of synapsins in human neuropathology. Synapsins have been proposed to maintain a reserve pool of SVs in the proximity of the active zone by tethering SVs to each other and to the presynaptic actin
cytoskeleton and to control the availability of SVs through their ability to dissociate from SVs and actin in a
phosphorylation-dependent manner. We have found that synapsin I and its phosphorylation by PKA play a pivotal role in membrane dynamics in growth cones as well as in synapse formation. These results provide new
clues as to the bases of the function of synapsin I in development and offer an explanation for the well known
actions of cyclic AMP in developing neurons. Moreover, they establish a conceptual framework for the development of therapeutic tools aimed at promoting the formation of nerve terminals after injury or in neurodegenerative diseases.
Mode of action of mood stabilizing agents on the activity of synaptic terminals.
F. Valtorta, C. Albertinazzi, A. Menegon
Bipolar disorder, also known as manic-depressive illness, is a common psychiatric disorder characterized by
cycling periods of extreme elation (mania) and depression. A variety of drugs are effective mood stabilizers
used for bipolar disorders. Although a number of pharmacological actions have been reported for these compunds, the molecular mechanisms at the basis of their activity as mood stabilizers are still unknown. We have
evaluated the dynamic properties of synaptic terminals of hippocampal neurons treated with therapeutic concentrations of the mood stabilizing agents lithium, carbamazepine and sodium valproate. The results show that
chronic treatments with all three drugs result in an increased synaptic vesicle recycling at the level of nerve terminals and that this effect is related to the ability of the drugs to inhibit inositol monophosphatases.
Snake presynaptic neurotoxins as tools for the study of the molecular mechanism of neurotransmitter
F. Valtorta, D. Bonanomi, M. Pennuto
Transfer of information in the brain occurs through the exocytotic release of neurotransmitters which are
contained in Synaptic Vesicles (SV). SV exo-endocytosis is a multistep process which involves a highly regulated interplay of soluble and membrane-associated proteins. Animal toxins are useful tools for dissecting the molecular steps involved in neuroexocytosis. Indeed, thanks to refinements in the course of evolution, some toxins
are exquisitely specific for (a) selected target step(s) in the process. The application of the snake neurotoxin
taipoxin to hippocampal neurons induced exocytosis of SV not balanced by endocytosis. Using digital imaging
videomicroscopy to measure Fluorescence Resonance Energy Transfer (FRET) in live neurons, we found that
taipoxin causes dissociation of the SV proteins VAMP2 and SypI at a stage preceding SV fusion, thus making
VAMP2 available for entering the fusion complex which drives exocytosis. Thus, we have validated in intact
neurons interactions which had been hypothesized based on experiments carried out with isolated proteins or
organelles, and have shown the relationship of such interactions with neurotransmitter release.
Mechanisms of synaptic vesicle biogenesis and recycling
F. Valtorta, L. Rusconi, D. Bonanomi
Synaptic Vesicle (SV) proteins are synthesized in the cell body and are transported down the axon in membrane precursors. In spite of their fundamental importance for neuronal function, the mechanisms underlying
sorting of proteins to SVs remain unclear. To address the issue of protein sorting to SVs and to investigate
whether the sorting determinants required for SV assembly are dependent on a neuronal context, fluorescent
chimeras of SV proteins have been expressed in both hippocampal neurons and HeLa cells. We have observed
that: i) the various SV proteins follow different trafficking routes; ii) Synaptophysin I has a strong targeting signal to SV, and it selectively controls the sorting of Vamp2; iii) the behaviour observed for a single SV protein in
neurons is reproduced in non-neuronal cells. Thus, common sorting mechanisms are effective in neuronal and
non-neuronal cells, indicating that the sorting determinants of SV proteins are independent of their functional
See: Imaging
Functional role of AFG3L2/paraplegin complex in mitochondria
L. Atorino, L. Silvestri, L. Cassina, F. Maltecca, L. Bardinella, G. Casari
The SPG7 gene, responsible for a recessive form of hereditary spastic paraplegia (HSP), encodes paraplegin,
a nuclear-encoded mitochondrial metalloprotease. Recently we showed that paraplegin co-assembles with a
highly homologous protein, AFG3L2, to form a 900 kDa functional complex in the inner mitochondrial membrane which is aberrant in HSP patients’ fibroblasts. To get insight on the functional role of the complex in
neuronal cells, we are generating a SPG7-/- cell line in the SH-SY5Y neuroblastoma cell line. Furthermore, we
are using this cell line for the identification of binding partners of the complex by crosslinking and mass spectrometry analysis. The Spg7-/- mouse model shows a slowly progressive phenotype. We are characterizing two
different Afg3l2 mutant mouse models showing an extremely severe neuromuscular phenotype. Preliminary results show that in mutant mice the number and the size of corticospinal fibers are significantly reduced. Moreover, a marked decrease of complex I activity has been observed. To better analyze the progression of the neuropathological damage in the adult mouse, we are generating an Afg3l2-/- conditional mouse model.
Characterization of mouse models for FHM2
V. Barone, L. Leo, G. Casari
Migraine is a common chronic pain syndrome with recurrent attacks (1-3 days) of disabling headache, associated symptoms and, in 1/3 of patients, neurological aura symptoms. Genetic factors are involved in the mechanisms for the attack, pain and aura. We are generating two transgenic mouse strains for the mutant Atp1?2 gene
linked to Familial Hemiplegic Migraine type 2: i) a knock-in mouse model to investigate the consequences of
expression of mutant Atp1?2 driven by its endogenous promoter ii) a conditional knock out of the Atp1?2 gene
to elucidate the effect of the loss of ATP1A2 expression in specific tissues and cell types.
Biological function and genotype-phenotype correlates of PINK1, a novel gene responsible for Parkinson’s
L. Atorino, L. Silvestri, L. Bardinella, G. Casari
Parkinson’s Disease (PD) is characterised by a massive degeneration of the dopaminergic neurons in the substantia nigra and the presence of typical cytoplasmic protein inclusions, the Lewy bodies. Although most cases
are sporadic, a number of genes responsible for rare familial forms of PD has been identified. Several evidences
suggest that the impairment of mitochondrial activity could represent an early, critical event in PD pathogenesis, even if a primary abnormality of mitochondria has not been yet established. In three PARK6-linked consanguineous families with early onset PD, E.M.Valente et al have identified two distinct missense mutations in the
PINK1 gene, which encodes a mitochondrial protein kinase. Preliminary results show that PINK1 mutations
cause mitochondrial impairment and increase cell susceptibility to oxidative stress. These findings link for the
first time a mendelian PD gene to mitochondrial dysfunction and suggest a protective role for PINK1 against
stress-mediated neurodegeneration. This project is focused on the physiological role and functional interactions
of PINK1 and how these functions are disrupted by pathogenic mutations. The characterisation of PINK1 and
its substrates is of paramount importance to elucidate the mechanisms leading to mitochondrial dysfunction in
Characterization of a conditional mouse model for ADNFLE
I. Manfredi, I. Bernascone, G. Casari
Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE) is a paediatric form of epilepsy characterised by clustered attacks of epileptic episodes originating from the frontal lobe. Six mutations are currently
linked to ADNFLE, involving a4 or b2 subunits of the nicotinic acetylcholine receptor (nAChR). We demonstrated in cells that this single amino acid substitution (V287L) dramatically alters the electrophysiological
properties of the receptor complex by retarding its desensitisation and causing neuronal hyperexcitability. We
generated a transgenic mouse using the TET-OFF conditional gene expression system: mouse Chrnb2 mutant
cDNA has been cloned under the control of the tetracycline-responsive promoter Ptet, which is activated by
binding of the protein tTA. Administration of tetracycline prevents tTA binding, therefore silencing the expression of the transgene. Responder Tg(TetO-CHRNB2VL) mice were crossed with a transgenic strain carrying
the tTA gene under the control of the brain specific Prion protein promoter (PrP) to produce double transgenics where the phenotype can be turned off by doxycycline administration.
See: Gene and stem cell therapy
See: Gene and stem cell therapy
HCN channel expression in the central nervous system
T. Lavazza, M. Ripamonti, D. Di Francesco, A. Malgaroli
HCN channels have a crucial role in the genesis of the cardiac rhythm. These channels which typically activate at hyperpolarizing potentials smallaer than -50mV, are highly expressed in the central nervous system. In
some neuronal populations this current has a clear pacemaker function, whereas in most other regions its role is
unclear. The relevance of HCN channels in the CNS can be inferred by the large number of pathologies such as
epilepsy and cerebellar ataxia where the function of these channels is altered. To better understand the functional diversity of the four HCN isoforms and their specific role, we developed isoform specific antibodies and
studied the distribution of these channels. At a cellular level we observed that HCN1, HCN3 and HCN4 isoforms are mostly present in neurites and synaptic terminals, whereas the HCN2 isoform is mainly expressed on
the somas of interneurons and some glial cells. We then focused our work on a big inhibitory terminal in the
cerebellum, the basket synapse, between basket and Purkinje cells. We found that HCN1 channels are higly expressed at this synapse which also contains some significant levels of the HCN3 isoform. By electrophysiological analysis and by [Ca2+]i imaging we have evaluated the role of HCN channels in synaptic transmission and,
more in general, in network activity in the cerebellum (Lavazza et al. in preparation).
Novel tools to label brain synapses in vivo
M. Cambiaghi, R. Ingrassia, D. Panzeri, A. Malgaroli, V. Zimarino
Despite modern brain imaging techniques have provided important results, they still infer quite indirectly
about the activity of neuronal cells and synapses. A valuable goal in neuroscience would be to develop novel
methodologies to track the functional activity of synapses inside the brain that in the future could be coupled to
in-vivo brain imaging. Since exocytosis exposes luminal epitopes of synaptic vesicle proteins, binding of fluorescent molecules to such epitopes represent a useful approach to monitor synaptic activity (Malgaroli et al.,
Science 1995). Because the size of these molecules restricts their access to synaptic sites deep in brain tissue, we
recently developed small constructs from the sequence of immunoglobulins against the vesicular protein
synaptotagmin –I. These, with a M.W. ranging between 24 kDa (SP6-VH.CH1) and 12 Kda (VH domain), bind
to synaptotagmin-1 and are effectively taken up by living synapses in brain slices (Ingrassia et al. Submitted).
More recently we begun to mutagenize the vesicle protein VAMP-2 to produce hybrid molecules with the ability to bind peptide sequences smaller than 10 Kda. The overall goal is to use these molecular tools to understand how neuronal cells and their synapses behave in situ while the brain is performing a specific behavioural
Quantal variability at central synapses
D. Panzeri, M. Bossi, A. Villa, A. Malgaroli
Central synapses transmit signals via fusion of synaptic vesicles with the plasma membrane. Variation in vesicular size is an important parameter which contributes to variance of synaptic communication. Vesicular volumes can be estimated by Electron Microscopy (EM). Although EM displays a great resolving power, it is also
susceptible to errors and imperfections which affect measurement of vesicle diameters. The main problem relates to tissue sectioning which introduces a significant amount of variability because a fraction of vesicles gets
always sectioned below vesicles’ equator. This produces artefactual vesicle images that are smaller than real. To
correct for this, we developed a mathematical treatment of the data which allowed us to extract the true distribution of vesicle sizes. Using this approach we compared the ultra-structure of type-I hippocampal synapses either fixed by standard chemical fixation or vitrified by a quick-freezing apparatus (which avoids changes in organelles’ shape and size by chemical fixation). This approach provided the first clear evidence that the variability of vesicles volumes is very small at resting hippocampal synapses. This variability was found to increase with
stimulation, in a graded manner with stimulus strength, and required time to be eliminated (Panzeri et al., submitted).
See: Imaging
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Gene and stem cell therapies are complementary areas of research, which share a great potential to fuel the
rapidly growing field of molecular medicine. Stem cells are optimal targets for genetic correction because their
capacity for self-renewal and long-term multi-lineage tissue repopulation ensures a stable supply of gene-corrected cells in transplanted hosts. Thus, gene and stem cell therapies are powerful approaches for the development of new ex vivo and in vivo therapeutic strategies to treat a number of genetic and acquired diseases, including hematopoietic diseases, neurodegenerative disorders, muscle dystrophies, cystic fibrosis, and cancer. In
addition, gene transfer technologies represent powerful tools to gain insight into the basic biology of stem cells
obtained from different sources and tissues.
The San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET) was created in 1995 in Milan as a joint
venture between the San Raffaele Scientific Institute and the Telethon Foundation for the implementation of
basic, pre-clinical and clinical research for genetic diseases. The mission of the Institute is to perform cutting
edge science in the field of gene therapy and to promote the translation of basic discoveries into therapeutic advances. Since its beginning the Institute’s approach to gene therapy research has been disease-oriented. The genetic diseases, which are presently under investigation (from those in advanced clinical experimentation to
those in early pre-clinical development) include primary immunodeficiencies, lysosomal storage disorders, type
I diabetes, hemophilias, thalassemias, and muscular dystrophies. Research on adenosine deaminase severe combined immunodeficiency (ADA-SCID) at HSR-TIGET has progressed from the initial studies of safety and feasibility to the recent demonstration that ADA gene transfer into human hematopoietic stem cells results in stable correction of the disease, with proven clinical benefits. Other research projects at HSR-TIGET have the
primary goal to improve the safety and efficiency of gene transfer and broaden its application to the treatment
of human disease by: a) design and validation of new vectors with improved safety and allowing one or more of
the following gene expression features: high-level, tissue-specificity, exogenous regulation, and coordinate dualgene expression; b) development of novel gene delivery strategies that broaden the reach of gene transfer in vivo; c) in vivo characterization of transduced cells; and d) modulation of immune responses to genetically-modified cells and gene therapy products. Gene transfer research is particularly focused on advancing the scope and
exploring the potential applications of lentiviral vectors, a new gene transfer technology. A Pediatric Clinical
Research Unit has also been established at HSR-TIGET for the translation of basic research into the treatment
of genetic diseases. The activity of this Unit focuses on the diagnosis, treatment and follow-up of patients with
primary immunodeficiencies and metabolic disorders, including those enrolled in the gene therapy trial for
The Stem Cell Research Institute (S.C.R.I.) was established in the fall of 2000 to create a centre of excellence
in the stem cell area. The mission of the Institute is to study different types of stem cells, including neural and
mesodermal stem cells, and to test their therapeutic potential in pre-clinical models of genetic and acquired diseases. The Physiopathology of Skeletal Muscle Development Unit has been working on the mechanism that
regulate the formation of skeletal muscle during embryonic development and, after birth, when the muscle tissue is damaged as a result of an injury or a primary myopathy. Recently, a class of vessel-associated, fetal stem
cells that can differentiate into most mesoderm cell types has been identified in mice and termed mesoangioblasts. The Unit has been evaluating new therapeutic strategies based on intra-arterial delivery of wild type
or gene-corrected mesoangioblasts for correcting the dystrophic phenotype of mouse models of muscular dystrophy. The distinct advantage of this strategy over previous approaches is the widespread distribution of donor
stem cells through the capillary network.
The work of the Neural Stem Cell Unit of the S.C.R.I. follows two major lines of investigation. The first one
concerns the development of novel therapeutic approaches to neurodegenerative diseases by means of neural
stem cells of both murine and human origin. The diseases that are presently under investigation include Parkinson’s and Alzheimer diseases, spinal cord injury, multiple sclerosis, lysosomal storage disorders. The Unit has
been developing new strategies for the manipulation, genetic engineering, and in vivo delivery of neural stem
cells. Systemic injection of neural stem cells is being evaluated for the experimental therapy of multiple sclerosis
in the mouse model. The second line of investigation concerns the discovery that human glioblastomas contain
a cell population, which displays features of tumor-founding neural stem cells. These cells are being characterized as in vitro models for drug screening, pre-clinical model of human glioblastoma, and by genomics/proteomics studies aimed at the identification of new candidate therapeutic genes (micro array-based comparative
analysis with normal human neural stem cells).
The new research unit on Angiogenesis and Tumor Targeting was established in 2003 to explore novel strategies to target the tumor vasculature via genetic modification of bone marrow-derived cells. Taking advantage of
the power of cell transplantation and gene-based delivery, these strategies are providing new insight into the
mechanisms governing angiogenesis, and are being applied to the screening and validation of candidate therapeutic targets in tumor angiogenesis. The outcome of these studies may highlight new crucial step in cancer
progression, and open the way to the design of innovative cancer therapies.
The Institute for Experimental Treatment of Cystic Fibrosis was born as a spin-off of the Local Cystic Fibrosis Association, which actively supports the Regional Centre for Cystic Fibrosis, in assisting patients affected by
this disease. The mission of the Institute is to develop new gene therapy vectors for Cystic Fibrosis through a
disease-oriented approach, which includes basic research on pathophysiology of Cystic Fibrosis airway bacterial infection and inflammation, pre-clinical applications of new vectors in animal models, and establishment of
surrogate end-points for the therapeutic treatments.
Hematopoietic stem cell gene therapy is an efficacious treatment for ADA-SCID
A. Aiuti, B. Cassani, U. Benninghoff, F. Cattaneo, L. Callegaro, G. Andolfi, M. Mirolo, S. Scaramuzza, A.
Tabucchi, F. Carlucci, M. Eibl, M. Aker, S. Slavin, M. Bregni, R. Miniero, C. Bordignon, M. G. Roncarolo
Adenosine deaminase (ADA)-deficiency is characterized by impaired immune functions, recurrent infections,
and systemic metabolic abnormalities. We have enrolled six children affected by ADA-SCID in a gene therapy
clinical protocol based on infusion of autologous BM CD34+ cells transduced with an ADA-encoding retroviral vector, combined with nonmyeloablative conditioning. Gene therapy resulted in stable engraftment of gene
corrected stem cells and lymphocytes, progressive increase of PB lymphocyte counts, normalization of T-cell
functions, and antigen-specific antibody responses. Molecular analysis revealed a profile of polyclonal integrations in T cells, with no evidence of clonal expansion. Sustained ADA activity in RBC and lymphocytes resulted
in the correction of purine metabolism and amelioration of systemic toxicity. None of the patients experienced
severe infections or adverse events after gene therapy, with the longest follow up at 50 months. All the children
are healthy and thriving, in the absence of enzyme replacement therapy. In summary, these results show that
gene therapy is safe and efficacious in correcting both the immune and metabolic defect of ADA-deficiency.
Role of altered purine metabolism in the immunological defect of ADA-SCID
B. Cassani, F. Cattaneo, U. Benninghoff, C. Bordignon, M. G. Roncarolo, A. Aiuti
ADA-deficiency leads to an accumulation of adenosine (Ado) and dAdo in plasma and cells, resulting in immune deficiency and systemic toxicity. Our goals are to identify the biochemical pathways linking the altered
purine metabolism to immune dysfunctions, as well as to investigate the efficacy of gene therapy. For this purpose, we are using as an in vitro model untransformed T-cell lines and dendritic cells from ADA-SCID patients
or healthy donors. We found that both Ado and dAdo induce apoptosis in ADA-deficient T-cell lines but Ado
acts faster and at lower concentration compared to dAdo. Remarkably, T-cell lines generated from gene therapy-treated patients were protected from apoptosis as control cells, in agreement with the expression of functional ADA. Proliferative responses and cytokine production were severely impaired after dAdo exposure in
ADA-deficient T cells, but they were restored to normal in gene corrected cells. These studies are providing
new insights into ADA-SCID pathogenesis and novel markers to evaluate the ability of ADA gene transfer to
restore normal purine metabolism and immune functions.
Efficient gene transfer into hematopoietic stem/progenitor cells with lymphoid potential
F. Ficara, R. Jofra Hernàndez, D. Superchi, S. Scaramuzza, S. Deola, C. Mocchetti, N. Carballido-Perrig,
G. Andolfi, A. Colombo, J. M. Carballido, C. Bordignon, M. G. Roncarolo, A. Aiuti
We are pursuing different approaches to improve ex vivo gene transfer and engraftment of Hematopoietic
Stem/Progenitor Cells (HSPC) for gene therapy of primary immunodeficiencies. To this aim, we have established a bone marrow stromal cell engineered to produce retroviral structural proteins, resulting in improved
gene transfer into primitive hematopoietic and lymphoid progenitors. We identified optimal conditions for ex
vivo gene transfer into HSPC which included the cytokines TPO, FLT3-Ligand, SCF, and IL-3. Under these
conditions, bone marrow CD34+ cells from normal subjects or adenosine deaminase (ADA)-SCID patients
transduced with retroviral vectors, engrafted efficiently into SCID-hu mice and retained B- and T-cell differentiation capacity. Finally, we developed a clinically applicable gene transfer procedure into mobilized peripheral
blood CD34+ progenitors, based on single exposure to retroviral vector and selection of engineered cells,
which allowed to retain multilineage reconstitution capacity in SCID-hu mice. In conclusion, retroviral vectors
can be used to efficiently target HSPC, preserving their engraftment and lymphoid reconstitution capacity in vivo.
Lentiviral-mediated ex vivo gene therapy in ADA-Deficient SCID mice
A. Mortellaro, R. Jofra Hernàndez, M. Guerrini, A. Tabucchi, F. Carlucci, A. Follenzi, L. Naldini, C. Bordignon,
M. G. Roncarolo, A. Aiuti
Lentiviral vectors are promising tools for application in gene therapy of ADA-SCID. We are investigating the
preclinical safety and efficacy of lentiviral-mediated Gene Transfer (GT) in the mouse model of ADA-deficiency. ADA KO mice were transplanted after non-myeloablative conditioning either with wild type Bone Marrow
(BM) cells or ADA KO BM cells transduced with a SIN HIV-1-based lentivector encoding human ADA cDNA. Both GT and BM transplant rescued ADA KO mice from lethal phenotype and restored their normal
growth. Donor-derived transduced cells were detected at high levels in transplanted mice and increased over
time in the lymphoid lineage, demonstrating their selective advantage in vivo. Two months after treatment,
ADA activity was normalized in RBC and toxic metabolites decreased to undetectable levels. GT resulted in
normal lymphoid differentiation in primary and secondary organs, as well as restoration of T-cell and B-cell
functions, similarly to BM transplant. These results indicate that lentiviral-mediated ADA gene transfer can
correct the metabolic and immune defect of ADA KO mice. The long-term safety of GT with lentiviral vectors
is currently under investigation.
Preclinical assessment of efficacy and safety of a gene therapy protocol for Wiskott-Aldrich Syndrome
(WAS) using lentiviral vectors
L. Dupré, F. Marangoni, S. Scaramuzza, S. Trifari, S. Martino, C. Bordignon, A. Aiuti, L. Naldini,
M. G. Roncarolo
Wiskott-Aldrich Syndrome (WAS) is a severe X-linked primary immunodeficiency characterized by infections, severe hemorrhages, and lymphomas. We investigated the efficacy and safety of WAS gene transfer in patients’ T cells and WASP-deficient (WKO) mice. Patients’ T cells were transduced with lentiviral vectors encoding WASP under the control of the ubiquitous PGK promoter or the hematopoietic-specific WAS promoter. The percentage of transduced T cells expressing WASP increased during long-term culture indicating that
WASP+ T cells have a selective growth advantage. Correction of functional defects including TCR-driven proliferation and IL-2 production was achieved with one to two vector copies of the vectors per transduced cell.
No toxicity was observed in long-term cultures of transduced cells. We developed a gene therapy protocol
based on the transplantation of non-lethally irradiated WAS-/- mice with WAS-/- HSC transduced with the
lentiviral vectors. The procedure resulted in high-level engraftment and restoration of WASP expression in T
cells, B cells and platelets. T cells harbored 1 to 2 vector copies and displayed reproducible correction of TCRdriven IL-2 production and proliferation. The treatment was well tolerated and no malignancy was detected by
systematic blood analysis and autopsy. The efficacy and safety of WAS gene transfer with the WAS promotercontaining lentiviral vector into patients’ T cells and HSC from the murine model of the disease provide preclinical data for the development of gene therapy for the treatment of WAS patients.
Role of SAP in T lymphocytes of patients with x-linked lymphoproliferative disease
L. Dupré, G. Andolfi, S. Tangye, R. Clementi, F. Locatelli, M. Aricò, A. Aiuti, M. G. Roncarolo
The adaptor protein SAP regulates signaling through SLAM-family receptors expressed on T and NK cells.
In patients affected by X-linked lymphoproliferative (XLP) disease, mutations in the SH2D1A gene result in
defective lytic activity. However, the mechanism by which SAP controls cytotoxic activity remains unclear. TCR
activation of CD8+ cytotoxic T cells (CTL) results in down-regulation of SAP, suggesting that this protein is involved in early activation events. Here, we show that SAP-deficient CTL from patients with XLP and hemophagocytic lympho-histiocytosis (HLH) display a specific lytic defect against autologous and allogeneic EBVpositive B cells. This defect is associated with the defective polarization of 2B4, perforin and lipid rafts at the
contact area of CTL with EBV-positive targets. Blockade of 2B4 in normal CTL reproduces the defects in lysis
and polarization observed in SAP-deficient CTL. Expression and regulation of the SLAM-family receptors
SLAM, CD84 and 2B4. as well as the lytic effectors perforin and granzyme-B are normal in SAP-deficient CTL.
In addition, TCR stimulation leads to normal proliferation and production of IL-2, IL-4 and IFN-γ. These results demonstrate that the SAP/2B4 pathway plays a key role in CTL lytic activity against EBV-positive targets
by promoting the polarization of the lytic machinery.
Bone marrow transplantation as successful treatment for severe IPEX: a case report (Pediatric clinical
research unit)
M. G. Roncarolo, F. Cattaneo, L. Callegaro, A. Aiuti, R. Bacchetta
IPEX (Immune dysregulation, polyendicrinopathy, enteropathy, X-linked) is a rare genetic disease due to different mutations in the human FOXP3 gene which is clinically characterized by severe secretory diarrhea, insulin-dependent diabetes mellitus and eczema. A severe case of IPEX was diagnosed at our Centre and successfully treated by a related HLA-identical nonmyeloablative bone marrow transplantation. At 10 days of age, the
patient was hospitalized for early onset insulin-dependent diabetes mellitus with IgG-antibodies against insulin.
At 4 weeks, an upper airways infection and eczema occurred with high IgE serum levels. A mutation located in
the forkhead (FHK) domain of the FOXP3 gene was identified. The same mutation was present at the heterozygous state in the patient’s mother. At 6 months of age severe enteritis with total villous atrophy was documented, confirming the diagnosis of IPEX. The patient underwent a non myelo-ablative allogeneic HLA-identical BMT from his brother after conditioning regimen with intravenous Fludarabine, Busulfan, rabbit anti-Tlymphocyte globulin. No transplant-related complications were observed. Mycophenolate Mophetil was administered for GVHD prophylaxis. Hepatic GVHD (grade I-II), which appeared at day +30, resolved when
Cyclosporine was introduced intravenously at day +44. The hematological reconstitution (neutrophils count >
500x109/l) occurred at day +17. A chimerism of 90% donor cells was detected by VNTR polymorphism-analysis in the peripheral blood granulocytes on day +24 and a full donor engraftment (>98%) in all lymphoid and
myeloid lineages in the bone marrow was detected starting from day +36 and confirmed still 1 year after transplantation. The chimerism in the peripheral blood T-lymphocytes increased progressively from 50% on day
+15 to 100% on day +50. After the transplant, we observed a total resolution of the enteropathy with increase
in the patient’s weight. Metabolic control of the diabetes was achieved with a normal intake of insulin. A dramatic decreased in anti-insulin antibodies the IgE levels to normal value was also observed. At present, the patient is at home, in good clinical conditions, with regular rate of growth in length and weight and good control
of the HbA1c level.
Functional characterization of regulatory and effector T cells in patients with FOXP3 mutations (Pediatric
clinical research unit)
R. Bacchetta, L. Passerini, E. Gambineri, S. Allan, M. Levings, A. Aiuti, F. Cattaneo, F. Dagna Bricarelli,
M. G. Roncarolo
Mutations of the FOXP3 gene in humans are responsible for a fatal X-linked autoimmune disease, called
IPEX, characterized by immune dysfunction, polyendocrinopathy and severe enteropathy, occurring in early
childhood. In the mouse, FOXP3 has a crucial role in the generation of naturally occurring CD4+CD25+ T
regulatory (Tr) cells. In IPEX patients, a clear correlation between FOXP3 mutations and a defect in Tr cells
has not yet been demonstrated. Our studies in two patients with FOXP3 mutations and either severe or moderate IPEX, showed that the number and cell surface phenotype of CD4+CD25+ Tr cells are comparable to those
of normal donors. These Tr cells suppress the in vitro proliferation of effector T cells from normal donors, but
are not able to suppress autologous effector T cells, unless they have been previously expanded in vitro in the
presence of exogenous IL-2. Furthermore, PBMC from IPEX patients have a significant impairment in IL-2
and IFN-γ production. This study indicates that FOXP3 mutations in humans do not lead to the absence of
CD4+CD25+ Tr cells, but that these cells are dysfunctional, possibly due to lack of activation-associated production of IL-2 by effector T cells. These results suggest that the functional role of FOXP3 gene in humans
might not be restricted to Tr cells.
Adoptive transfer of il-10 anergized T cells/regulatory t cells in patients transplanted with haploidentical
hematopoietic stem cells (Pediatric clinical research unit)
R. Bacchetta, F. Ciceri, L. Callegaro, K. Fleischhauer, E. Biral, G. Serafini, M. Andreani, M. Bregni,
M. G. Roncarolo
Graft-versus-host disease (GVHD) and lack of immune reconstitution are still major limitations for the success of allogeneic hematopoietic stem cell transplantation (HSCT). Adoptive transfer of donor lymphocytes
rendered tolerant in vitro to the host’s alloantigens (alloAg) could ameliorate the immunodeficiency status without the risk of fatal GVHD. IL-10 is a potent anti-inflammatory and suppressive cytokine. Addition of IL-10 in
primary mixed leukocytes reactions with total PBMC from mismatched or haploidentical donors, induces alloAg specific unresponsiveness, defined as anergy. IL-10 anergized T cells become unable to respond to a
rechallenge with the same alloAgs, but can still respond to nominal or viral Ags. Importantly, IL-10 also induces
the differentiation of T regulatory type 1 (Tr1) cells, which produce IL-10, TGF- γ, IL-5 and low levels of IFNγ, and inhibit Ag-specific responses of naive T cells in vitro and in vivo, and have been shown to be important in
peripheral tolerance. These data provided a strong rationale for the development of a clinical protocol of adoptive transfer of ex-vivo IL-10 anergized T cells of donor origin in patients after haploidentical HSCT. This cellular therapy protocol is currently approved for patients with hematologic malignancies and will be extended to
patients with genetic hematologic diseases, such as Thalassemia, for whom allogenic HSCT is often the only
possibility of cure.
Cure of established disease and direct evidence of cellular cross-correction in the nervous system of
Metachromatic Leukodystrophy (MLD) mice after HSC-based gene therapy
A. Biffi, A. Capotondo, A. Quattrini, U. Del Carro, R. Brambilla, S. Fasano, S. Marchesini, C. Bordignon,
L. Naldini
MLD is an inborn lipidosis caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA), characterized by progressive demyelination. We showed that transplantation of LV-transduced HSC allowed preventing
functional and neuropathological manifestations of MLD in the mouse model, when applied at early pre-symptomatic stage (A. Biffi et al., J. Clin. Invest 2004, 113(8): 1118-29). We then assessed whether this approach
could be effective in correcting already established MLD manifestations. Neurophysiological and behavioural
studies demonstrated that HSC-based ex vivo gene therapy corrected already established functional neurological deficits when applied to already symptomatic animals. Immunohistochemical studies of the brain of transplanted mice showed the occurrence of ARSA in the lysosomal compartment of HSC-derived microglia cells
and resident CNS neurons, oligodendrocytes and astrocytes, proving the occurrence of in vivo enzyme transfer.
Being these findings associated to reversal of the focal neuronal degeneration which affects aging MLD mice, in
vivo enzyme transfer achieved robust metabolic cross-correction of the cell targets of the disease. These results
demonstrate that transplantation of LV-transduced autologous HSC represents a promising therapeutic strategy for correcting MLD in humans.
Neonatal cell therapy with oligodendrocytes prevented central deficits in mice affected with metachromatic
M. I. Givogri, F. Galbiati, L. Perani, D. Superchi, S. Amadio, P. Morana, U. Del Carro, S. Fasano, R. Brambilla,
E. Bongarzone
We conducted experiments to assess the value of this cell-based therapy to prevent the natural progression of
disease-related central deficits in mice affected by dysmyelinating Metachromatic Leukodystrophy (MLD).
Oligodendrocyte progenitors (OLPs) showed to migrate extensively throughout the neonatal MLD brain after
intraventricular injection, integrating within white matter areas and surviving as long as one year after trans-
plantation. One-year-old transplanted MLD mice showed normalization of central conduction time and central
motor evoked potentials. Furthermore, MLD mice treated with OLPs displayed a complete prevention of the
cerebellar-related locomotor deficit, characteristic of sick old MLD mice.
Our results show for the first time that the brain of neonatal MLD mice is plastic and receptive for neural
transplantations and that OLP grafting during presymptomatic stages aids in preventing the natural evolution
of MLD central impairment, likely by contributing to the pool of myelinating cells as well as by enhancing neuroprotection. Our findings open a new scenario in central cell-based therapy for individuals affected by dysmyelinating MLD.
HSC-based ex vivo gene therapy prolongs survival and improves the phenotype of Globoid Cell
Leukodystrophy (GLD) mice
A. Biffi, I. Visigalli, S. Letterio Politi, A. Quattrini, U. Del Carro, D. Wenger, G. Scotti, L. Naldini
We evaluated the therapeutic potential of the HSC-based ex vivo gene therapy in the rapidly progressive and
lethal evolution of GLD, an inborn lipidosis caused by the deficiency of the lysosomal enzyme galactocerebrosidase, in the mouse model. Neonate Twitcher mice were transplanted with HSC transduced with the
GALC gene, supplemented with accessory cells to reduce short-term toxicity of lethal conditioning. We observed a significant improvement in long-term survival of transplanted mice, in the phenotype (absence of leg
palsy and delayed, minor twitching) and prevention of mayor histopathological signs of the disease (demyelination, axonal loss). Brain MR imaging showed clear improvements of the massive CNS demyelination in the
cerebellum and corpus callosum of the Twitcher mouse by treatment. These results are providing evidence, in a
severe disease setting, of the therapeutic potential of ex vivo gene therapy for lysosomal storage disorders.
Neural stem cells are insufficient to prevent the natural evolution of Globoid Leukodystrophy in Twitcher
F. Galbiati, D. Superchi, G. Clementi, M. I. Givogri, D. Dolcetta, L. Perani, C. Cavazzin, A. Gritti, S. Amadio,
P. Morana, U. Del Carro, A. Vescovi, E. Bongarzone
We studied the therapeutic capacity of Neural Stem Cells (NSCs) to prevent central disease evolution in
Twitcher pups. NSCs were delivered directly in the brain, infused intravenously or intraperitoneally between
P2 to P4. NSCs grafted in the brain and those infused through the parietal vein but not those infused within the
peritoneal cavity showed profuse distribution within the central nervous system of Twitcher mice. Most of the
cells showed to retain an intermediate maturation stage without evidence of full terminal differentiation in neurons or oligodendrocytes. Even though NSCs appeared to survive throughout the evolution of the disease, they
were no able to ameliorate or prevent central dysmyelination, overall body decay, worsening of central and peripheral nerve conduction and death around 45 days.
The low impact of these unilateral strategies on preventing the progression of the disease prompted us to
study the value of combined peripheral and central therapies. We are evaluating combined approaches including bone marrow transplantation –historically the only treatment to enhance the survival of Twitcher mice- with
the systemic delivery of lentiviral vectors to reconstitute the expression of galactocerebrosidase (the deficient
enzyme in this disease) and the central delivery of NSCs to improve central neuroprotection.
Successful expression of transgenes in the adult mouse cerebellum without compromise of neurological
function after in vivo lentiviral gene delivery
C. Croci, S. Fasano, A. Martellosio, D. Superchi, L. Perani, L. Naldini, R. Brambilla, E. Bongarzone
We have characterized the capacity of lentiviruses to transduce cerebellar cells and the impact of this gene
transfer method on locomotor abilities in adult mice. Using the GFP gene for gene marking, we found important levels of transduction in several cell types, including Purkinje cells, granular neurons and glia. This expression showed to be sustainable for long periods of time and was not accompanied by any significant inflammatory reaction. Of fundamental importance, treated mice showed absence of any impairment on motor coordination and motor learning and open field locomotor activity recordings showed that treated mice behaved indistinguishably from non-injected controls.
Our results demonstrate that lentiviral vectors can transduce with an excellent efficiency different cerebellar
cells, including Purkinje neurons -for long considered non-transduceable- and that this gene transfer approach
per se does not diminish cerebellar performance.
Olig1 factor improved the in vivo remyelination potential of adult neural stem cells
M. I. Givogri, A. Gritti, A. Corio, C. Cavazzin, F. Galbiati, L. Perani, D. Rowitch, A. Vescovi, E. Bongarzone
Expression of Olig1 basic helix-loop-helix transcription factor has been shown to play an essential role in
oligodendrocyte formation during early embryogenesis. Aiming at creating experimental conditions to facilitate
the progression of neural precursors into the oligodendroglial lineage, we have generated neural precursors in
which the expression of Olig1 transcription factor has been constitutively forced after retroviral transduction.
Olig1+ neural cell lines showed to maintain their clonogenic capacity and to self renew without any significant
compromise under ectopic expression of Olig1. Upon differentiation, we found that Olig1+ progenitors contained about twice the number of cells with oligodendrocyte characteristics in comparison to mock-transduced
and non-transduced neural precursors. We further tested the repairing capacity of these Olig1+ precursors in a
mouse model of focal demyelination of the corpus callosum. Olig1+ cells were grafted near the lesion and
showed to migrate preferentially towards the wounded site, leading to faster recovery of the demyelinated area,
without signs of tumorigenesis and in a non-disruptive manner.
Our work gives the experimental demonstration to envision the use of Olig1 as a target molecule to enhance
the oligodendroglial potential of human derived neural stem cells for cell therapies of myelin disorders.
Clinical Study of Metachromatic Leukodystrophy (Pediatric clinical research unit)
M. Sessa, A. Biffi, F. Fumagalli, M. Cesani, U. Del Carro, C. Baldoli, S. Gerevini, S. Amadio, M. G. Roncarolo
The Pediatric Clinical Research Unit follows patients affected by metachromatic leukodystrophy (MLD), an
inborn lipidosis caused by the deficiency of the lysosomal enzyme arylsulfatase A (ARSA), which results in storage of sulfatides in the CNS and PNS, leading to severe progressive demyelination and early death. No effective
treatment is currently available. Because of the rarity little is known on the factors determining the different
clinical phenotypes of MLD and their natural history. To increase our knowledge, 21 patients with different
variants were biochemically and molecularly characterized and monitored along a 3-year follow up period. Our
data show that the clinical evolution of MLD is highly variable and only partially influenced by the age of onset,
especially among the juvenile patients. No clear-cut correlation exists between clinical phenotype and residual
enzymatic activity. On the contrary, a complete molecular characterization based on screening for common mutations or on sequencing of the entire coding region of the ARSA gene revealed a genotype-phenotype correlation. The presence of peripheral neuropathy at disease onset seems a reliable prognostic index, being associated
with a severe evolution. MR pattern of demyelination, which greatly differs in infantile as compared to juvenile
and adult forms, could represent an additional tool to differentiate the various clinical phenotypes. In conclusion, results from this longitudinal study not only allowed the identification of reliable prognostic markers but
also represent a fundamental prerequisite for the selection of the best therapeutic option, the identification of
candidate patients eligible to gene therapy, and the evaluation of the clinical benefits.
Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters
M. Amendola, M. A. Venneri, A. Biffi, E. Vigna, L. Naldini
Transfer of multiple genes within the same cell allows combining genetic correction with marking, selection,
and conditional elimination of transduced cells, and reconstitutes multi-subunit components and synergistic
pathways. However, it poses significant challenges to gene transfer technologies. To overcame these limitations,
we developed synthetic bidirectional promoters that mediated coordinate transcription of two mRNA’s in ubiquitous or tissue-specific manner, based on the finding that some cellular promoters were intrinsically capable of
promoting divergent transcription. Lentiviral vectors incorporating the new promoters enabled efficient dualgene transfer in several tissues in vivo after direct delivery or transgenesis, and in a human gene therapy model.
Because divergent gene pairs, likely transcribed from shared promoters, are common in the genome, the synthetic promoters that we developed may mimic a well-represented feature of transcription, providing the basis
for their robust performance. Vectors incorporating these promoters should increase the power of gene-function studies and the reach and safety of gene therapy.
Lentiviral Gene Transfer into HSC is Enhanced by Early-Acting Cytokines without Impairing Stem Cell
Properties and Involves Cellular Responses Distinct from Cell Cycle Control
F. R. Santoni de Sio, L. Naldini
Hematopoietic Stem Cells (HSC) are attractive targets for gene therapy. HIV-derived Lentiviral Vectors (LV)
are promising tools to obtain high transduction efficiency, without affecting stem cell features. We demonstrated that LV transduce efficiently SCID/NOD mouse repopulating cells (SRC), by a short ex vivo incubation and
that IL6, SCF, TPO and Flt3L enhanced transduction. To determine if cytokines affected SRC repopulating
ability, we performed a competitive repopulation assay and we found that stimulation did not impair it. We
then asked if cytokines enhanced transduction triggering the cells through the S-phase of the cell cycle, a condition that could be detrimental for SRC. Using an SRC S-phase suicide assay, we found that cytokine enhancement of transduction was not due to progression into S-phase. We then evaluated viral entry, transducing progenitors with LV pseudotyped with different envelopes; we found that cytokine effect was not linked to a specific entry pathway. Preliminary data indicate that susceptibility to LV transduction is regulated by a specific
enzymatic complex in the cytosol. Our results will help to establish a new effective protocol for HSC gene therapy.
Development of transcriptionally-targeted viral vectors for expression of human β globin
A. Miccio, G. Facchini, F. Lotti, R. Cesari, C. Rossi, G. Ferrari
Correction of blood genetic disorders requires in most cases permanent gene transfer into self-renewing,
long-term repopulating stem cells, and regulation of transgene expression in specific cell lineages. Globin gene
transfer for the treatment of hemoglobinopathies represents a relevant model in which to study strategies aimed
at transducing Hematopoietic Stem Cells (HSCs) and restrict transgene expression to a single differentiated cell
lineage. We have developed different lentiviral vectors containing transcriptional control elements derived from
erythroid specific genes (GATA-1, LCR elements, beta globin promoter) to drive human beta globin expression. Transcriptional targeting strategies were based on both the generation of chimeric LTR and the cloning of
erythroid enhancer/promoter elements as internal transcriptional unit. Replacement of the HIV U3 region with
an enhancer (HS2) of the erythroid-specific GATA-1 promoter generated a chimeric LTR driving the expression of a cDNA coding for an epitope-tagged beta globin, while a reporter gene (EGFP) was cloned under the
control of an internal constitutive promoter (PGK). In a different vector design, the beta globin gene was
cloned in reverse orientation to the LTR-driven transcript and its expression was under the control of the beta
globin promoter and two elements of the Locus Control Region (HS2 and HS3). Gene expression was assayed
in human and murine erythroblastic cell lines by Western blot and HPLC analysis. The activity of these vectors
will be analyzed by transducing human thalassemic cells, and by testing the therapeutic potential of transduced
cells in appropriate pre-clinical models, such as bone marrow transplantation in beta-thalassemic mice.
Effect of retroviral and lentiviral vector integrations on transcription of flanking genes
E. Montini, M. Fabbri, D. Cesana, F. McBlane, L. Naldini
In mouse, retroviral infection triggers leukemias by activation of cellular proto-oncogenes upon integration.
This is usually mediated by promoter/enhancer sequences in the proviral LTR that up-regulate expression of
flanking genes. The actual frequency and extent of transcriptional de-regulation occurring at randomly picked
retroviral integration sites is currently unknown. Thus, we embarked in testing and comparing the effect of
non-selected Retroviral Vector (RV) and Lentiviral Vector (LV) integration on the expression of flanking genes.
We generated two panels of hematopoietic cell clones, each carrying a single insertion of a conventional RV or a
third-generation SIN-LV. We retrieved genomic DNA flanking the proviral insertion by linear amplificationmediated PCR allowing in silico mapping of 40 single integrations. We then selected the genes contained in a
600 kb interval centered on each integration site and compared their relative expression levels by Q-PCR. No
significant changes of gene expression around the site of integration have been detected. These data indicate
that the transcriptional perturbation around RV and LV integration, is neither frequent nor strong.
Testing the oncogenic potential of retroviral and lentiviral vector integrations
E. Montini, D. Cesana, F. Sanvito, L. Sergi Sergi, F. Benedicenti, M. Ponzoni, C. Doglioni, L. Naldini
Retroviral Vectors (RVs) expressing therapeutic genes have been used to transduce Hematopoietic Stem Cells
(HSCs) and treat hematological diseases. Unfortunately, RVs may trigger oncogenesis by insertional mutagenesis. Our aim was to generate a mouse HSC transplantation model sensitive to genotoxic stress to measure the
tumorogenic potential of RVs and LVs in vivo. We transduced HSCs from leukemia-prone Ckdn2a-/- mice with
LV and RV and transplanted them into wild-type recipients. In this model, genotoxic stress can be measured as
degree of tumor onset acceleration with respect untreated control groups. Mice transplanted with untransduced Cdkn2a-/- HSCs, or transduced with low dose RV, or all doses LV, developed hematopoietic malignancies without acceleration in tumor onset. Mice transplanted with Cdkn2a-/- HSCs subjected to high dose RV
transduction displayed a significant earlier tumor onset. Indicating a detectable tumorigenic risk caused by RV
transduction in a dose dependent fashion. Our data also indicate that LV transduction at any dose did not result in any acceleration on the tumor onset. The described model provides an important tool to compare the
risk of insertional mutagenesis of different integrating vectors and a platform to test safety improvements on
vector design.
Development of a new lentiviral vector for site-specific integration into mammalian genomes
A. L. Lombardo, M. Calos, L. Naldini
Gene therapy is an effective strategy for the correction of inherited diseases. However, recently reported adverse events in one clinical trial have highlighted the risk of retroviral vector-mediated insertional mutagenesis
in Haematopoietic Stem Cells (HSC). For a gene therapy approach based on HSC, vector integration in target
cell chromatin represents a necessary step to allow stable long-term transgene expression and its propagation to
the cell progeny. Therefore, to endow Integration-Defective Lentiviral Vector (IDLV) with site-specific integration, we take advantage of the previous described phage phiC31 integrase system. Coupling these systems in
human target cells, we show that IDLV represents a suitable substrate for phiC31 integrase activity. Importantly, using different genomic analysis approaches, we show effective override of the lentiviral vector integration
machinery and preferential integration of circular IDLV forms in selected human genomic sites. Our results also form the basis for the development of new IDLV systems based on other site-specific integrases, and is currently used to explore gene correction of X-linked SCID using engineered zinc-finger proteins.
Improved lentiviral vectors for the treatment of hemophilia B
B. D. Brown, E. Hauben, A. L. Lombardo, L. Sergi Sergi, M. G. Roncarolo, L. Naldini
Lentiviral Vectors (LVs) are an attractive candidate for gene therapy of monogenic diseases such as the hemophilias. LV administration can achieve stable expression of a transgene. Unfortunately, studies in immunocompetent mice indicate that vector delivery of a neo-antigen can result in an immune response. Thus, we set out to
improve our system by studying the mechanisms of immune activation and developing improved expression
cassettes for preventing induction of immunity. Initially, we determined if LV transduction induces maturation
of dendritic cells (DCs), a key event in activation of the immune system. Transduction of DCs was shown to be
highly efficient (>80%), but did not trigger upregulation of CD83 or CD86, nor did it induce secretion of IL-6,
TNF-α or IL-12. Concordant with this work, we developed LVs with improved expression cassettes, which do
not express the transgene in DCs. In vivo testing of a synthetic hepatocyte-specific promoter enabled us to
achieve high levels of Factor IX (>2microgram/mL) in a mouse model. We are now developing an additional
layer of regulation, utilizing RNA interference, that will prevent further transgene presentation in mature DCs.
In vivo modulation of the immune responses to gene therapy derived products
M. Battaglia, A. Annoni, A. Follenzi, A. L. Lombardo, L. Naldini, M. G. Roncarolo
Stable gene replacement by in vivo administration of lentiviral vector (LV) has important therapeutic applications. However, successful gene therapy is often limited by the immune response to the transgene products.
Therefore, induction of tolerance to a transgene mediated by T regulatory cells (Tr) may be a successful strategy.
We previously demonstrated that immunocompetent mice injected with LV-GFP develop an immune response, mediated by both CD8+ cytotoxic T cells and antibody production. This response leads to clearance of
the GFP-expressing cells. Adoptive transfer of purified CD4+CD25+ Tr cells isolated from syngeneic wild type
or GFP transgenic (tg) mice did not down-regulate the transgene specific immune response. On the contrary,
the co-administration of LV-GFP and highly purified GFP tg splenic Antigen Presenting Cells (APC) signifi-
cantly modulates the immune response to GFP. These results demonstrate that APC isolated from GFP tolerant mice can reduce the immune response to GFP in vivo. Therefore, mode of transgene presentation by APC
is crucial in order to activate an immune-regulatory response in vivo.
Induction of tolerance by targeting CD45
S. Gregori, E. Tresoldi, B. Migliavacca, F. Capra, G. Aversa, J. M. Carballido, J. E. de Vries, U. Korthauer,
M. G. Roncarolo
CD45 is a transmembrane protein tyrosine phosphatases that plays a critical role in regulating T-cell activation. Several studies demonstrated that anti-CD45RB mAbs are a potent immunomodulant that prolongs allograft survival in several murine, and non-human transplantation models. We investigated the effects of a monoclonal antibodies (mAb) chA6, which has a unique specificity for the RO and RB isoforms of CD45 on human
T cells. We demonstrated that chA6 mAb potently inhibited allogeneic, polyclonal, and Ag-specific responses
and induced activation independent apoptosis in CD4+A6bright cells in vitro. In addition, chA6 mAb induced anergic Ag-specific CD4+ and CD8+ T cells that displayed a Tr1-like phenotype, and suppressed IFN-γ production and proliferation of Ag-specific T cells. ChA6 mAb also modulated the effector function of mature Dendritic Cells (mDCs). Although chA6 mAb did not significantly change the expression of costimulatory molecules on mDCs, it modified their function. ChA6 mAb-treated mDCs drived the differentiation of Tr1 cells
which are phenotypically and functional similar to those generated with immature DCs. ChA6 mAb was also effective in vivo in a model of human islet allograft in NOD-SCID mice. ChA6 mAb treatment prevented human
islet allograft rejection. Taken together, these results show that chA6 mAb is a new immunomodulant agent
with multiple mechanisms of action.
Induction of CD4+ regulatory T cells by IL-10 modulated dendritic cells
S. Gregori, M. Levings, E. Tresoldi, M. G. Roncarolo
Dendritic cells (DCs) are professional APCs which are crucial for the initiation of T cell immunity. After antigen uptake, inflammatory stimuli drive the maturation of DCs, which ultimately promote the activation of effector T cells. However, depending on their maturation state, biological properties or localization, DCs may
have different functions. Several studies indicate that tolerogenic DCs can drive the differentiation of Tr cells.
We demonstrated that repetitive stimulations of naive CD4+ T cells with immature DCs (iDCs) induce the differentiation of human Tr cells in vitro. The resulting Tr cells are phenotypically and functionally identical to
type 1 Tr (Tr1) cells since they are anergic, produce high level of IL-10 and TGF-β significant amounts of IFNγ and IL-5, low IL-2 and no IL-4, suppress T-cell responses via an IL-10- and TGF-β-dependent mechanism,
and autocrine production of low amounts of IL-10 by iDCs drives their generation. Addition of exogenous IL10 on iDCs induces the differentiation of a unique subset of iDCs (IL-10/iDCs) characterized by the expression
of CD14, CD11c, CD11b, CD83 and HLA-DR, but not CD1a. IL-10/iDCs secrete higher levels of IL-10 and
display lower stimulatory capacity, compared to iDCs. Interestingly, a single stimulation of naive CD4+ T cells
with allogeneic IL-10/iDCs results in induction of anergic Tr1 cells with suppressive activity, indicating that IL10/iDCs are more powerful than iDCs in generating Tr1 cells. Thus, IL-10/DC can be used to generate large
number of alloantigen-specific Tr1 cells for clinical use as a cellular therapy to restore peripheral tolerance.
Long-term tolerance in autoimmune diabetes is induced by rapamycin and IL-10 via both CD4+CD25+ Tr
and T regulatory type 1 (Tr1) cells
M. Battaglia, A. Stabilini, E. Draghici, B. Migliavacca, E. Bonifacio, M. G. Roncarolo
Avoiding immune responses to self-antigens while maintaining responses to foreign-antigens is a complex
process mediated by multiple mechanisms including active suppression of T effector cells by T regulatory (Tr)
cells. CD4+ Tr cells comprise the naturally occurring CD4+CD25+FOXP3+ cells and the inducible Tr type 1
cells (Tr1) cells. The Tr1 cells differentiate from CD4+CD25- T cells activated in the presence of IL-10. Both
CD4+CD25+ Tr and Tr1 cells play a key role in inducing and maintaining tolerance to self-antigens. However,
the precise contribution of these two Tr cell subsets in regulating self versus non-self antigens is still elusive. We
demonstrate that prolonged administration of rapamycin and IL-10 efficiently treats autoimmune diabetes and
induces long-term immunotolerance, which is maintained after treatment withdrawal and in the absence of immunosuppression in nonobese diabetic (NOD) mice. Rapamycin mediates expansion and accumulation in the
pancreas of suppressive CD4+CD25+FOXP3+ Tr cells, which prevent diabetes. IL-10 induces Tr1 cells, which
reside in the spleen and prevent migration of diabetogenic T cells to the pancreas. These two Tr cell subsets act
in concert to control diabetogenic T cells, which are still present in long-term tolerant mice. Our results demonstrate that treatment with rapamycin and IL-10 can induce both CD4+CD25+ Tr and Tr1 cells, and may constitute a novel and potent tolerance-inducing protocol for autoimmune-mediated diseases.
Rapamycin+IL-10 treatment induces long term tolerance to allogeneic pancreatic islets transplantation via
induction of antigen-specific T regulatory type 1 (Tr1) cells
M. Battaglia, A. Stabilini, E. Draghici, B. Migliavacca, E. Bonifacio, M. G. Roncarolo
Allogeneic pancreatic islet transplantation is a potential cure for type 1 diabetes, however an immunosuppressive therapy is required to prevent allograft rejection with consequent high risk for infections. One valid alternative is the induction of transplantation tolerance mediated by T regulatory (Tr) cells. Tr type 1 cells (Tr1)
are characterized by production of high levels of IL-10, which is crucial for their differentiation and suppressive
function. We demonstrated that rapamycin and IL-10 treatment induces stable long-term tolerance in diabetic
BALB/C mice transplanted with B6 pancreatic islets. In rapamycin and IL-10 treated mice, endogenous IL-10
mediates an active state of tolerance since blockade of IL-10 activity rapidly induces graft rejection more than
100 days after transplantation. Tr1 cells that produce IL-10 in an Ag-specific way and with suppressive ability in
vitro can be isolated only from mice treated with rapamycin and IL-10. Furthermore, CD4+ T cells from rapamycin and IL-10 treated mice transfer Ag-specific tolerance in newly transplanted mice. Thus, a pharmacological approach, based on rapamycin and IL-10, not only prevents allograft rejection but also induces Ag-specific Tr1 cells that mediate stable long-term tolerance.
Differentiation fate of hematopoietic progenitors in regenerating muscle.
N. Belmonte, B. Camisa, G. Ferrari
Bone Marrow (BM)-derived cells have the potential to differentiate into a number of non-hematopoietic cell
progenies (muscle, liver, blood vessels or epithelia) when assayed in appropriate tissue regeneration models in
vivo. We and others had previously shown that fully differentiated muscle fibers can be formed by the progeny
of transplanted hematopoietic cells, in models of acute or chronic muscle regeneration. The role and fate of
these cells in the homeostasis of blood or muscle tissue is still poorly understood. We used transplantation of
bone marrow (BM) from transgenic donors expressing EGFP constitutively (B6/GFP), or nuclearly-localized
beta galactosidase under muscle-specific control (Myf5-nlacz), to follow the engraftment and fate of
CD45+/Sca1+ hematopoietic progenitors in the BM and skeletal muscle under normal conditions or after acute
tissue damage. Analysis of GFP+ cells co-expressing different cell lineage markers (hematopoietic, myogenic
and endothelial) revealed that hematopoietic transplantable cells contribute to different muscle-residing cells,
depending on the molecular signals originating from the tissue. Additional clues on the molecular program of
CD45+/Sca1+ cells isolated from either BM or muscle will come from the ongoing analysis of their transcription profile.
MyoD activation during somitogenesis
S. Brunelli, F. Relaix, M. Buckingham, G. Cossu
Skeletal myogenesis occurs in the paraxial mesoderm through the activation of myogenic regulatory factors
such as Myf5 and MyoD. In particular, Wnt1, produced by the neural tube, signals via a b-catenin dependent
pathway and lead to the transcription of Myf5. MyoD transcription can be activated by either Myf5 or by Pax3,
in a Wnt signalling non-canonical pathway. We dissected somites and presomitic mesoderm from Myf5KO
mice, where myogenesis is driven mainly by MyoD, and have they differentiated ex-vivo in presence or absence
of different drugs that specifically inhibit component of this signal transduction pathway. We observed a drastic
reduction of MyoD activation in the presence of inhibitors of trimeric G proteins and Protein Kinase C. We
postulated that Pax3 could be transcriptionally activated after phosphorylation by PKC. Drug treatment of
UPM or somites from Pax3GFP mice show that Pax3 transcription is not affected (as seen with the GFP), even
if MyoD activation is. We are planning other experiments to further investigate this pathway, including biochemical analysis of Pax3 phosphorylation and phosphorylation and activation profiling of several signalling
The role of Necdin in skeletal myogenesis
S. Brunelli, S. Baesso, D. De Ponti, V. Broccoli, F. Muscatelli, E. Clementi, G. Cossu
Necdin, a member of the MAGE family, is a DNA binding protein, involved in the Prader Willis Syndrome
(PWS). We have demonstrate that necdin, as well as msx2, are co-expressed in all SMCs and tissues (gut and
vessels), are induced by TGF and, when co-expressed induce a number of smooth muscle markers in mesoangioblast, fibroblast and endothelial cell lines. These data support the hypothesis that Msx2 and Necdin act as
master genes regulating smooth muscle differentiation in at least a subset of SMCs. Necdin is also expressed in
the developing somites, and later in muscle fibers and satellite cells. Necdin KO embryos at 10.5 display an increased level of apoptotic cells in the somites. A role of necdin in myogenesis is also suggested by the fact that
differentiating C2C12 overexpressing necdin form myotubes whose size in much increased respect to controls,
and when necdin is downregulated by RNA interference differentiation is affected. We have produced transgenic mice overexpressing necdin under the control of MLC1 muscle specific promoter. Preliminary results
show that mice carrying the transgene show an increased capacity to regenerate damaged muscle fibres after injury.
Mesoangioblast differentiation into skeletal muscle
G. Messina, S. Brunelli, E. Tagliafico, S. Ferrari, G. Cossu
Mesoangioblast cannot undergo myogenesis spontaneously but do so when co-cultured with skeletal myoblasts and not with fibroblasts. We decided to investigate the signaling molecules involved in the myogenic
differentiation of mesoangioblast cells with a functional genomic approach and to compare the gene expression
profile of myoblasts (proliferating, at the beginning of differentiation, at myotube stage), of fibroblasts and
mesoangioblast cells. We looked at genes coding for membrane receptors, ligands and secreted molecules
specifically expressed at high level in myoblast (but not fibroblasts) at the beginning of differentiation, and
whose interacting protein (ligand, receptor etc) is expressed in mesoangioblasts. We have restricted the candidate molecules to four categories: Wnts/Frizzled, Ephrine/Eph receptors, Semaphorins/Neuropilin-Plexin, inhibin/Follistatin. We are now testing if the inhibition/activation of the different pathway can affect the differentiation of mesoangioblasts using various approaches such as the over-expression of receptor/ligand and the use
of dominant negative constructs.
Myf5 activation is independent from cell division in newly formed somites
A. Innocenzi, G. Messina, L. Latella, P. L. Puri, G. Cossu
Myf5 is activated in the dorso-medial lip of newly formed somites and drive epaxial (dorsal) myogenesis while
MyoD is activated later in the lateral somite and drives hypaxial myogenesis (trunk and limbs). Blocking cell division with Mitomycin C has no effect on Myf5-dependent epaxial myogenesis, but drastically inhibits MyoDdependent hypaxial myogenesis. Labeling somites with BrdU revealed that most epaxial myoblasts did not incorporate the nucleoside before differentiating while most hypaxial myoblasts did. Embryos null for Myf5 (MyoD only) do not undergo myogenesis in the presence of Myto C, while embryos null for MyoD (Myf5 only) differentiate normally in the presence of the drug. Transfection of 10T1/2 fibroblasts with MyoD converted them
to myoblasts that however could not differentiate in the presence of Myto C. In contrast, Myf5-converted myoblasts differentiated in the presence of the drug. Many of the regulatory important tyrosine of MyoD are characterized. Swapping tyrosines between MyoD and Myf5 should reveal the aminoacid(s) whose mitogen-dependent phosphorilation prevents induction of terminal differentiation.
Molecular phenotype of embryonic and fetal myoblasts
S. Biressi, E. Tenedini, M. G. Cusella, G. Lamorte, S. Tajbakhsh, S. Ferrari, G. Cossu
During embryonic development (E 11,12 in the mouse) embryonic myoblasts differentiate and produce primary fibres. After a period in which no new myotubes are formed, fetal myoblasts begin to fuse (E 15, 16) fuse
to form a new generation of fetal fibres. Several phenotypic differences between embryonic and fetal myoblasts
cells have been reported. However the heterogeneity of primary cultures has until now prevented more detailed
analysis. A cell-sorting method for isolating fresh myoblasts at different developmental stage was developed using a mouse strain with the GFP gene targeted into the Myf-5 locus. Almost pure embryonic and fetal populations were analyzed for gene expression with microarray technology. Many genes involved in the TGF-β signal
transduction pathway appeared to be differentially transcribed in embryonic and fetal myoblasts. Specifically,
the proteoglycan decorin and bigycan, protein kinase C ϑ and the transcriptional co activator CITED-1 seem to
be expressed at high level in fetal myoblasts. Moreover the inhibitory SMAD6 is present at high levels in embryonic myoblasts in their fetal counterparts.
Preclinical trial of mesoangioblast transplantation in Golden Retriever Muscular Dystrophy (GRMD)
M. Sampaolesi, A. Innocenzi, R. Tonlorenzi, S. Blot, N. Granger, G. Cossu
The Golden Retriever dystrophic dog (GRMD) develops a very severe muscular dystrophy and is the closest
counterpart of DMD. This animal model has been used to explore the effect of cell therapy with i) canine wild
type mesoangioblasts and ii) genetically dystrophic mesoangioblasts, transduced with a lentiviral vector encoding for human micro-dystrophin. We injected 108 mesoangioblasts in the femoral artery of 2 dogs for 3 times.
In the biopsies of the muscles analyzed we detected 2-3% of dystrophin positive fibers (for the dog receiving
autologous, engineered cells) and 4-8% (for the dog receiving donor cells). We also noted a significant amelioration of the morphology of treated muscles in comparison with the contralateral ones. Western blot analysis revealed low expression of dystrophin or micro-dystrophin. However, physiological analysis of single skinned
fibers isolated from the gastrocnemius muscles showed a normal absolute force. On the basis of these results, a
second experiment is in progress aiming to reach clinical efficacy in the treated leg.
Cell therapy in mdx mice by delivery of dystrophic mesoangioblasts, genetically corrected through exon
skipping or with micro-dystrophin
L. Calvillo, B. Gonzalez Galvez, R. Tonlorenzi, M. Sampaolesi, F. De Angelis. I. Bozzoni, G. Cossu
Most of the DMD mutations consist in deletions and point mutations in the 2,5 Mb dystrophin gene that induce stop codons and consequently premature translation termination. The consequence is the absence of cytoskeletal dystrophin and severe and progressive muscle deterioration. Mdx mice, commonly used as model for
DMD. Were used to compare cell mediated gene replacement and exon-skipping for dystrophin. We treated 17
mdx mice by injection through the femoral artery with different types of mesoangioblasts: wild type (D16
clone), mdx dystrophic mesoangioblast engineered with lentiviral vectors expressing micro-dystrophin or small
RNAs desdigned to skip the mutated exon. Preliminary results indicate efficient dystrophin expression through
exon skipping.
A possible role of alpha sarcoglycan in proliferation of satellite cells
A. Dellavalle, A. Salvadè, G. Messina, M. Sampaolesi
Sarcoglycans are Dystrophin-Associated Proteins (DAPs), and mutations in the relative genes produce cardiomyopathy and/or muscular dystrophy in animal models and humans (LGMD). Alpha sarcoglycan null mice
develop muscular dystrophy, more severe than that observed in mdx mice which is caused by a point mutation
in dystrophin gene. Satellite cells are a population of muscle progenitors that are activated, proliferate and differentiate into new muscle cells in response to muscle damage; therefore they play a key role in muscle regeneration in patho-physiological conditions. Satellite cells from alpha-SG null mice produced a similar number of
clones than wt or mdx cells, but with a dramatically reduced number of cells for each clone. However their ability to differentiate into myotubes was similar to the wild type satellite cells. We isolated and cloned satellite cells
from dystrophic and control muscle in the presence of different growth factors (FGFb, PDGF, SCF, HGF).
Our results suggest a possible involvement of alpha-sarcoglycan in FGF signalling, and our future investigations will be focused to understand the nature of this interaction.
Expression profiling in mouse neural development and differentiation
A. Bulfone, F. Napoletano, M. Gentilini
The vertebrate telencephalon is comprised of many architectonically and functionally distinct areas and structures, and of billions neurons which are precisely connected. It is believed that this complexity is fined tuned
during development by the activity of a large set of genes.
In the year 2004 we have identified, through a functional genomics approach, a set of 832 unique trancripts
mainly corresponding to uncharacterized genes (including 48 new non-coding RNAs) that are preferentially expressed in the embryonic mouse telencephalon.
This selected transcriptome has been annotated, mapped for chromosome loci, and arrayed for gene expression profiling experiments during embryonic development and neural differentiation. The novel genes were also analyzed for their expression on embryonic and postnatal tissue by in situ hybridization, which has demonstrated that they are specifically expressed in the embryonic central nervous system. The full information has
been organized into a searchable database (http://tess.tigem.it) that has been linked to other genomic resources, which provides the scientific community with new molecular players and potential targets in forebrain
Cellular and molecular characterization of human brain tumor-derived stem cells
R. Galli, E. Binda, C. Foroni, B. Cipelletti, A. Cannavale, A. Vescovi
By exploiting the same methodology routinely employed to isolate and expand normal Neural Stem Cells
(NSCs) in vitro, we have established different Tumor Stem Cell (TSC) lines from human glioblastoma multiforme (GBM). These TSCs can be grown in the presence of mitogens, for up to 80 passages in vitro, thus displaying a wide self-renewal capacity. Upon removal of growth factors, TSCs give rise to the three major CNS
lineages and, importantly, are tumorigenic.
Two different groups of TSCs have been identified based on specific cellular and molecular characteristics, as
differences in proliferation, and self-renewal as well as reciprocal expression of genes as EMX2 and HOXA10.
TSCs are currently being characterized by micro-array technology, aiming at the identification of novel molecular targets, differentially expressed in normal human fetal NSCs and in the two different clusters of GBM-derived TSCs.
Since TSCs give rise to experimental tumors, which faithfully recapitulate the invasive behaviour of the human disease, we are characterizing the expression of several mediators of cell motility and migration as matrix
metalloproteinases (MMPs), uPA and cathepsin B.
Development of novel therapeutic approaches to neurodegenerative diseases by means of Neural Stem
Cells (NSCs).
A. Gritti, C. Cavazzin, M. Neri, D. Ferrari, C. Maderna, G. Lamorte, A. Vescovi
Our group focuses on the development of novel therapeutic approaches for brain disorders, particularly demyelinating diseases and lysosomal storage disorders. We have developed procedures to deliver neural stem
cells at the site of lesion in adult mice showing a Multiple Sclerosis (MS)-like syndrome. The same experimental
approach is ongoing using a) human NSCs in a non-human primate model of MS; b) eterologous and autologous (genetically corrected) cells in a model of widespread demyelination (Shiverer mouse) and in a model of
Tay-Sachs disease (Sandhoff mouse). We have also developed procedures allowing for the expansion of the
oligodendrocyte pool generated by human Neural Stem Cells (NSCs). Further, we have accomplished effective
in vivo gene delivery into adult NSCs, establishing widespread, long-lasting chimerism of wild type and transduced cells. This allows for effective cell replacement in the adult brain while avoiding in vitro cell expansion
and transplantation and establishes a novel system for combined gene-cell therapy. The same approach wiil be
used to introduce potentially therapeutic genes in endogenous NSCs using the mutant animal models available
in the laboratory.
Non-viral vectors for cystic fibrosis lung disease
M. Conese, S. Carrabino, S. Di Gioia, A. Bragonzi, J. Rejman
Non-viral vectors for the transfer of CFTR (cystic fibrosis transmembrane conductance regulator) gene into
human respiratory epithelial cells have been shown to have low efficiency. The aim of this study was to evaluate
whether albumin may increase the efficiency of polyethylenimine (PEI) complexes in mediating gene transfer
into respiratory epithelial cells in the presence of CF mucus.
The ternary PEI-HSA complexes increased luciferase expression in confluent cultures in a dose-dependent
fashion up to 500-1000 times as compared to PEI-DNA. The number of GFP-expressing cells, as evaluated by
epifluorescence, was augmented by six fold. No significant cytotoxicity was observed with PEI-HSA polyplexes. The ternary complexes determined detectable CFTR gene transfer and expression at the apical membrane
in polarized CF CFT1-C2 cells, as evaluated by confocal microscopy. CF sputum inihibited PEI-mediated gene
transfer by 7-186 times. Although luciferase expression mediated by PEI-HSA was still inhibited by CF sputum, these levels were 18-83.8 fold higher than with PEI.
Our results demonstrate that PEI-HSA complexes display a higher efficiency than PEI also in the presence of
CF sputum, indicating that albumin-containing polyplexes may help overcoming barriers imposed by CF airway secretions. It is under investigation whether PEI-albumin efficiency is hampered by nasal instillation into
the airways of Pseudomonas aeruginosa, the bacteria most responsible for colonization and infection in CF patients and for mucus overproduction. This model will be thus applied to CF mice, which have already been
characterized in our laboratory as bacterial clearance in the lung and histopathological responses to the infection.
Evaluation of efficiency and efficacy of CFTR gene delivery mediated by lentiviral vectors in model
systems of CF airway epithelium
M. Conese, M. Penzo, L. Palmieri, S. Castellani, E. Copreni
HIV-based lentiviral vectors appear to be promising gene transfer vehicles for the respiratory epithelium by
virtue of their ability to infect non-dividing cells and mediate long-term persistence of transgene expression.
Our study aims to evaluate the efficiency and efficacy of lentiviral vector mediated gene transfer in the CF
murine lung. A lentiviral vector pseudotyped with the envelope of the Vesicular Stomatitis Virus (VSV-G), carrying the green fluorescent protein (GFP) as a reporter gene and the cytomegalovirus promoter (PPT-GFP),
was injected in the lung via trachea in C57BL/6 mice at the dose of 1 X 108 TU. Immunohistochemistry studies
show that PPT-GFP transduced the murine airway epithelium with good efficiency at the level of bronchial
compartment at 2 weeks post-injection. No epithelial damage was observed, neither peribronchial and perivascular cellular infliltrates. It is under investigation whether the GFP expression maintains over time (up to 3-4
We have achieved the cloning of CFTR cDNA fused to the C-Myc tag in the SIN transfer lentiviral vector.
The C-Myc tag is necessary in order to distinguish the exogenous protein from the endogenous one, which is
expressed but at low levels. The CFTR-lentiviral vector will be analysed in vivo in the murine airways and then
in CF mice to study the recovery of the bacterial clearance.
Tie2 expression defines an integrated system of cell types specifically involved in angiogenesis, and
provides a platform for targeted gene delivery to tumor angiogenesis
M. De Palma, M. A. Venneri, R. Galli, M. Sampaolesi, L. Naldini
The role of Bone Marrow (BM)-derived cells in angiogenesis is the subject of intense scrutiny. We showed
that a monocyte subset expressing the angiopoietin receptor Tie2, and not endothelial progenitors, account for
the major BM contribution to tumor angiogenesis. These cells i) are a distinct hematopoietic lineage of pro-angiogenic cells functionally determined in the circulation, ii) are specifically recruited to tumors, including spon-
taneous and orthotopic models, and regenerating tissues, iii) promote angiogenesis in a paracrine manner, iv)
account for most of the pro-angiogenic activity of myeloid cells in tumors. By targeting Tie2-expressing cells
with a suicide gene, we generated angiogenesis-defective transgenic mice displaying remarkable tumor resistance, and show that TEM cells are required for tumor angiogenesis. Unexpectedly, beyond TEM and endothelial cells, vascular mural cell progenitors also express Tie2. Thus, Tie2 expression defines an integrated system
of distinct cell lineages specifically involved in angiogenesis. Combined targeting of these cell types by interfering with the angiopoietin-Tie2 system provides a platform for improving anti-angiogenic therapies.
G.1. Aiuti, A. Advances in gene therapy for ADA-deficient SCID.
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Surgical treatment of atrial fibrillation
S. Benussi, S. Nascimbene, G. La Canna, O. Alfieri
We were the first center to report the results of epicardial ablation of atrial fibrillation in 1999. Our center is
part of the steering committee of the European Database on Atrial Fibrillation Surgery. We extensively studied
the results of diverse forms of unipolar ablation, mainly focusing on radiofrequency, analysing both the electrophysiological and the functional outcome on patients. Since 2003 we are extensively adopting the bipolar radiofrequency technology, which allows transmural lesions in almost all patients, with reduced operative times
and with simplified surgical technique. We are now analysing the results of the two different approaches, not
only clinically, but also in the animal model, working on the development of minimally invasive strategies to explore the feasibility of a surgical approach for lone atrial fibrillation.
Surgery for heart failure
M. De Bonis, L. Torracca, E. Lapenna, S. Benussi, G. La Canna, F. Maisano, A. Castiglioni, J. J. Schreuder,
O. Alfieri
In our center, we adopted (and in some cases developed in-house) novel techniques and technologies to improve the results of heart failure surgery, including new techniques for mitral reconstruction, new algorithms
for patient selection, new IABP systems (IABP has been very frequently used as a prophylactic system to decrease mortality in this high risk subgroup of patients), new imaging modalities to study and select patients,
new anastomotic devices, and techniques of myocardial repopulation via cell autotransplantation (skeletal myoblasts to the left ventricular scar). Functional results of such therapies have been evaluated with state of the art
imaging modalities including MRI, 3D echo and analysis of the pressure-volume relationships. In particular, using a conductance catheter, we studied the effects of surgery on dyssynchrony, a novel mechanism of left ventricular failure. We also adopted mathematical models to simulate left ventricular remodelling, and functional
mitral regurgitation, and used these models to test the new surgical therapies.
Percutaneous approaches to treat valve disease
O. Alfieri, F. Maisano, A. Blasio
Percutaneous Valve Interventions (PVI) is an emerging area of cardiovascular medicine in which heart valve
replacement and repair procedures are completed using catheter-based techniques rather than via the traditional open surgery. Specifically, heart valve replacement products and procedures are being developed to treat aortic and pulmonary valve stenosis and heart valve repair products and procedures are being developed for mitral
valve regurgitation. Most procedures will simulate the surgical ones, while some new approaches will be a better solution for the percutaneous environment. Our group has been involved in the pioneering phase of PVI,
since we are working on animal models since the year 1998. More recently, in cohoperation with the interventional cardiology unit (Dr Antonio Colombo and Dr Matteo Montorfano), we developed a program intended to
explore the clinical opportunities offered by PVI for the treatment of mitral valve regurgitation and aortic valve
disease. First human aortic valve implantation has been done in the year 2004, while first mitral repair is expected in the first half of the current year.
Outcome research
F. Maisano, A. Blasio, A. Verzini, F. Sorrentino, O. Alfieri
Biology of disease is the next opportunity to investigate the mechanism of heart valve disease and heart failure. Taking the unique opportunity of surgery, we collected clinical data and biological samples from patients
undergoing elective valve surgery and developed a biological data bank for an outcome-based research to investigate the biological bases of disease. Our unit performs approximately 400 mitral repairs, and 150 replacements per year. A dedicated outpatient clinic (Ambulatorio per la ricerca e cura delle malattie valvolari) has
been opened. The patient is followed-up in this facility, and undergoes clinical assessment, echo-Doppler, MRI
according to the specific protocol. As clinical course deviates from average, patients are selected as case index
and studied using sophisticated techniques including: differential gene expression, proteomics, confocal microscopy, immunofluorescence, etc. We will address issues such as the mechanism of development of mitral regurgitation in degenerative disease, the mechanisms of development of heart remodelling and failure, insights
on valve remodelling, and the role of the left atrium in the clinical course of the disease.
Pharmacological preconditioning and myocardial damage
G. Landoni, A. Leoni, A. M. Scandroglio, G. Crescenzi, T. Bove, M. G. Calabrò, E. Cerchierini, M. De Luca,
A. Zangrillo
The release of cardiac biomarkers after cardiac surgery, coronary stenting and non cardiac surgery is associated with higher mortality during follow-up. Even detection of a very small myocardial necrosis/infarction in this
setting augurs a worse prognosis for the patient. Volatile anesthetics, commonly used for general anesthesia
during surgery to induce and maintain hypnosis, analgesia, and amnesia seems to reduce myocardial infarction
and improve postischemic recovery at the cellular level. We’re studying preventive measures (pharmacological
preconditioning with halogenates) that could be transferred to all ischemic patients undergoing procedures
that could trigger ischemia and myocardial damage using the most recently described and preferred biomarker
for myocardial damage: cardiac troponin I. We’re also identifying normality ranges of cardiac biomarkers after
different cardiac operation and the relationship with ECG alterations. Electrocardiography has been the topic
of extensive studies to understand the relationship between cardiopulmonary bypass and myocardial stunning
and the role of Q waves after cardiac surgery
Acute renal failure after cardiac surgery
T. Bove, M. G. Calabrò, G. Landoni, G. Marino, A. Zangrillo
Acute renal failure is a serious complication following cardiac surgery as it is associated to high morbidity and
mortality. A prospective, single-center, randomised, double blind trial to evaluate the usefulness of fenoldopam
mesylate, a specific agonist of the dopamine-1 receptor in patients at high risk of perioperative renal dysfunction was conducted. Even if there is an increasing number of reports pointing to the renal protective properties
of fenoldopam we observed no difference in clinical outcome with respect to dopamine in a high-risk population undergoing cardiac surgery. An observational study evaluated the outcome and risk factors of acute renal
failure in a surgical population (5.000 consecutive adult patients) confirming that acute renal failure is one of
the major complications of cardiac surgery, identifying risk factors, and suggesting that optimizing cardiac output and reducing cardiopulmonary bypass time could improve the outcome of high risk patients. Long term
survival and quality of life has been investigated in patients who had renal replacement therapy and in patients
with prolonged ICU stay.
Interactions between cardiopulmonary bypass (CPB) and coagulation system
G. Crescenzi, F. Pappalardo, A. Franco, M. G. Calabrò, A. Zangrillo
We have thoroughly investigated interactions between coagulation system and cardiopulmonary bypass, particularly the effects of foreign surfaces (coating) and reduction of priming volume. These studies were supported by use of specific devices namely Thromboelastography and Platelets Function Analyzer 100 to evaluate the
interaction between coagulation factors and platelets and the activity of platelets themselves. Potential benefits
of these researches might encompass reduction of thromboembolic postoperative events and use of blood
Off-pump coronary artery bypass grafting surgery
G. Landoni, G. Crescenzi, A. Leoni, F. Pappalardo, A. Franco, A. Zangrillo
Off-pump coronary artery bypass grafting surgery has been studied extensively to understand whether the
theoretical advantages of this “less-invasive” surgical technique could translate into an improved clinical outcome. A prospective randomised study on prevention of atrial fibrillation in this subgroup of patients using
high doses Magnesium has been conducted. A prospectively data collection on the “conversion rate” from offpump to on-pump is currently going on. Data on high risk patients have been collected. Normothermia, maintained through different techniques (fluid warming and mattresses), has been studied with a RCT. To improve
patients’ outcome and Intensive Care Unit resource utilization, “fast track” technique is progressively implemented and applied. Anaesthesia for minimally invasive cardiac-surgery has been studied and implemented.
Long-term symptomatic and functional prognosis in patients with cardiac syndrome X
G. Fragasso, A. Margonato, A. Maseri
Studies focusing on the long-term course of cardiac syndrome X (angina pectoris, positive exercise test results
and normal coronary arteriograms) have provided conflicting results. Some reports indicate persistence of angina and deterioration of left ventricular function, while others evidence a sustantially benign prognosis in these
patients. The objective of the study is the evaluation of the symptomatic and functional prognosis in syndrome
X patients.
We will recall all patients among those referred to our department between 1987 and 2004 and diagnosed as
having cardiac syndrome X. We will review the records of their clinical and functional characterization. Followup of these patients will be obtained by telephone interview, administering them a specific questionnaire we
have designed to assess present status and compare it to that at first presentation. Patients still symptomatic for
angina will undergo exercise test. All patients will also undergo echocardiographic evaluation of left ventricular
function (calculation of ejection fraction). Appropriate statistical analysis of data will then subsequently be performed.
Effects of selective and non-selective beta-blockade on left ventricular function, exercise tolerance and
cardiac energetics, as assessed by magnetic resonance spectroscopy in patients with heart failure
G. Fragasso, A. Margonato
The metabolic effects of beta-blockers in patients with heart failure have not been elucidated. It is well
known that fatty acid oxidation by the heart uses more oxygen per unit of mechanical work performed. Because
catecholamines, increased in heart failure may stimulate the utilization of fatty acids (FFA), beta-blockade may
alter the ratio of FFA to carbohydrate utilization. Although FFA oxidation provides the highest yield of adenosine triphosphate (ATP) (130 ATP per mole FFA versus 38 ATP per mole glucose), its metabolism requires
more oxygen than glucose oxidation. The ATP yield for FFA per oxygen atom is 2.83 compared to 3.17 for glucose. Therefore, FFA oxidation is less energy efficient than glucose oxidation. Previous studies showed differential effects of different beta-blockers in term of myocardial metabolic function, in patients with LV dysfunction. Our aim is to compare the metabolic effects of B1 selective beta-blockade with bisoprolol and non selective, with A block properties, carvedilol in patients with chronic heart failure, on exercise tolerance, LV function, glucose metabolism and magnetic resonance spectroscopy of cardiac high energy phosphates.
Integrated e-system for the management and the monitoring of international multicenter clinical studies
D. Cianflone, N. Cristell, N. Pozzoli, A. Pessina, F. Grassi, S. Pagani, A. Haddas
Management, monitoring and quality control of multicenter, international clinical studies and trials are complex and resource consuming. Electronic (off-line) and web-based on-line Case Report Forms (CRF) are sometimes used thus shifting the burden of data entry at the level of individual participating center. Still, study monitoring relies on human resources that have to travel to the study sites and follow complex data verification procedures. To overcome these limitation, time constraints and related costs we developed a real-time, electronic
only, on-line solution for the collection, control and verification of CRFs and study management forms, as well
as for real-time study progress monitoring and quality control procedures. No single data management or office
automation package is capable of fulfilling all the complex requirements for these tasks. We hence used a set of
commercially available platforms and tools. The core of the system is an standard SQL database/management
platform made available on-line through a web-based Oracle Portal.
This combination provides electronic CRFs. Investigators have access only to their cases and receive immediate warning (inconsistencies, lack of data, upcoming and overdue deadlines). Data are accessible to the study
monitor in real-time via the same web based portal. Warnings and recalls are then made via e-mail with receipt
requests. A higher level of integration has been achieved by developing real-time control procedures, reports
and charts that query the on-line database through a secure ODBC connection. Front-end and development
tools include a querying and reporting application that triggers stored and ad-hoc procedures for quality control purposes, a set of highly integrated “executive” reports, tables and summary charts. Security throughout is
guaranteed by server access control policies, SSL, data codification and encription. Real-time reports on ongoing activities are available to study monitors and the steering committee.
An integrated, “visual” database and reporting tool for vascular doppler labs.
D. Astore, E. M. Marone, F. Sioli, S. Ventura, A. Haddas, A. Zappalà, R. Chiesa, D. Cianflone
The Vascular Doppler Lab is a high volume facility that produces over 160 tests per day, on a tight schedule.
In order to provide a database and reporting tool we developed a customized application in the framework of
the existing clinical and research database for the Cardio-thoracic and Vascular dept. After in-depth workflow
and process analysis development was accomplished using a graphical front end and DB tool (dataEase 6. X
RADD) and an industry standard SQL back-end engine (Oracle 9.x). The application currently provides forms
and reporting tools for all kind of tests performed in the labs: carotid, upper limbs (arterial and venous), lower
limbs (arterial and venous), aortoiliac. To integrate this application with the existing hospital information system (SIO) for booking and planning the Doppler exams we developed additional SW layers. These layers,
jointly developed with the Information Systems Dept at HSR, allow a continuous communication flow from the
SIO and the application in order to transfer demographic, booking and registration data and, ultimately, retransfer reports back to the SIO. As of December 2004 the application contained over 10,000 Doppler reports.
A framework-study for the precipitating and predisposing components of Acute Coronary Syndromes
D. Cianflone, N. Cristell, M. Banfi, M. Magnoni, S. Coli, A. Maseri
Patients (PTS) with Acute Coronary Syndromes (ACS) encompass a variable spectrum of presentations: at
one extreme PTS with an unheralded single occurrence of ACS followed by complete and persistent remission
(Maseri Type I) and at the other extreme PTS with multiple recurrences of instability and/or myocardial infarction (Maseri Type II). To study the potential precipitating inflammatory components and the possible predisposing genetic factors, all PTS admitted in the CCU for an ACS are classified in homogeneous groups according to clinical presentation, risk factors and biological profiles to undergo a blood sampling for serum, plasma,
DNA (whole blood) and RNA (Quiagen) for subsequent analysis. Data are stored in dedicated databases along
with follow-up data. PTS with a first myocardial infarction without previous history are also included in the
First Acute Myocardial Infarction (FAMI) Study of the environmental, inflammatory and genetic causes of myocardial infarction in Italy, China and Scotland. This is an observational multiethnic prospective study designed
to analyse environmental, inflammatory and genetic mecchanisms that trigger Acute Myocardial Infarction
(AMI). PTS with AMI occurring as the first manifestation of Coronary Disease within 6 hours of onset of symptoms are recruited as well as age, gender and ethnic matched controls. Our aim is to develop novel and accurate
screening modalities and targeted therapeutic strategies. Three novel approaches will be used: 3000 PTS
case/control study in 3 populations with extreme differences in the incidence of AMI, environmental and genetic background; the selection of PTS unaware of having coronary disease with AMI as the first manifestation
of disease; the use of post-genomic nanotechnologies to obtain: gene-expression profiling in platelets, leucocytes; proteomics to assess concentration of key determinants of lipid, coagulation and inflammatory profiles;
broad genetic characterisation (expression profiling of candidate gene clusters and the pertinent proteomics
studies) to identify unsuspected genetic defects and polymorphisms and gene-function interactions. In 2004,
247 Chinese, 87 Italian and 60 Scottish PTS + controls per country were recruited and a thorough clinical
work-up was performed using identical questionnaires, entered on-line by each participating center.
Characterization in lymphocyte subsets in atherosclerotic plaques and peripheral blood from patients with
acute and chronic atherosclerotic disease
E. Ammirati, M. Banfi, D. Cianflone, A. Maseri, C. Monaco
The aim of the study is to investigate the phenotype of T cells from peripheral blood and atherosclerotic
plaque-infiltrating lymphocytes in patients with diverse manifestation of the atherosclerotic disease, with a particular focus on the representation of specific T cell subsets, namely CD28null, TCRz dim, and NK T cells.
CD28null. The role in atherosclerosis of these subsets, and in particular in Acute Coronary Syndrome, have not
been yet investigated. Recruitment has already started, to include patients with 1.chronic stable angina, 2. unstable angina, 3. acute myocardial infarction, 4. asymptomatic carotid artery disease, 5.symptomatic carotid artery disease and 6.Healthy age- and sex- matched controls. PBMC isolated from peripheral blood, and stored in
liquid nitrogen, will be analyzed by FACS, for CD3, CD4, CD8, CD28, TCRz, CD16, CD25, CD69 markers.
Immuno-histochemistry for the same markers will be carried out on the atherosclerotic plaques. Cells will be
isolated from the atherosclerotic plaques (fresh) via enzymatic dissociation. FACS analysis will be carried out to
evaluate the phenotype of intra-plaque cells.
UHSR BIOBANK: Interdepartmental Biological Bank
D. Cianflone, G. Ruotolo, M. Banfi, O. Carandente, F. Sioli, S. Ventura, A. Haddas, N. Lanzi, S. Coli, A. Maseri
The aim of the project is to implement an operational framework for the collection, storage and management
of biological speciments (blood, serum, plasma, cells, tissues, DNA, RNA, biopsies, etc.)
We developed a standardized, anonymized, barcoded and ISO-compliant sampling procedure.
We also developed a set of management procedures on a dedicated database application. This application is
used for the allocation and retrieval of the aliquotes in the array of –80°C freezers.
The array of freezers is monitored via a computerized control station, capable of sending the complete status
through the Institute intranet and the alarm warnings also through e-mail and GSM-SMS messaging.
In 2004 the Biobank has actively added cooperation with the Electrophysiology Unit and the Neurology dept,
along with the support already provided to Coronary Care Unit, Vascular Surgery, Clinical Cardiology, Cardiac
Surgery and the Cath Labs. As of December 2004 the Biobank contains biological samples from patients categorized in 37 homogeneous groups: over 2000 aliquotes from the Cardio-thoracic and Vascular Dept. and over
25,000 aliquotes from the CCUs in China and Scotland (FAMI study).
Myocardial production of chromogranin A in hypertrophic cardiomyopathy: a new regulatory peptide of
myocyte function
M. Pieroni, C. Chimenti, F. Curnis, B. Colombo, M. A. Russo, A. Maseri, A. Corti, A. Frustaci
Chromogranin-A (CgA) is a protein diffusely present in secretory granules of neuroendocrine cells. CgA levels are increased in heart failure and experimental studies showed a negative inotropic effect exerted by CgA
fragments on myocardium. In this study we assessed whether myocardial tissue may produce CgA. Twentythree patients with hypertrophic cardiomyopathy (HCM) (15M/8F, mean age 45.1±14.4 ys) underwent cardiac
catheterization with left ventricular endomyocardial biopsy. Myocardial samples were processed for histology
and immunohistochemistry with anti-CgA antibody. Large myocardial specimens from three patients undergoing surgical septal reduction were used for polymerase chain reaction and ELISA studies. In all patients plasma
CgA and brain natriuretic peptide (BNP) levels were measured. All patients showed increased left ventricular
end-diastolic pressure (LVEDP); histology confirmed the diagnosis of HCM in all cases. CgA and BNP plasma
levels were increased in all patients (229.4±137.1 ng/ml and 162.6±99.7 pg/ml respectively) with a positive correlation between CgA and BNP (R=0.81), CgA and LVEDP (R=0.93), BNP and LVEDP (R=0.81) (p<0.001).
Immunohistochemistry showed positive staining for CgA, and co-localization with BNP at confocal microscopy, in HCM patients but not in controls. ELISA of tissue extracts showed a significant amount of CgA
(>1 mg/g of myocardium) in HCM patients but not in normal myocardium. Left ventricular myocardium is a
source of CgA in patients with HCM. The correlations between CgA, BNP and LVEDP suggest a stretch-induced mechanism of release. Given the negative inotropic action of its fragments, CgA may play a central role
in neuroendocrine regulation of myocyte function.
HMGB1 is secreted by human atherosclerotic plaque and promotes smooth muscle cell migration and
A. Porto, R. Palumbo, M. Pieroni, G. Aprigliano, R. Chiesa, A. Maseri, M. E. Bianchi
High Mobility Group Protein 1 (HMGB1) is a chromatin protein leaked out by necrotic cells and secreted by
activated myeloid cells. The extracellular protein is a potent mediator of inflammation. We tested whether cells
within human atherosclerotic plaque could secrete and respond to HMGB1.Human carotid endarterectomy
specimens (n=25),obtained from patients with carotid stenosis >70% and normal internal mammary arteries
specimens from patients (n=10) undergoing coronary artery by-pass surgery were put into culture. By western
blot we found a large amount of HMGB1 in the supernatant of atherosclerotic but not of normal arteries. The
absence of lactate dehydrogenases, a marker of necrosis, in the supernatants indicated an active secretion of
HMGB1 by diseased arteries. We showed that the origin of extracellular HMGB1 in atherosclerotic plaque was
not only endothelial and neointimal foam cells, but also mostly Smooth Muscle Cells (SMCs) because all these
cells expressed HMGB1 in the cytosol wheras cells of normal arteries contained HMGB1 only in the nuclei.
Significantly, we demostrated in vitro that HMGB1 was secreted by SMCs, isolated from normal and atherosclerotic arteries, when acquired a macrophage-like state as after cholesterol loading. We also showed that human aorta SMCs proliferate and migrate in response to HMGB1.
HMGB1 is expressed and secreted by cells within the human atherosclerotic plaque and promotes SMC migration and proliferation. Human SMCs can also secrete HMGB1 when acquiring a macrophage-like phenotype. HMGB1 may be a key player in neointimal hyperplasia and restenosis after angioplasty.
Mitral valve regurgitation and atrial fibrillation
C. Chimenti, F. Maisano, A. Meris, S. Benussi, O. Alfieri, A. Maseri, A. E. Becker
The aim of the study is to compare the clinical, echocardiographic and histologic findings and to analyze the
neurohumoral activation in patients with mitral valve regurgitation with and without atrial fibrillation. The
study population consists of 42 consecutive patients (22 with chronic atrial fibrillation and 20 in sinus rhythm)
with degenerative Mitral Regurgitation (MR), either with flail leaflets or with simple mitral valve prolapse, who
underwent cardiac surgery between March and October 2004 at San Raffaele Hospital. In all cases the severity
of MR was assessed semiquantitatively as 3+ or 4+ by color-Doppler echocardiography. In all patients non invasive (ECG, color-Doppler echocardiography) and invasive (coronary angiography, ventriculography) data were
collected. Tissue specimens of left atrium and left ventricle were obtained during cardiac surgery, embedded in
paraffin wax for histologic, immunohistochemistry and morphometric analysis, or snap frozen in liquid nitrogen for molecular biology analysis. Blood samples were collected before cardiac surgery and stored at -80°C to
evaluate neurohumoral activation.
Multifocal cardiac uptake of [18 F]-FDG PET in patients with acute infarction in ischemic and nonischemic heart regions, persistant at 10 weeks
C. Godino, C. Messa, D. Cianflone, A. Margonato, V. Leccese, N. Cristell, F. Fazio, A. Maseri
We investigated cardiac [18 F]-flurodeoxyglucose uptake by positron emission tomography ([18F]- FDGPET) in fasting patients (pts) with their First Acute Myocardial Infarction (FAMI) and at 3 months follow-up. 2
pts (1 female, age 59±10) with single vessel disease, soon after successful primary PTCA and 12 pts (CSA pts,
control, 3 females, age 60±8) with uncomplicated chronic stable angina and 3-vessel disease were investigated.
Baseline left ventricular ejection fraction (LVEF) of FAMI and CSA pts were 52±10% and 61±3%, respectively. Myocardial [18F]-FDG-PET scan was performed in FAMI pts within 48 hrs after primary PTCA and repeated after 9.6±4.3 weeks, and in CSA pts only at baseline. Plasma levels of glucose, free fatty acid, insulin, hsCRP, IL-6 and HOMA index were determined. [18F]- FDG uptake was below intraventricular background
(Bg) in all circumferential or longitudinal ventricular slices in CSA pts (mean FDG uptake index, FUI:0.70±0.2
of Bg). By contrast, 10 out of 12 FAMI pts had an average wall uptake, 1.3 to 3 fold higher of Bg
(FUI:1.73±0.40 p<0.0001). In FAMI pts, FDG uptake was heterogeneous, being limited to the culprit area (infarct-related vascular district) in 2 pts, to the non-culprit areas in 2 pts and involving both culprit and non-culprit areas in the remaining 6 pts. On average 60% of segments had an uptake >Bg. At follow-up, regional cardiac [18F]-FDG uptake remained unchanged in 7 pts, was reduced in 4 and became positive in multiple segments in 1 pt who was negative in the initial study. No FAMI pts developed recurrent instability, during followup LVEF was unchanged (55±9%). Statistically significant differences was only found in IL-6 plasma levels, between FAMI and CSA pts(8.47±6.59 pg/mL vs 2.17±1.18 pg/mL; p<0.003), and in FAMI pts between basal
and follow-up (8.47±6.59 pg/mL vs 3.24±1.83 p<0.02). Massive multifocal, persistent cardiac glucose uptake
was observed in the majority of FAMI pts, mostly in areas perfused by non stenosed coronary arteries and normocontractile, possibly related to increases GLUT1 glucose transporter expression and function in ischemic
and nonischemic cardiac regions, as a protective response to ischemic injury previously reported.
Gene expression profiling in platelets of patients affected by acute coronary syndromes (subgroup of
FAMI population)
L. Nanni, G. Vallanti, D. Vozzi, A. Maseri, G. Lanfranchi
The aim of our research program on coronary syndromes is to study the role of inflammation in causing a
sudden and unexpected transition from stable chronic forms of coronary artery disease (extremely frequent in
the adult population over 50 years of age) to unstable forms with impending myocardial infarction or directly to
an acute myocardial infarction. We are identifying the differential gene expression profiles of whole blood and
platelet population collected from selected patient groups (FAMI subgroup), using oligonucleotide microarray
with 55,000 human transcripts. Our secondary aim is the analysis of those proteins coded by deregulated transcripts detected through microarray analysis, which are involved in inflammatory processes.
Paradigmatic phenotypically homogenous patients are being selected by the Cardiological Clinical Research
Unit and divided into 4 clinically and biologically homogeneous groups: patients with acute infarction as their
very first manifestation of coronary disease (subdivided into those in whom the infarction was preceded by unstable angina and those in whom the infarction was totally unheralded); patients with persisting coronary instability as their first manifestation of coronary disease; patients with chronic stable angina. As positive control, we
also enroll patients with rheumatoid arthritis who show a stimulation of the immune cascade. We are also collecting blood from 18 donors, grouped by sex and age, representing our negative experimental control.
MRI-histopathological correlation of carotid plaques features in carotid artery occlusive disease
C. Foglieni, N. Anzalone, A. Giazzon, G. Ruotolo, R. Castellano, M. Sessa, A. Maseri
The carotid artery occlusive disease is an atherosclerotic disease, susceptible of thrombo-embolic complications and acute ischemic syndromes. Symptomatic patients (pts) with carotid stenosis (Cst) >50%, assessed by
US, undergo endoarterectomy; the indication to surgery for asymptomatic pts is controversial. The symptoms
are not prognostic for the evolution from a stable carotid plaque (CP) (with fibrocalcific features) to a vulnerable CP (with cup-ruptures, inflammatory infiltrates, or thrombi). Due to the number of causal factors (cellular
and molecular composition, inflammatory mediators, flow rate) the CP structure vary not only from pts to pts
but along CP ax. Our aim is to correlate in vivo MRI with post-endoarterectomy histopathology, to characterize
the inflammatory elements inside the CP. We planned to enroll 20 pts with Cst > 50%, 10 with TIA or ischemic
stroke omolateral to Cst, 10 with asymptomatic Cst. MRI focus on neo-vessels, cell infiltrates, thrombo-hemorrhages, cup-ruptures, through resolution improvements. Histology validates the MRI: CPs are dissected in
rings, frozen, sectioned and stained with Hematoxylin/Eosin or Movat.
Morphology analysis follows modified Virmani’s classification. Immunohistochemistry will be performed. In
the pilot phase (n=5) we verified the comparability MRI/histology by co-identifying inflammation-related CP
MHC class II is highly expressed in endothelium and vasa vasorum of internal mammary arteries of
patients with acute coronary syndrome suggesting a widespread vascular inflammation
C. Foglieni, F. Maisano, A. Castiglioni, O. Alfieri, L. Livolsi, A. Giazzon, S. Coli, E. Dal Cin, G. Santambrogio,
A. Maseri, G. Ruotolo
Patients (pts) with recurrent Acute Coronary Syndromes (ACS) are characterized by multiple coronary unstable plaques, coronary inflammation, complex carotid plaques elevated activated blood cells and soluble inflammatory mediators. The markers levels are lower in pts with severe coronary disease (CSA). We assess the activation of IMA in paradigmatic ACS or CSA (n=10-15/group). ACS includes pts with unstable angina undergoing
urgent CABG, pts with myocardial infarction w/wo ST elevation. CSA includes pts scheduled for CAGB because of chronic stable angina with stable symptoms for the last 6 months, no elevation of blood inflammatory
markers. Clinical data, risk factors, selected cytokines and chemokines will be collected. IMA morphology is
evaluated by Hematoxylin/Eosin. Endothelium (END) preservation and IMA activation are graded at confocal
microscopy upon labeling for CD31, E-selectin, CD54, CD106, MHC I, MHC II, iNOS, tissue factor, TLR-4.
(+++bright, ++ good, +visible, +/-weak, -none). MHC II is more expressed by END of IMA lumen (L) and
vasa vasorum (vv) of ACS (n=8) compared to CSA (n=14).
An intensity >++ was seen in 50% ACS, but only in 7% of CSA L, and in 75% ACS but 21% CSA vv. Smaller differences were found in L END for TLR-4 and E-selectin. The IMA expression of MHC II, TLR-4 and Eselectin on L and vv END is compatible with a widespread endothelial inflammatory activation in ACS pts.
Role of epicardial adipose tissue in the etiopathogenesis of acute coronary syndromes
S. Langheim, F. Maisano, G. Sinagra, H. Li, E. Ferrero, G. Vallanti, A. Maseri, G. Ruotolo
While plasma inflammatory biomarkers may not adequately reflect local tissue inflammation, epicardial adipose tissue has been demonstrated as a source of several inflammatory mediators in high-risk cardiac patients.
We have therefore initiated a study in male patients with Acute Coronary Syndromes (ACS) vs those with
Chronic Stable Angina (CSA), aimed at: comparing the expression of inflammatory mediators in epicardial adipose stores; evaluating the association between the inflammatory pattern of epicardial adipose stores and red
blood cell w-3 fatty acid content; evaluating the role of inflammatory mediators and/or adipokines from epicardial adipose tissue on endothelial cells in vitro. A group of patients operated for valvular defects will serve as a
control group. The study will be performed in 2 distinct ethnic groups: Caucasians (Milano and Trieste), and
Chinese (Bejing). In each center, 3 groups of 20 patients will be collected over a 1-year period.
Cell-derived microparticles in blood as markers of thrombosis and inflammation in cardiovascular patients
L. Mendolicchio, Z. M. Ruggeri
Inflammation and thrombosis are potential contributors to acute coronary artery occlusion. We consider
three potential sources of inflammation and thrombosis markers relevant for our studies: endothelial cells,
platelets and leukocytes. These markers may differ in patients at immediate risk of coronary artery occlusion
compared to the normal population, and in the same patient may vary with disease evolution. We are studing
cell-derived microparticles as markers of thrombosis and inflammation; in particular, we are defining which microparticles express Tissue Factor (TF). We study patients with different types of acute and chronic coronary
syndromes and normal volunteers as control group. To detect microparticles in blood we use a flow cytometric
approach. In essence, microparticles varying in size between 100 and 500 nm are being detected on the basis of
their forward and side light scattering. Specific membrane glycoprotein markers are being used to ascertain the
origin of different microparticles. We expect to find an increase in the number of blood-borne microparticles in
patients with acute coronary syndromes.
The microparticles are of different origin, endothelial cells, leukocytes and platelets. Some of the microparticles express tissue factor. We anticipate that the occurrence of an acute coronary event will be related to an increase in the number of TF-bearing microparticles and, possibly, in the number of microparticles originating
from inflammatory cells. Depending on progress in basic research on the biology of TF, we expect to be able to
differentiate, in the near future, between expression of active (prothrombotic) versus inactive TF.
Heterogeneous innate responses to toll-like receptor activation and apoptotic cells in patients with acute
coronary inflammation
A. A. Manfredi, L. Ferri, I. Dumitriu, P. Rovere Querini, C. Monaco, D. Cianflone, A. Maseri
Patients with acute coronary syndromes have an unpredictable course and inflammation appears to play a
major role in the unstable phases of the disease. The molecular events upstream are poorly identified. A well
defined primary anti-inflammatory signal is delivered by the recognition of apoptotic cells, a frequent component of atherosclerotic lesions. To verify whether a failure in decoding this signal is involved, we purified
mononuclear phagocytes from 22 consecutive patients with acute coronary syndromes and 40 healthy donors,
matched for age and sex. We then analysed the response to apoptotic cell recognition upon stimulation with
microbial ligands for Toll-like receptors 2, 3, 4, 7 and 9. Cytokines and chemokines release was then measured
by multiparametric ELISA. The data suggest that the net effect of innate stimuli and apoptotic cells varies from
an anti- to a pro-inflammatory outcome in different subjects. The response was conserved when cells from identical donors were re-challenged at 8 weeks intervals and correlated in selected patients with C reactive protein
levels, revealing individual innate patterns of response to microbes and injury.
Down-regulation of the CD91 heat shock protein receptor reflects monocyte activation and acute coronary
A. A. Manfredi, S. Heltai, A. Calabrese, L. Ferri, G. Augello, S. Coli, D. Cianflone, C. Monaco, A. Maseri
Acutely symptomatic phases (unstable angina, myocardial infarction) and long periods of quiescence (chronic stable angina or asymptomatic disease) characterize ischemic heart disease. Leukocyte activation in particular
is an hallmark, and may provide hints to identify patients that will experience unstable disease. We concentrated on scavenger receptors, the receptor for advanced glycation end-products and the CD91 common receptor
for heat shock proteins. These molecules decipher tissue injury signals, which are possibly involved in inducing
and maintaining systemic inflammation. We set an efficient whole blood flow cytometry assay, which allow the
simultaneous independent assessment of different receptors on neutrophils, monocytes and lymphocytes of patients with acute coronary syndromes (myocardial infarction and unstable angina) and control subjects, including patients with chronic stable angina and healthy volunteers. The results indicate that the CD91 receptor is
selectively down-regulated upon activation, and suggest that its level can reflect the previous in vivo exposure
to long-lasting tissue injury signals.
Effects of pulmonary involvement in progressive systemic sclerosis on exhaled nitric oxide levels
G. Cremona, G. Donadoni, A. Fumagalli
Background: Exhaled nitric oxide (FENO) has been widely used as a measure of inflammation and has been
reported to be increased in patients with progressive systemic sclerosis. The onset of pulmonary fibrosis (ILD)
and pulmonary hypertension (PH) in these patients has been associated both with decreased FENO and or
with unchanged FENO but with increased alveolar concentrations of NO (CANO). Objective: To investigate
the role of pulmonary involvement in PSS on the dynamics of NO exchange independently of inflammation.
Measurements of FENO at various expiratory flow rates in patients with systemic sclerosis with PH, and/or
ILD and in healthy age-matched controls in order to partition FENO into alveolar and airway compartments.
These values will be related to the diffusion coefficient of nitric oxide, pulmonary capillary volume as well as
humoral and radiological markers of disease. The study should help to clarify the source and mechanisms of
pulmonary exchange of nitric oxide in systemic sclerosis contributing to the validation of the measurement of
FENO as a clinical tool.
The role of exhaled nitric oxide measurements in asthma management
G. Cremona, E. Baraldi
Despite being a condition characterised by inflammation, asthma treatment is currently guided by either
symptoms related to airway mechanics and/or by indices of lung function. Assessments of asthma induced inflammation, such as cell counts in induced sputum, have remained largely unavailable to the clinician because
of their complexity or cost. Increased quantities of nitric oxide have been measured in exhaled air (FENO) in
asthmatic patients which have been associated with up-regulation of inducible nitric oxide synthase as well as
through nitrite protonation in the acid environment of inflamed airways. FENO levels are invariably increased
in allergic or uncontrolled asthma and tends to decrease with antiinflammatory therapy. Unfortunately levels of
FENO are affected by a host of other variables such as expiratory flow rate, age, height, atopy, airway infections
and smoking. Over the last 14 years, exhaled nitric oxide has been widely studied as a simple non-invasive
marker of inflammation but has yet to achieve a defined role in routine clinical asthma management. This prosepective multicentre observational study is aimed at determining whether sequential measurement of FeNO in
an outpatient setting provides a clinically useful tool to monitor asthma and predict exacerbations. The study
involves 10 Italian centres who will each enrol 30 patients with asthma and 10 healthy controls. The patients requiring treatment according to GINA guidelines will be treated for two weeks and all the patients will be followed up for 1 month. Weekly assessments will be carried out monitoring FENO measurements, spirometry
and asma severity score (ASS) which includes twice daily peak flow measurements. Outcome variables will include FENO levels, number of exacerbations, and ASS. The study should be able to evaluate the predictive value of FENO on the short term course of asthma, provide a correlation of FENO values with other parameters
of asthma control and provide reference values for FENO healthy subjects with a negative allergy skin tests.
Ch.E.S.T. in NSCLC, chemotherapy for early stages trial. randomized phase iii trial of surgery alone or
surgery plus preoperative gemcitabine-cisplatin in clinical early stages (T2N0, T1-2N1, T3N0 and T3N1)
Non-Small Cell Lung Cancer (NSCLC)
P. Ciriaco, M. Pansera, L. Libretti, M. Casiraghi, P. Zannini
An european multicenter randomized study to assess whether preoperative chemotherapy with gemcitabine
and cisplatin for 3 cycles improves progression-free survival compared to surgery alone in previously untreated
patients with clinical stage IB II and selected IIIA Non-Small Cell Lung Cancer (NSCLC). The objectives study
are also to compare the survival and sites of relapse in the two study arms, the operative mortality and other
toxicities, to evaluate the response rates and to obtain samples for correlation of radiologic, pathologic, molecular and biologic factors with outcome.
MAGE-A, BAGE, GAGE, and MAGE-B genes in non-small cell lung cancer: frequency of expression and
prognostic significance
G. Melloni, P. Ciriaco, A. Carretta, P. Zannini, V. Russo, C. Bordignon
MAGE, BAGE and CAGE genes encode T cell-defined tumor-associated antigens (TAA), which are expressed by various human tumors and are silent in normal tissues. Because of their expression pattern these tumor-associated antigens have received attention as potential target for active immunotherapy, and molecular tumor markers. Both of these tools should be useful for therapeutic improvement in non-small cell lung cancer. In
this study, we analyze the frequency of expression of some members of the MAGE, BAGE and GAGE gene
families in non-small lung cancer to determine the fraction of patients that are potential candidates for TAAspecific immunotherapy approaches. Moreover, we assess the correlation between TAA genes expression and
different clinicopathologic parameters. In addition, the prognostic role of TAA genes expression is evaluated.
Prognostic significance of microsatellite instability determined by immunohistochemical staining in
colorectal cancer lung metastases
G. Melloni, G. Negri, A. Puglisi, L. Libretti, P. Zannini
Surgical resection for colorectal cancer (CRC) lung metastases is a well-accepted treatment modality with a
reported 5-year survival ranging from 30 to 60%. Currently, the International Registry of lung metastases
(IRLM) classification is the only tool used during the selection process to determine which patients with CRC
lung metastases might be eligible for surgery. Identification of new tumor markers to supplement standard clinical and pathological staging may make possible to subdivide patients with CRC lung metastases, thus identify-
ing those at highest risk of recurrence following surgery. Microsatellite instability (MSI) has been identified as a
factor with good prognosis in CRC but not in CRC lung metastases. A recent study showed that patients with
stage III CRC who had MSI-positive tumors had a 74% 5-year survival rate compared to 46% in patients with
MSI-positive tumors. The purpose of this study is to evaluate the use of immunohistochemical analysis to identify colorectal cancer lung metastases with MSI and assess the prognostic value of this analysis.
Evaluation of total-body ct-pet in the differential diagnosis and staging of non-small cell carcinoma of the
A. Carretta, G. Negri, P. Ciriaco, L. Libretti, P. Zannini
Surgery remains the treatment of choice of early-stage non-small cell lung cancer. Preoperative staging is of
utmost importance in order to adequately select patients for surgical treatment. Standard staging techniques include total-body CT scan, bone scan and invasive procedures as mediastinoscopy. CT-PET has been recently introduced in clinical practice, and preliminary reports show that this non-invasive technique seems to be associated with an improved accuracy both in the differential diagnosis of pulmonary lesions and in the staging of
lung cancer. The aim of this study is to assess the diagnostic accuracy of CT-PET in the differential diagnosis of
pulmonary lesions and in the staging of lung cancer, also evaluating the cost-effectiveness of this technique.
Autofluorescence bronchoscopy in the screening of lung cancer
A. Carretta, G. Melloni, P. Carretta, A. Puglisi, P. Zannini
Early diagnosis of lung cancer is essential to obtain favourable results in the treatment of this disease. The detection of pre-neoplastic lesions and early-stage tumors of the airway is therefore of utmost importance to improve therapeutic results. The aim of this study is to assess the role of autofluorescence bronchoscopy as a
screening tecnhique in the detection pre-neoplastic and neoplastic lesions of the airway in patients at high risk
for the development of lung cancer.
A phase I trial investigating gene therapy after surgical maximal cytoreduction for malignant pleural
G. Melloni, P. Ciriaco, P. Zannini, V. Gregorc, A. Bolognesi, E. Villa, V. Russo, C. Bordignon
Although Malignant Pleural Mesothelioma (MPM) remains a rare tumor, epidemiologic studies confirm the
increasing incidence of this malignancy. The need for innovative treatment is clear because there are no universally accepted standard treatments for MPM, and the efficacy of current therapies remains disappointing with a
median survival of 12 months. Because of its localized nature, extremely poor prognosis and lack of effective
therapy, MPM has been identified as a reasonable target for gene therapy. Recent studies support the idea that
delivery of an adenovirus vector containing the herpes simplex thymidine kinase gene driven by an RSV promoter (Ad.RSVtk) followed by the administration of Ganciclovir may be an effective approach in the treatment
of MPM. The purpose of this Phase I trial is establish the safety, side effect profile and the maximally tolerated
dose of administered the Ad.RSVtk virus in patients with MPM after surgical maximal cytoreduction.
Evaluation of clinical and histopathological prognostic factors after surgical treatment for solitary fibrous
tumors of the pleura
A. Carretta, C. Voci, A. Puglisi, M. Casiraghi, P. Zannini
Solitary fibrous tumors of the pleura are neoplasms with peculiar clinical and histological features. Although
frequently benign, these tumors may also have a malignant and metastatic potential. Surgical treatment remains
the mainstay of treatment of these tumors. The aim of the study is to identify clinical and pathological prognostic factors to select patients at high risk of recurrence, in whom the efficacy of adjuvant treatments should be
Assessment of clinical and pathological prognostic factors in patients with large cell neuroendocrine
carcinomas of the lung
A. Carretta, P. Ciriaco, C. Voci, M. Pansera, P. Zannini
Neuroendocrine tumors are associated to an extremely poor prognosis. According to the 1999 WHO classification these tumors, considered as a variant of large cell carcinoma, have been further subdivided between LC-
NEM, LCNEC and LCNED according to microscopical and histopathological features. The aim of the study is
to analyse the results of surgical treatment of LCNEC and to assess the prognostic role of clinical and histological factors in order to identify patients at high risk of neoplastic recurrence who could benefit from combined
oncological and surgical treatment.
Endovascular surgery for thoracic aortic aneurysms. Evaluation of new devices
R. Chiesa, G. Melissano, E. Civilini, Y. Tshomba
New devices for the endovascular treatment of thoracic aortic diseases were recently approved for clinical use
by the European authorities, obtaining the CE mark. The aim of the project is clinically to evaluate the characteristics and performance of these new devices. The most popular commercially manufactured thoracic stentgrafts were the Talent (AVE/Medtronic Inc. Santa Rosa CA) graft and the Excluder TAG (WL Gore and Ass.,
Flagstaff AZ.) graft. The Excluder was withdrawn from the market for structural problems, and a new modified
version is now clinically available and being evaluated. Other stent-grafts that obtained the CE mark were the
Endofit (Endomed Inc. Phoenix, AZ) and the Zenith (William Cook Europe Bjaev-erskov, Denmark) graft. Results recorded in a total of 11 patients treated in 2002 with the Endofit were disappointing. From 2003 the
Zenith and New Gore TAG devices are employed for patients undergoing an endovascular procedure for a
thoracic aortic disease, with encouraging results A new modular device from Zenith (the TX2) is also being investigated at our center as part of an FDA trial. Overall, more than 100 thoracic devices have been implanted at
our centre.
Endovascular treatment of aortic arch disease
R. Chiesa, G. Melissano, E. Civilini, L. Bertoglio, F. Setacci
Traditional repair of aortic arch aneurysms requires cardiopulmonary bypass, hypothermia and circulatory arrest. Endovascular repair is an attractive, less invasive alternative that may change our therapeutic approach. In
the last six years, we treated 36 patients for aortic arch pathology with an “off-pump” endovascular repair, consisting in a combined hybrid open surgical and endovascular approach. Endovascular treatment of aortic arch
pathology is feasible even in elderly patients, but challenging due to the involvement of supra-aortic vessels and
anatomical peculiarities of the arch. Further research is still required for the optimal surgical technique and
stent-graft, as well as prospective analysis of data collection at follow-up.
Optimal cerebral and myocardial protection during circulatory arrest for aortic surgery through left
R. Chiesa, G. Melissano, E. Civilini
Circulatory arrest and deep hypothermia (14-18 °C) is the preferred method to treat “unclampable” descending thoracic aneurysms that involve the distal aortic arch. In cooperation with the Cardiac Surgery department,
from 2003, in an effort to benefit from the advantages of mild (24-26 °C) hypothermic circulatory arrest with
cerebral protection, in patients with a thoracic aneurysm involving the distal arch operated through a left thoracothomy, we employ selective perfusion of the supra aortic trunks. Cardiac protection with cardioplegia is accomplished satisfactorily in spite of the inaccessibility of the ascending aorta from this route, employing an aortic occlusion/perfusion catheter. We achieve an excellent cerebral and myocardial protection during circulatory
arrest using only moderate hypothermia; this allows us to save time and avoid the disadvantages of deep hypothermia, especially coagulopathy. Prospective data collection and follow up of patients undergoing this innovative surgical technique are in progress.
Technical adjunct to increase safety in thoraco-abdominal aortic aneurysms (TAAA) surgery
R. Chiesa, G. Melissano, E. Civilini
Despite of recent advances surgery, for TAAA has still important complications, involving particularly the
spinal cord and the kidneys. The principal determinant of these complications is the transient ischemia caused
by aortic cross-clamping. The aim of this study is to evaluate the impact of technical adjuncts such as partial left
heart by-pass, cerebro-spinal fluid drainage, epidural analgesia, on the clinical outcome of these patients.
Frenotomy and one-lung ventilation are important in the development of postoperative respiratory complications. Different surgical and anaesthesiological techniques are also being evaluated for the prevention of respiratory complications.
The role of magnetic resonance angiography in endovascular treatment of thoracic aortic disease
R. Chiesa, G. Melissano, Y. Tshomba, E. Civilini, F. Setacci, L. Bertoglio
Many patients with thoracic aortic aneurysms have renal insufficiency and may be at increased risk when conventional imaging modalities (contrast-enhanced computed tomography and arteriography) are used for aortic
endograft design and follow up. To determine if magnetic resonance angiography (MRA) could be used as the
sole imaging modality for endoprosthetic design and follow up, a total of 30 patients selected for endovascular
grafting of thoracic aorta presenting renal insufficiency or a history of severe contrast reaction will be included
in the study. Data will be collected prospectively. Gadolinium-enhanced MRA will be used preoperatively in
place of conventional imaging. The control group will undergo conventional imaging. Intraoperative access;
unplanned proximal and distal extensions; conversion to open, aborted procedures; and intraoperative endoleaks will be compared in the MRA-designed and control groups. In the MRA-designed group the creatinine
level after endograft implantation will be assessed and the follow up evaluation with MRA will be compared to
Computed Tomography imaging.
Evaluation of a clinical flowchart for carotid endarterectomy
R. Chiesa, G. Melissano, R. Castellano, B. Catenaccio, S. Frigerio
Between 1992 and Jan 2005 we performed 6842 carotid endarterectomies. Last year a strict clinical protocol
has been implemented and is currently being evaluated: 1. Indications according to AHA guidelines; 2. Duplex
scan performed at a validated Lab; 3. Contrast angiography + CT in selected cases; 4. Echocardiography +
dobutamine if increased cardiac risk; 5. Loco-regional anesthesia; 6. Exposure extended to atheroma free ICA;
7. Systemic heparinization; 8. ICA clamped first and unclamped last; 9. Neurological monitorization during
cross clamping; 10. Selective shunting with Javid shunt; 11. Choice of surgical technique (patching, eversion endarterectomy); 12. Intraoperative completion ar-teriography; 13. Intraoperative stenting if distal flap is detected; 14. Availability of postoperative ICU, selective use; 15. Immediate surgical revision if cervical hematoma is
diagnosed; 16. Ascertain ICA patency with the quickest available diagnostic test if a new neurologic deficit is
detected postoperatively; 17. Relieve ICA thrombosis immediately; 18. Early discharge safe in uncomplicated
cases; 19. All patients followed; 20. Recurrent stenosis are treated only if symptomatic or very tight.
Role of inflammation in carotid atherosclerotic plaques
R. Chiesa, G. Melissano, R. Castellano
In cooperation with the CCVB (Director Prof. A. Maseri) and with other european institutions, this project
aims at providing a clinical database and biological samples from peripheral blood and carotid atheroma from
patients with surgically relevant atherosclerosis of the supraaortic trunks. The gathered data will help elucidating the role of inflammation, immunity and infective agents in the pathogenesis and progression of carotid
plaques and in the development of instable active plaques.
Efficacy of remifentanil conscious sedation during regional anaesthesia for carotid endarterectomy
R. Chiesa, M. Marrocco
Carotid endarterectomy (CEA) is a well-established technique for the prevention of stroke in selected patients. However, controversy persists concerning the optimal anaesthetic modality. Regional anaesthesia (RA)
has been advocated because in a conscious patient cerebral perfusion during carotid artery cross-clamping can
be monitored with mental status evaluation, avoiding the use of other monitoring techniques that yield a significant number of both false positive and negative results. On the other hand, during RA the patient’s co-operation and ability to remain still during the surgical procedure are required. In fact, the main objections to RA are
patient’s anxiety, fear, pain, discomfort, and awareness during surgery. In order to reduce anxiety and improve
analgesia, intravenous sedation with benzodiazepines and hypnotics has been used. However, these drugs may
impair the patient’s coherency and co-operation and if the patient becomes oversedated measures to control the
airway should be taken. Remifentanil is a potent µ-opioid agonist with an ultrashort half-life and duration of effect. If administered at low doses, it has no effect on haemodynamic variables, and in contrast to other opioid
analgesics it does not influence cerebral capacity. Preliminary data showed that Remifentanil as an adjunct to
RA for CEA provides adequate comfort and analgesia while preserving respiratory rate above the threshold of
respiratory depression and maintaining haemodynamic stability, and did not hamper mental status evaluation.
The aim of this study is to prove its efficacy by means of a prospective, randomized, placebo-controlled study.
In vivo magnetic resonance imaging characterization of complicated carotid plaques in patients with
symptomatic carotid stenosis
R. Chiesa, M. Marrocco, G. Melissano
The results of the ECST and NASCET trials showed that there was no benefit of carotid endarterectomy for
symptomatic carotid stenoses of <70%. However, there is a large stroke burden from stenoses that are currently not recommended for surgery according this criteria. Complicated plaques, defined by the presence of intraplaque hemorrhage, thrombus, and surface disruption, largely account for morbidity and mortality related to
atherosclerotic extracranial carotid disease and Magnetic Resonance Imaging (MRI) studies have recently focused on the characterization in vivo of these plaques considered at high-risk for cerebral ischemia. The aim of
our study is to validate the MRI criteria for complicated carotid plaque by means of a histological correlation
analysis and to assess the prevalence and the predictive significance of plaque characteristics in patients with
cerebral ischemic events, other than the degree of carotid stenosis.
Chronic venous insufficiency in italy: the 24-cities cohort study
R. Chiesa, E. M. Marone
Venous disease is very common in Italy, in particular in people living in the South. In cooperation with Alpha
5 (Switzerland) and Boehringer Ingelheim (Milan), under the auspices of the Italian society of Vascular Surgery
(SICVE), to define epidemiology of varicose veins in an Italian sample, we started a cross-sectional population
study in 24 cities in the north, center and south of Italy. A spontaneous sample of 5,247 people were selected
during spring and summer 2003 and selection of another sample of people during spring and summer 2004 is in
progress by advertising in television, newspapers, and leaflets in the urban areas. Results are collected by mean
of a questionnaire on subjective symptoms of chronic venous insufficiency in the lower limbs, and clinical examination, including color-coded duplex ultrasonography to determine the presence and severity of varicose
So far, in the group of people selected in 2003, only 22.7% of the subjects examined were free of visible signs
of disease, with approximately 53% of the population over 50 years of age showing some disturbances in the reflux. Correlation between varicose veins and venous incompetence is more marked in men, while minor objective and subjective symptoms prevail in women. Finding from this non-random sample closely matches results
from previous studies, in which the sampling was random. Results from 2004 sample of people are in progress.
A novel biochemical diagnostic method for diagnosis of aortic dissection
R. Chiesa, G. Esposito, L. Bertoglio, F. Calliari
Multicenter study, in cooperation with the Istituto Scientifico Biomedico Euro-Mediterraneo (ISBEM), to
evaluate diagnostic value of blood samples in patients admitted to E.R. for suspected or confirmed acute aortic
dissection (AAD). AAD is the most frequent catastrophic event involving the aorta. Without treatment, 33% of
patients die within 24 hours, 50% within 48 hours and 75% of patients with undiagnosed aortic dissection die
within 2 weeks. Even with common signs and symptoms of AAD that pathology is difficult to evaluate. Nowadays AAD is diagnosed with TEE, Ct-scan and MRI. In this study on plasma will be determined miosine chains
(MCH) relieve from muscular wall of vessels in blood. Previous studies reported MCH increase in patients with
aortic dissection. Aim of this study is to determine the sensibility of MCH serum level for AAD diagnosis.
Chlamydia Pneumoniae and aortic dissection
R. Chiesa, G. Esposito, L. Bertoglio, F. Calliari
International, multicenter prospective study, in cooperation with Prof. Luigi Allegra (Institute of Respiratory
disease, University of Milan, IRCCS, Ospedale Maggiore, Milan) and Prof. A. Distante (National Research
Council, Insitute of Clinical Physology – Lecce). Aortic dissection is an acute disease (AAD) with high mortali-
ty and morbidity. Current body of evidence establishes Clamydiae Pneumoniae (CP) as a plausible, emerging
and potentially and modifiable risk factor in cardiovascular disease. However its role in ADD remains still unknown and to be demonstrated. Patients with AAD type A and type B will be enrolled. Aims of the study are: a)
Investigate if CP is present in the wall of patients with AAD. b) To investigate the relation between immune response to CP and detection of CP in the wall of patients with AAD.
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Role of cellular death, epidermal growth factor, vascular growth factor and immunity response cells in the
chemoradio resistance in stage III rectal cancer
A. M. Tamburini, E. Orsenigo, S. Di Palo, A. A. Manfredi, V. Russo, L. Albarello, C. Doglioni, C. Staudacher
In the last decade we faced an improvement of the preoperative chemo-radiotherapy for stage III rectal cancer. In our series we showed an effective down-staging after this treatment in about 80% of patients. The others
were no responders despite the introduction of new chemotherapic regimens. The survival and disease-free
curves of down-staged patients were similar to no-treated patients (stage I and II), so we decided to set up a
study to explain the resistance to adjuvant treatment to cellular death induced by the treatment either in the expression of growth factors or in immunity response. In one year we will recruit 80 patient with cancer of lower
or mid rectum at stage III and analyze their serum and bioptic specimens for death cell factors, EGF, EGFR,
VEGF-A, VEGF-C, COX2, microsatellite instability, immunitary response cells. These analyses will be performed after surgical treatment and in the oncologic follow up for two years. The aim of this study is to identify
factors inducing tumour resistance, in order to discover no responders patients before treatment and add immunological treatments to allow response to the treatment.
Long-term survival after kidney and kidney-pancreas transplantation in diabetic patients (IDDM)
E. Orsenigo, C. Socci, E. La Rocca, P. Maffi, A. Secchi, V. Di Carlo, C. Staudacher
Aim: to evaluate the benefits of kidney and kidney-pancreas transplantation for diabetic patients with end
stage renal disease. Among 270 renal transplants performed from 1985 to 2002, 204 (75%) were diabetic patients. In these patients, in 161 cases (60%) the kidney has been transplanted simultaneously with a pancreatic
graft (SKPT). The mean time on dialysis was 31 +/- 21 months (range 0 to 126) and the mean duration of diabetes was 24 +/- 7 years (range 5 to 55). 99% of the patients were on renal replacement therapy. Both patient
and kidney graft survival rates were worse in diabetics. Patient survival was 82% at 5 years among patients undergoing SKPT, 60% in diabetics receiving kidney alone transplant, and 88% in non-diabetic transplanted patients. Kidney graft survival at 5 years was 77% in diabetics receiving SKPT, 68% in diabetics receiving kidney
alone transplant, and 82% in non-diabetic patients. Overall survival was significantly higher in non-diabetics (p
=0.002) or in diabetics who received SKPT when compared to diabetics receiving kidney alone transplant (p
=.001). This study confirms that pancreas and kidney transplantation is the treatment of choice for diabetic patients with end-stage renal disease.
Could laparoscopic colon and rectal surgery become the gold standard? A review and experience with 599
S. Di Palo, A. Vignali, A. M. Tamburini, E. Orsenigo, C. Staudacher
The aim of this study was to analyse our experience in laparoscopic colorectal surgery and evaluate complications rate and oncologic impact. We considered 599 consecutive unselected patients who underwent laparoscopic operation for cancer from 1998 to 2004. Mean age was 65.8 ±11.7 years. The following operations were
performed: 248 left colectomies, 131 right colectomies, 26 sigmoid resections, 164 rectal resections, 21 abdominal resection and 9 total colectomies. Conversion rate was 7,2%. The overall morbidity rate was 23,3%. The
mortality rate was 0,3%. Anastomotic leak occurred in 7,3% patients. Re-operation rate was 4,6%. Mean number of lymph-nodes collected was 16,7 ±9.8 Median time of follow up period was 20.2 months. One port-site
metastasis was found at 18 months after surgery. Overall five years survival was 81%. In conclusion, laparoscopic colectomy can be considered a surgical procedure with rates of complications similar to traditional surgery. From an oncologic point of view it can be considered a valid procedure, though a median follow up not
yet completed, with a survival rate similar to traditional surgery. These results lead us to propose a perspective
randomized trial on hand assisted laparoscopy aimed to reduce postoperative complications and to improve patients’ outcome.
Prominent tumour-infiltrating lymphocites improved disease free survival in early stage gastric carcinoma
A. M. Tamburini, E. Orsenigo, L. Albarello, V. Tomajer, C. Doglioni, C. Staudacher
Previous studies have identified high levels of tumour infiltrating lymphocytes (TILs) to be associated with
good prognosis in various types of cancer. On the basis of this statement we analyzed the value of intraepithelial
(IEL), peritumoral (PTL) and Crohn’s-like (CRL) lymphoid aggregates in predicting the outcome of
T1/T2N0M0 (R0) gastric cancer. Specimens were taken from 44 pts (24 m-20 w, mean age 63 ±12.39).The median follow-up was 48±32 months. Local relapse was observed in 6 patients (13%. Patients with high levels of
IEL, and CRL had a better clinical course, with significantly higher 5-year disease free survival (p=0.0003 and
p=0.005, respectively). No PTL and CRL infiltration was observed in patients with recurrence. This preliminary study suggests that TILs may be useful as predictors of patient survival in surgically treated T1,T2N0M0
gastric cancer. Based on preliminary results we will continue our trial with 150 patients with early stage gastric
cancer, in association with bio-molecular analysis so to identify clusters of patients at risk of recurrence though
an early stage of disease, allowing us to identify adjuvant therapy in patients at risk.
Laparoscopic gastric resection with extended lymph node dissection for gastric cancer
E. Orsenigo, R. Castoldi, M. Carlucci, A. M. Tamburini, V. Tomajer, C. Staudacher
BACKGROUND: Laparoscopic surgery has been used in the treatment of gastric cancer with low mortality
and morbidity and improvement in patients’s quality of life. AIM: to evaluate the results of laparoscopic gastric
resection. AIM: to evaluate the feasibility and safety of minimally invasive techniques in the treatment of gastric
cancer. METHODS: A retrospective review of 59 patients after laparoscopic surgery for gastric cancer was performed. The patients were 31 males and 28 females with a mean age of 67 (±11) years (min 39, max 90). RESULTS: Tumour stage was IA in 15 patients, IB in 10, II in 9, IIIA in 6, IIIB in 9, and IV in 10. In 15 cases the
tumour was an early gastric cancer. The mean number of dissected lymph nodes was 29 +/-10. Conversion rate
was 16%. Morbidity rate was 37%. The median length of hospital stay was 10 days. Operative mortality was
3%. The mean time of follow-up was 23 months. Two-year survival was 75%. CONCLUSIONS: Laparoscopic
radical total or subtotal gastrectomy with extended lymphadenectomy for gastric cancer is a feasible, safe, and
oncologically effective procedure. The learning curve has been concluded. We are planning a randomised study
comparing laparoscopic gastric resection with open surgery.
Relationships between lymph node metastases and gastric cancer progression: experience on more than
1000 cases treated at a single western centre
E. Orsenigo, M. Carlucci, M. Braga, V. Tomajer, V. Zuber, V. Di Carlo, C. Staudacher
The benefits of extended lymphadectomy for gastric carcinoma are still debated. In order to evaluate the relationships between lymph node metastases and gastric cancer progression, we reviewed 1074 hospital records
of patients who underwent curative resection for gastric cancer from 1980 to 2004. The surgical procedure consisted of 289 total and 785 subtotal gastrectomies. The patients were analysed by univariate analysis to predict
implications of lymph node metastasis on survival. The extent of lymph-node dissection was limited D1 376 or
extended D2 578 and D3 12; no lymphadenectomy was performed in 108 cases. The pathological N status has
been defined in this way: N1<6 nodes; N2: 7-15 nodes; N3> 15 nodes. The distribution of N stage was:
N0=278, N1=344; N2=215; N3=129. No lymph nodes have been retrieved in 108. The median number of
lymph nodes was 17. In the univariate analysis all were found to be significant factors. In the multivariate analysis T stage, N stage and extent of lymphadenectomy were all independent predictors. The mean time of survival
was 111, 90, 47, and 5 months in N0, N1, N2, and N3 stage, respectively. Overall survival at 5 years was 80%,
53%, 24%, and 0% in N0, N1, N2, and N3 stage, respectively (p<0.001). In conclusion, the stage of node
metastases is well correlated with tumour progression.
Sexual dysfunction as a complication of abdominoperineal resection for carcinoma of the rectum
C. Staudacher, F. Montorsi, A. Vignali, P. Baccari, E. Orsenigo, S. Di Palo, N. Suardi, A. Salonia
Urinary and sexual dysfunctions are common complications after rectal cancer surgery, and this is more likely
due to damage of pelvic autonomic nerves during surgery. In a number of cases such lesions are necessary as
bulky masses clearance is not possible without pelvic nerves injury. Besides neoplasm evolution, patients suffer
a clear worsening in their quality of life. Efforts have been made to reduce the incidence of such complications
and adjuvant radiotherapy and chemotherapy have increased the likelihood of sphincter preservation though
accomplishing radical surgery (R0). In this study we evaluated 20 patients twice in a year and we are going to
continue for further 24 months.
Laparoscopic pancreatic resection: preliminary experience
E. Orsenigo, M. Carlucci, A. M. Tamburini, T. Casiraghi, S. Di Palo, C. Staudacher
The aim of our study was to evaluate the results of laparoscopic pancreatectomy for pancreatic tumours. Four
women and three men underwent laparoscopic pancreatectomy from June 2002 to February 2004. Pancreaticoduodenectomy (n=4), intermediate pancreatectomy (n=1) and distal pancreatic resection with splenectomy
(n=2) were successfully performed: operative mortality was nil. The postoperative morbidity included two low
output pancreatic leaks. The mean operating time, blood loss and hospital stay was 342 minutes, 289 mL and
14 days, respectively. Pathological diagnosis were ductal adenocarcinoma in one, neuroendocrine tumour in
five and metastatic melanoma in one. All patients remain well at median follow up of 10 months (range 1-20).
Patients appear to benefit from laparoscopic pancreatectomy for pancreatic tumours. Minimally invasive approach ensures an adequate treatment despite it requires the expertise of highly skilled laparoscopic surgeons.
The effect of margin involvement on outcome after pancreatic cancer resection
G. Balzano, A. Zerbi, S. Rocchetti, A. A. Beneduce. V. Di Carlo
In this retrospective study we assessed: 1. the incidence of margins involvement after pancreatic cancer resection 2. the pattern of failure after non-radical resection, 3. the value of Intraoperative Radiotherapy (IORT) in
this setting, 4. the effect of margins invasion on survival. Patients were classified according to the UICC-R classification: R0: no margins involvement; R1: microscopic involvement, R2: macroscopic residue. 175 out of 296
pts (59%) underwent R0 resection, 68 pts (23%) and 53 pts (18%) underwent R1 and R2 resection, respectively. Local relapse (LR) was detected in 40% of cases after R0 resection, 39% after R1 and 55% after R2 (p=0.4).
IORT was applied in 127 pts: it was not effective to reduce LR neither after R0 resections (42% vs 40% p=0.9),
nor after R1 or R2 resection (33% vs 53 p=0.3 and 55% vs 55%, p=1) The microscopic margin involvement
did not significantly influence survival, with respect to patients with radical resection (p=0.2), whereas patients
with a macroscopic residue had a worse postoperative survival (p<0.001). Two- and five-years survival rates
were 38.6% and 14.8% in R0 patients, 32.7% and 8.6%, in R1 patients; 13.9% and 6.9% in R2 patients.
Endocrine pancreatic tumours
A. Zerbi, G. Balzano, V. Capitanio, F. Gavazzi, V. Di Carlo
These are heterogeneous neoplasms, presenting with a wide spectrum of symptoms as well as a wide spectrum of prognosis. To improve our knowledge in this setting, we have ideated a prospective, multicenter Italian
study, started in 2004. Aims of this study are to describe the prevalence of endocrine pancreatic tumours in
Italy and to define their natural history. Furthermore we have recently analysed the results of our treatment of
89 patients with insulinoma. According to WHO classification, 67% of tumors were classified as benign, 21%
as uncertain behaviour, 6% as well differentiated carcinoma. 5 patients had MEN-1. Preoperative imaging
identified 95% of insulinomas. Endoscopic ultrasonography and MR showed the higher sensitivity (87.5% and
80%%), followed by ultrasounds (72.2%), angiography (63.5%) and CT scan (53.4%). Intraoperative handling and ultrasounds allowed to identify 2/4 occult insulinomas. Surgical procedures were: 54 enucleations, 22
distal pancreatectomies, 7 median pancreatectomies, 6 pancreatoduodenectomies. No mortality was observed.
Morbidity was 36,3%. No recurrence of symptoms was observed in patients with benign disease.
The management of a difficult pancreatic stump during pancreaticoduodenctomy
G. Balzano, A. Zerbi, M. Cristallo, M. Frasson, V. Di Carlo
Pancreatic fistula is the most feared complication after pancreatoduodenctomy (PD), especially when the
pancreatic stump is frail. To identify a technique that could reduce the fistula’s rate in case of a difficult pancreatic stump, we performed a retrospective analysis of 285 patients, operated between 2000 and 2004. 68 patients
with soft parenchyma and non-dilated duct were identified. Three techniques were applied: A. standard reconstruction (pancreatic, biliary and duodenal anastomoses on the same loop) (22 patients); B. pancreato-jejunostomy on a separate Roux-en-Y loop with external Wirsung stenting (30 patients); C. duct occlusion with prolamine without anastomosis (16 patients). Pancreatic fistula rate was 41% in group A, 46% in group B and
62% in group C (p=0.3). The severity of fistula was measured by the fistula-related mortality and relaparotomy
rates: in different groups they were: 22% and 33% (A); 0% and 0% (B); 10% and 30% (C). The reduction of
major complications in group B was significant (p<0.01). From these data we can conclude that in case of difficult stump, the Roux-en-Y anastomosis with external stenting may reduce the sequelae of an anastomotic leak.
Combined morphological and metabolic studies using Computed Tomography and Positron Emission
Tomography (CT-PET) of pancreatic carcinoma
L. Piemonti, F. Fazio, C. Messa, A. Zerbi, G. Balzano, G. Perseghin, M. Picchio, C. Doglioni, V. Di Carlo
Aim: to acquire functional data on tumor and general metabolism in vivo in patients with pancreatic cancer
using Positron Emission Tomography (PET) and indirect calorimetry. We are studing in vivo: 1) energy homeostasis by means of indirect calorimetry, 2) hormonal profile (insulin, c-peptide, proinsulin, glucagon, growth
hormone, IGF-1, ghrelin, cortisol) with a special emphasis on the endocrine functions of the adipose tissue
(leptin, adiponectin, alpha-TNF and circulating alpha-TNF receptor II), 3) circulating metabolites, 4)CT-PET
imaging using [18F]FDG. PET data quantification (SUV = standardize uptake value) will be correlated to
serum data (exploring energetic homeostasis, lipocytes function, energetic metabolism and immunological parameters) and histological data (providing information on tumor proliferation (MIB), apoptosis (DNA fragments) and neoangiogenesis (VEGF). This should allow us to provide: 1) a better understanding of the molecular basis underlying the mechanisms of tracers uptake, such as [18F]FDG, in pancreatic tumors 2) add possible
biologic predictors of therapeutic response.
Pancreatic development and ductal pancreatic cancer
L. Piemonti, A. Mercalli, V. Sordi, V. Lampasona, C. Doglioni, L. Albarello, E. Bonifacio, A. Scarpa, V. Di Carlo
Objective: to understand whether pancreatic ductal cancer is a cancer of pancreatic stem cell. Summary: We
are studing in vitro the expression of transcriptional factor involved in pancreas development in ductal pancreatic cancer cell. This should permit to examine the expression of candidate transcription factors in cellular lines
and to understand whether they are differentially expressed. This is relevant to provide new markers for classification of the lines and to identify in which stage of the pancreas development the line are blocked. After this
first step (characterization) we will study the possibility to modify the stage of differentiation by exposure to
soluble factors playing a role in pancreatic development. This should permit to identify factor/s and pathways
able to modulate the differentiation of pancreatic precursor.
Pancreatic cancer treatment
M. Reni, G. Balzano, A. Zerbi, E. Villa, V. Di Carlo
Most pancreatic cancer patients are not resectable at diagnosis. Our recent efforts were mainly directed to
identify more effective chemotherapeutic treatments for these patients. We compared two regimens in a multicenter randomized study. Patients with advanced cancer were randomized to receive either cisplatin and epirubicin 40 mg/m2 on day 1, gemcitabine 600 mg/m2 on days 1 and 8 and 5-FU as continuous infusion 200
mg/m2/day on days 1 to 28 of 4-week cycles (PEFG regimen, arm A) or gemcitabine 1 g/m2 for 7 of 8 consecutive weeks in cycle 1 and for 3 of 4 weeks thereafter (arm B). 104 patients were enrolled. Outcomes for patients in arm A and B, respectively were as follows: 4 months progression-free survival 60% and 28% (P=.003),
1-yr overall survival 38.5% and 21% (P=.05), response rate 40% and 8.5% (P<.001), and clinical benefit 65%
and 25% (P=.01). We concluded that PEFG improved outcome when compared to gemcitabine. The same
regimen (PEFG), followed by external RT, was tested in an adjuvant setting in 51 patients who underwent cancer resection. Our results were encouraging, with a one-yr failure free survival of 67+7% and two-yr overall survival of 53+7%.
The problem of fistula after left pancreatectomy
G. Balzano, A. Zerbi, M. Cristallo, E. Ortolano, V. Di Carlo
Aim of the study was to identify factors related to the onset of pancreatic fistula after left pancreatectomy
(LP). 123 patients underwent LP since 1996. Pancreatic closure was made by a hand-sewn technique (39 pts)
or two kinds of staplers: proximate (46pts) and Endo-GIA: (38 pts). In case of fistula, drain removal was scheduled at a daily output <5 ml. Mortality was 0%, morbidity was 48%, pancreatic fistula rate was 34%. Fistula
rate was 38% after hand-sewn closure, (39%) proximate (NS). None of the further factors, after Endo-GIA
and 26% after Proximate (separate duct ligation, hand-sewn suture in addition to stapler, spleen preservation,
use of pledgetted suture, sex, age, indication for surgery), proved to reduce the fistula rate. All fistulas healed
spontaneously. Mean fistula duration was 36 days; 92.8% of patients with fistula were discharged with drain.
The policy of delayed drain removal allowed a low-rate of fistula associated morbidity (16%) and of readmission (4.7%). In conclusion, fistula is an unsolved problem of LP. However, a careful drain management allows a
good outcome in patients with fistula.
Laparoscopic gastric banding for morbid obesity
M. Paganelli, G. Ferla
Laparoscopic Adjustable Gastric Banding (LAGB) is performed in case of primary obesity with Body Mass
Index (BMI) >40 or >35 with concomitant serious medical obesity-related conditions, unresponsive to dietary
treatment. Preoperative evaluation included screening for endocrine disease, endoscopic and radiological study
of the upper digestive tract and ultrasound study of the upper abdomen. The efficacy of the procedure was analyzed in terms of percentage of excess body weight loss. Between June 1996 and December 2003, 291 patients
(50 males/241 females, ranging 18-65 years) received LAGB. Mean body weight was 116.6 ± 19.3 Kg (range 90195 Kg), with a BMI ranging between 35 and 65.7 (mean 43.5 ± 6.0). The laparoscopic procedure was completed in 282 patients (97.2 %). Eight cases (2.8%) had to be converted to the laparotomic procedure either because of hepatomegaly (1 case) or because of gastric lesion during the laparoscopic approach (7 cases). During
the follow up period (53,9 ± 24.3 months), percentage of excess body weight loss was 15.7 ± 8.3 after 1 month,
25.6 ± 11.6 after 3 months, 39.4 ± 13.9 after 6 months, and 63.3 ± 1.7 after 1 year.
Role of neoadjuvant chemotherapy on hepatic resections for metastases from colorectal cancer
M. Arru, F. Milani, L. Aldrighetti, M. Ronzoni, M. Catena, R. Finazzi, G. Ferla
Liver resection is the first treatment for patients (pts) with metastases from colorectal cancer and chemotherapy (CT) can increase resectability rate. The impact of preoperative CT on the outcome of liver resections has
been evaluated. 61 liver resection for colorectal metastases performed in 57 pts divided in two groups: 22 pts
(Ch+ group) treated with preliminary CT and subsequent liver resection; 39 pts (Ch- group) treated only with
liver resection. No significant differences were observed in postoperative course except for the length of hospitalization, shorter in Ch+ group (median 8 vs 11 days; p=.002). No significant differences were noticed in median disease free survival (15 vs 13 months; p=.501), disease free survival at 1 and 3 years (54% and 28% vs
49% and 17%), median overall survival (32 vs 30 months; p=.373) and overall survival rate at 1 and 3 years
(93% and 29% vs 88% and 45%). In pts with unresectable disease at first observation, neoadjuvant CT allows
a surgical treatment with disease free and overall survival rates similar to those of the pts treated immediately
with resection.
Impact of advanced age on the outcome of liver resections
L. Aldrighetti, M. Arru, M. Catena, R. Finazzi, F. Milani, C. Pulitanò, G. Ferla
A total of 155 consecutive liver resections was divided into two groups: ≥75 years old [old group (O-group)]
and < 75 years old [young group (Y-group)].The outcome of liver resections was evaluated in terms of postop-
erative mortality, morbidity, transfusions, and length of hospitalization. The Y-group included 133 resections in
131 patients, aged 57.7 ± 11.0 years (mean ± SD; range: 23–74 years), and the O-group included 22 resections
in 22 patients, aged 77.8±1.9 years (mean ± SD; range: 75–82 years). The O-group included more hepatomas
(45.4% versus 26.3%, p=0.11), chronic liver (31.8% versus 29.3%, p=0.4) and cardiovascular diseases (22.7%
versus 3.7%, p=0.004). As regards the postoperative outcome, the length of hospitalization and the need for
postoperative transfusions were not statistically different. The 30-day overall mortality rate was 1.9%, with
2.3% in the Y-Group and none in the O-Group. Postoperative morbidity was higher in Y-group than in Ogroup (29.2% versus 4.5%, p = 0.03). In conclusion, the age factor itself should not be a contraindication to
liver resection, but it entails more stringent selection of the elderly patient to be considered for surgery.
Liver resection for hepatocellular carcinoma (HCC) in elderly cirrhotic patients
M. Arru, L. Aldrighetti, M. Catena, R. Finazzi, C. Pulitanò, F. Milani, G. Ferla
Sixty-two liver resections for HCC in 60 cirrhotic patients (pts) were divided into: Elderly group (E-gr) including 27 pts aged between 70 and 83 years (mean±DS: 74,6±3,8), and Young group (Y-gr) including 35 pts
aged between 36 and 69 years (mean±DS: 59,7±8,7). The analysis of the variables potentially affecting the perioperative course revealed a significant difference in concomitant diseases (55,5% in E-gr vs 28,6% in Y-gr,
p=0,032) and length of portal clamping [median 23 (range 10-52) minutes in E-gr vs 31 (range 12-82) minutes
in Y-gr, p=0,013]. Surgical complications were more frequent in Y-gr (41,2% vs 11,1%, p=0,005). No significant differences were recorded in cardiopulmonary postoperative complications. Mortality included one patient among elderly pts and two pts among young group with an overall mortality of 4,8%. No significant differences were recorded in postoperative transfusions and in length of postoperative hospitalization. In conclusion liver resection for HCC in cirrhosis has to be considered a relatively safe procedure even in elderly patients
as long as surgery is preceded by an accurate selection of the patient.
Benefits of preoperative corticosteroid administration on the hepatic ischemia-reperfusion injury and
cytokine response in patients undergoing hepatic resection
L. Aldrighetti, C. Pulitanò, M. Arru, R. Finazzi, M. Catena, G. Ferla
Preoperative steroid administration has been advocated to reduce ischemia-reperfusion (I/R) injury and surgical stress following hepatic resection. 43 patients undergoing liver resection were assigned to a steroid group
(MP group) or to a control group (C group), patients in MP group received 500 mg of methylprednisolone just
before surgery whereas those in C group did not. Postoperative serum levels of ALT, AST, bilirubin, were significantly lower in the MP group at postoperative day (POD) 1, POD2 and POD5. Values of inflammatory cytokines as IL-6 and TNF-alpha were significantly lower compared to C group. The incidence of postoperative
complications in the C group was higher than in the MP group (p .010). In conclusion, preoperative administration of methylprednisolone resulted in significantly lower hepatic enzyme elevation and inflammatory cytokine levels following hepatic resection, suggesting that steroid pre-treatment represents a potentially biologic
modifier of I/R injury and may improve the postoperative outcome.
Inihibition of cytokine response by methylprednisolone attenuates Antithrombin III (AT-III) reduction
following hepatic resection
C. Pulitanò, L. Aldrighetti, R. Finazzi, M. Arru, M. Catena, G. Ferla
Ischemia reperfusion (I/R) injury is a significant complication of hepatic resection. Effects of preoperative
glucocorticoids on the coagulant/anticoagulant system in pts undergoing liver resection are still unclear. To assess the value of intravenous steroids pulse (500mg of methylprednisolone just before surgery) on hepatic I/R
injury, 32 consecutive pts undergoing major hepatic resection under intermittent hepatic ischemia were classified into 2 groups: a control group (n=16) and a steroid group (n=16). Postoperative levels of AT-III were significantly lower in the control group (ANOVA P<.01). AT-III levels were negatively correlated with IL-6 levels,
lower in the steroid group on POD 1 (P=0.001), 2 (P=0.001), and 5 (P=0.001). Similarly, postoperative levels of
TNF-alpha in the steroid group were lower on POD 1 (P=0.034), 2 (P= 0.006) and 5 (P=0.010). Methylprednisolone effectively inhibited the postoperative increases in TNF-alpha and IL-6, limiting the decrease in ATIII. AT-III may have a significant role in the prophylaxis of surgical complications whereas cytokine elevation,
reperfusion injury and thrombosis contribute to the morbidity of the surgical procedure.
The role of hepatic resection in the treatment of non-endemic hepatolithiasis
M. Catena, L. Aldrighetti, M. Arru, R. Finazzi, G. Arzu, G. Ferla
The safety and the efficacy of hepatic resective surgery in the treatment of single lobe hepatolithiasis has been
analysed by retrospective comparison between hepatic resections in patients (pts) with hepatolithiasis [Hepatolithiasis Group (HG)] and liver masses [Control Group (CG)]. Fourteen consecutive pts with hepatolithiasis
(HG) and 18 pts with liver masses without chronic liver disease (CG) underwent major hepatic resection during a 4-year period. Intraoperative blood losses in CG was higher than in HG (1047±650 ml vs 570±489 ml,
p=0.03). The other variables considered were not statistically different. No mortality was registered. Postoperative complications occurred in 5 pts (15.6%) in HG and 4 pts (12.5%) in the CG, p=0.12. Difference in postoperative transfusions and median hospitalization was not significant. Histopathology showed cholangiocarcinoma in 2 cases (14.2%). One patient had lithiasis recurrence and 1 patient died for the coexisting cholangiocarcinoma. In conclusion, hepatic resection is the treatment of choice in pts with single lobe hepatolithiasis; an
early indication for surgery may reduce the mortality/morbidity rates.
Prognostic factors of long-term outcome of hepatic resection for colorectal liver metastases
L. Aldrighetti, R. Castoldi, S. Di Palo, M. Arru, M. Stella, E. Orsenigo, G. Ferla, V. Di Carlo, C. Staudacher
Prognostic factors of 297 liver resections for colorectal metastases were retrospectively analysed. The 1, 3, 5
and 10-yr overall survival rates were 90.6%, 51.0%, 27.5% and 16.9%, respectively. The univariate analysis revealed a statistical difference in 5-yr overall survival in pts with high differentiation grade of primary cancer
(30.7% in G1-G2 vs 14.4% in G3-G4, p=.0016), high preoperative CEA level (CEA<5ng/ml vs >5ng/ml:
51.1% vs 15.5%, p=.0016; CEA<200ng/ml vs CEA>200ng/ml: 27.9% vs 17.4, p=.0001), diameter of major lesion (30.0% in <5cm vs 18.8% in >5cm, p=.0074), time between primary tumor and metastases (<12 months vs
>12 months: 23.0% vs 36.1, p=.042), high MSKCC-CRS (0-1-2 vs 3-4-5 class: 36.4% vs 16.3%, p=.017). The
multivariate analysis showed 3 independent negative prognostic factors: G3-G4 differentiation, CEA level
>5ng/mL, and high MSKCC-CRS class. No single prognostic factor was found to identify patients to be excluded from resection. However, in the presence of specific prognostic factors, enrolment of patients in trials
exploring new diagnostic tools and adjuvant treatments may improve the pre-operative staging and reduce the
tumor recurrence after resection.
Fetal DNA in maternal plasma: physiological aspects of fetomaternal transfer
A. Ferrari, M. Smid, D. Gambini, L. Valsecchi, L. Cremonesi, S. Galbiati, M. Ferrari
We are collaborating with the Unit of Genomics for Diagnosis of Human Pathologies on the project: Fetal
DNA in maternal plasma: physiological aspects of fetomaternal transfer. An abnormal placentation has often
been found to be involved in the pathogenesis of intra uterine growth restriction (IUGR and preeclampsia. Our
aim was to evaluate whether IUGR is associated with high levels of fetal DNA in maternal circulation, similarly
to preeclampsia, and whether fetal DNA is related to altered uterine and/or umbilical artery Doppler velocimetry. Our study showed that fetal DNA content was significantly elevated in IUGR pregnancies similarly to
preeclampsia and correlated with altered umbilical Doppler velocimetry while no correlation was found with
uterine Doppler status. High fetal DNA levels in maternal plasma may be indicative of placental or fetal pathology even in absence of abnormal uterine Doppler velocimetry and may help in establishing a more precise diagnostic evaluation. If elevated fetal DNA in IUGR pregnancies correlates with abnormal umbilical Doppler velocimetry, fetal DNA release is associated more with fetal hypoxia than with fetal size.
“Three-dimensional reconstruction of human corneas by tissue engineering” 6th Framework Programme
EU NMP2-CT-2003-504017
P. Rama, S. Matuska, G. Paganoni, A. Spinelli, M. Viganò
The goal of the research project is to reconstruct a human cornea in vitro, for use both in corneal grafting and
as an alternative to animal models for cosmeto-pharmacotoxicity testing. The originality of the project lies in
the use of recombinant human extracellular matrix proteins to build a nano-engineered scaffold to support
growth of the different cell types found in the cornea, cells to be derived from human adult stem cells. The development of a reconstructed human cornea with cells of the recipient (autologous) will represent a real breakthrough, allowing diseased or damaged corneas to be replaced by tissue-engineered human corneal equivalents
without risk of rejection. This tissue engineered corneas will provide also a non-animal alternative for cosmetopharmacotoxicity testing which will therefore alleviate animal suffering. The project started in January 2004.
Six patients affected by limbal stem cell deficiency have already been operated on at San Raffaele Hospital. The
procedure was a reconstruction of the limbus (site of the corneal epithelial stem cells) by means of transplantation of autologous stem cells (from the fellow eye), expanded in vitro.
Pharmacologic therapy studies 1) Parallel Group Study of long-term safety of Travoprost 0.0015% and
Travoprost 0.004% eyedrops compared to timolol 0.5% eyedrops in patients with open angle glaucoma or
ocular hypertension
R. Carassa, P. Bettin, M. Fiori, R. Brancato
An increased intraocular pressure is the main risk factor for developing glaucoma, which is thus treated with
hypotensive topical drugs. In order to better control the disease, new highly effective and tolerated molecules
are tested. Prostaglandin analogues were introduced few years ago and showed high efficacy in reducing intraocular pressure with few local side effects. The Glaucoma Service was involved in major prostaglandin analogue multicenter phase three studies, and concluded in November 2004 a 3-year extension research aimed at
evaluating safety and efficacy of Travoprost which has been introduced in the market in 2002. Data will be soon
available and published.
Pharmacologic therapy studies 2) The effect of Latanoprost and Timolol on the blood retinal barrier in
eyes undergoing phacoemulsification and intraocular lens implant: an optical coherent tomography study.
R. Carassa, C. Ciampi, P. Bettin, L. Pierro, M. Fiori, R. Brancato
Latanoprost is the first choice hypotensive agent in glaucoma due to its high efficacy and tolerability. As a
prostaglandin analogue it might affect blood retinal barrier especially in operated eyes. Experimental and clinical studies using fluorangiography did not showed any clinically significant effect on the retina. Optical coherent tomography (OCT) is a non invasive new imaging technique capable of producing histological-resolution
pictures of the retina. In 2003-2004 we conducted a clinical study aimed at evaluating subclinical effects of
blood retinal barrier disruption by using this new technology. Patients scheduled for cataract surgery and under
latanoprost therapy were randomized to keep their therapy or to switch to the beta-blocker timolol. The results,
presented at ARVO meeting in USA, clearly showed the safety of Latanoprost therapy in cataract operated
eyes, confirming that the drug needs not to be withdrawn in case of uneventful anterior segment surgery.
Pharmacologic therapy studies 3) A 48-month, multicenter, randomized, double masked, placebo
controlled clinical study to evaluate the effectiveness and safety of oral memantine in daily doses of 20 mg
and 10 mg in patients with chronic open angle glaucoma at risk for glaucomatous progression
R. Carassa, P. Bettin, M. Fiori, C. Ciampi, R. Brancato
The glaucoma service is involved in this pioneering multicenter study which is first addressing the possibility
of neuroprotection in the treatment of glaucoma. The study is evaluating the memantine molecule (an NMDA
receptor antagonist) which has already been shown to be effective as a neuroprotectant in different degenera-
tive neurological disorders. One-thousand-fifty patients will be recruited in 50 Hospitals worldwide, and will
be randomized in 3 groups (placebo, memantine 20 mg and 10 mg) and will be followed for 48 months. If memantine will show a protective activity on the retinal ganglion cells in glaucoma, a totally new approach in the
management of the disease will arise. We recruited 4 patients that are at their 3rd-year follow-up without significant side-effects. The study will end in 2006.
Surgical therapy studies 1) A morphological and histological study on the mechanism of action of
E. Tamm, R. Carassa, D. Albert, B. Gabelt, S. Patel, C. Rasmussen, P. Kaufman
The glaucoma service conducted together with the University of Madison (USA) and the University of Erlangen (Germany) an original experimental study aimed at evaluating the mechanism of action of viscocanalostomy. Eight rhesus monkeys were operated with viscocanalostomy. At 2 months, 4 eyes were perfused with gold
tracer and all animals were sacrificed. All eyes were then analized by optic and electronic microscopy. Results
showed morphological changes at all aqueous outflow structures: at Schlemm’s Canal level patent micro-openings both facing the sclera and the trabecular meshwork were detected. The original study was presented at major international meetings and published on the Archives of Ophthalmology journal.
Surgical therapy studies 2) Viscocanalostomy in uveitic patients
E. Miserocchi, R. Carassa, P. Bettin, R. Brancato
Management of uveitic glaucoma is very challenging and often requires a surgical approach, which most of
the time is disappointing due to marked wound healing response. We conducted a clinical study in order to assess efficacy and safety of viscocanalostomy in these complex cases. Eleven patients were recruited and operated on in a pilot study and received a follow up ranging from 23 to 56 months. Results showed complete success
(intraocular pressure below 21 mmHg without medication) in 54.5% of the eyes, while qualified success was
present in 90.9% of the cases. No relevant complications were noted. This study showed that viscocanalostomy
is a safe and valid surgical option in uveitic patients. The study was published in the Journal of Cataract and Refractive Surgery.
Uveitis intravitreal implant study
G. Modorati, E. Miserocchi, A. Colucci, R. Brancato
A multicenter, randomized, controlled study was performed to evaluate the safety and efficacy of an intravitreal fluocinolone acetonide (0.5mg) implant compared to standardized therapy in patients with non infectious
uveitis affecting the posterior segment of the eye. The aim of this study is to test the possibility to implant intraocular devices releasing small and continuous amount of corticosteroids. This may represent the potential
benefit of sparing patients with chronic intraocular inflammation from the systemic and severe steroid related
side effects.
Uveitis epidemiological study
G. Modorati, E. Miserocchi, A. Colucci, R. Brancato
A retrospective review of 725 cases of uveitis (430 F, 295M) seen in the Ocular Immunology Center of the
University Hospital San Raffaele in Milan from 1997 to 2003 was performed. The evaluated parameters were:
epidemiological data of patients (sex, race, age at presentation of uveitis), anatomical location of the uveitis, etiology and association with systemic diseases, ocular condition, treatment received and ocular complications.
These data may help our approach to diagnosis and management of anterior, posterior and panuveitis at Ocular
Immunology Center.
Ocular diseases correlated with AIDS; epidemiological study
G. Modorati, E. Miserocchi, A. Colucci, R. Brancato
In this retrospective study we reviewed the charts of 286 HIV positive patients consecutively seen in the Ophthalmology Service, of the Infectious Diseases Clinic (San Raffaele Hospital Milan, Italy) from March 2003 to
March 2004. In the Infectious Disease Clinic 3500 HIV positive patients are followed every year. In this study
62.3% patients did not show any evidence of ocular disease. The most frequent anterior segment diseases were
ocular surface disorders and conjunctivitis (51.9%). The most frequent posterior segment disease was Cytomegalovirus retinitis (44.6%). The introduction of highly active antiretroviral therapy has modified the incidence of ocular manifestations in HIV positive patients but CMV retinitis remains the major vision-threatening
Herpes Virus Keratitis study
G. Modorati, E. Miserocchi, A. Colucci, R. Brancato
A prospective randomised study was performed. Fifty two immunocompetent patients with a history of recurrent ocular HSV disease were included in the study. Twenty five were assigned to the Valacyclovir group and
27 to the Acyclovir group. Patients randomized to the Valacyclovir group received one 500mg tablet daily and
patients randomized to the Acyclovir group received one 400mg tablet twice daily. Patients received oral treatment for 1 year. Eligible patients were older than 18 years of age, and had an episode of ocular HSV disease in
the preceding 12 months but their disease had been inactive and untreated during 1 month before the beginning of the study. The study outcome was the rate of recurrences of ocular HSV disease during 12 month of
treatment. Recurrences were classified as infections of the ocular surface (blepharitis, conjunctivitis or epithelial
keratitis), stromal keratitis (corneal infiltrate or corneal edema), or iritis. In this study 1-year suppression therapy with oral valacyclovir (500 mg tablet daily) has shown to be effective and well tolerated as acyclovir (400 mg
tablet twice daily) to reduce the rate of recurrent ocular HSV.
Potential benefits of the once daily regimen of valacyclovir include improved patient compliance.
Gamma Knife radiosurgery for uveal melanoma, ten years experience
G. Modorati, E. Miserocchi, A. Colucci, R. Brancato
Uveal melanoma represents the most common form of primary intraocular malignancy in adults. The estimated incidence of this disease in United States is 7.9 cases per million per year and 10 cases per million per year in
Europe. We evaluated sixty patients with uveal melanoma treated with Gamma Knife Radiosurgery (GKR)
from 1994 to 2004 at the Ophthalmologic Department of the San Raffaele Hospital, Milano. The Kaplan-Meyer 5-year cumulative survival rate was 80.1%. Five patients showed tumor recurrences; three of them were enucleated, one patient was retreated with Transpupillary Thermotherapy and one patient already presented with
liver metastases when the recurrence was diagnosed. In this group of five patients, four patients died from
metastatic disease and one enucleated patient is alive without metastases. The eye retention rate was 91.6%
(55/60 patients). We enucleated 5 patients: 3 patients with melanoma regrowth and 2 patients because of late
painful complications such as neovascular glaucoma and total retinal detachment and phthisis. In conclusion
Gamma Knife Radiosurgery may represent an alternative to enucleation in uveal melanoma treatment.
Retinal thickness modification assessed with the combined OCT/SLO instrument in patients with age
related macular degeneration after photodynamic therapy and its relations with visual acuity
U. Introini, L. Pierro, S. Donati, R. Brancato
A prospective observational non randomized one year study was applied to 30 patients with essudative subfoveal age related macular degeneration (AMD) treated with photodynamic therapy. The aim of this study is to
evaluate macular thickness variation during the follow up with the OCT/SLO (Optical Coherence Tomography/ Scanning Laser Opthalmoscopy), an high resolution technique compared to the traditional fluorangiography. The visual acuity is determined at each follow up visit and related to macular thickness. The anatomical
and morphological data from OCT/SLO and the functional response (Visual acuity) will give more information
on the efficacy of the therapies of macular degeneration diseases.
A phase II prospective, randomized, double masked dose ranging, multicenter trial to assess the effect of
MACUGEN (pegaptanib sodium) on macular thickening in patient with exudative subfoveal age related
macula degeneration
R. Brancato, U. Introini, S. Donati
The aim of the study is to assess the effect of the anti VEGF (vascular endothelial growth factor) MACUGEN
on macular thickness (measured by Optical Coherence Tomography OCT) in patients with a choroidal neovas-
cularization minimally classic in exudative AMD. Intravitreal injection of Macugen (0.3 or 1 mg) versus sham
was made every 6 weeks at a proportion of 1:1:1. We enrolled 5 patients for a 54 weeks follow up evaluated at
baseline with ETDRS visual acuity, fluorangiography and OCT. At each follow up visit, patients will be evaluated on their AV and with OCT as the principal outcome of this study, to assess macular thickness reduction on
the 6 weeks after the injection.
An evaluation of efficacy and safety of posterior juxtascleral administrations of anecortave acetate for
depot suspension (15 mg or 30 mg) versus Sham administration in patients (Enrolled in Study A or study
B) at risk for developing sight-threatening choroidal neovascularization due to exudative age-related
macular degeneration
R. Brancato, U. Introini, M. Setaccioli
Approximately 80% of people affected by Age Related Macular Degeneration present the early stage, characterized by presence of drusen and dystrophy of retinal pigment epithelium. 20% of them progress to the exudative phase, characterized by the presence of choroidal neovascularization (CNV). The exudative AMD is
the leading cause of legal blindness in older people of industrialized countries. Anecortave Acetate is a steroidderived with anti-angiogenetic effects administered as posterior juxtascleral depot onto the outer scleral surface. The efficacy of Anecortave Acetate has already been demonstrated for exudative AMD. Approximately
50% of patients with dry AMD in one eye and CNV in the second eye will develop bilateral CNV within 5
years; eyes with dry AMD are considered to be at high-risk. Aim of this new clinical phase III study is to
demonstrate that Anecortave Acetate is safe and effective in arresting the progression of non-exudative AMD
in eyes at high-risk for progressing to exudative AMD.
PDT in occult only lesions
R. Brancato, U. Introini, A. Ramoni
Age-related macular degeneration (AMD) is the leading cause of legal blindness in people over 50 years in the
developed countries. The neovascular (wet) form is responsible for 80% of the severe visual loss attributable to
AMD. Recent multicentric randomized case control studies, like TAP and VIP, have demonstrated the efficacy
of photodynamic therapy with Visudyne® in the treatment of neovascular form. The purpose of this Phase 4,
non controlled, open-label study is to broaden the experience with Visudyne® therapy in a target sub-population of subjects initially presenting with non classic subfoveal choroidal neovascularization secondary to AMD.
Imaging of the retina and macula with a combined scanning laser ophthalmoscope and optical coherence
system study
L. Pierro, U. Introini, L. Idone, S. Di Simplicio, R. Brancato
The OCT Ophthalmoscope (OCT/SLO) is a new imaging system designed to visualize retinal layers. It combines a digital fundus camera, confocal scanning laser ophthalmoscope and optical coherence tomograph. The
OCT / SLO operates in two scanning modes: B-Scan Longitudinal OCT which is analogous to ultrasound BScan and produce a longitudinal cross sectional image of the retina and C-Scan Traversal OCT Scanning Mode,
which produces simultaneously dual display of C-scan and a Confocal Ophthalmoscope fundus image. The aim
of this study is to evaluate the utility of C-scan OCT for a variety of pathologies including diabetic retinopathy,
choroidal neovascularization membrane, macular pucker. This research may play a significant role to recognize
remarkable new aspects of clinical anatomy in retinal pathology.
Familial retinitis pigmentosa in the northeastern area of Italy: epidemiological, clinical and genetic study
M. P. Manitto, S. Bianchi Marzoli, E. Martina, L. Melzi, M. Ferrari, L. Cremonesi, S. Stenirri, R. Brancato
Retinitis pigmentosa is a term currently used to indicative a heterogeneous group of inherited retinal degenerations, which is an important cause of acquired visual impairment. The natural course of the disease can be
highly variable: loss of central vision occurs later, due to macular involvement, but the progressive deterioration
of the peripheral visual field is a leading cause of legal blindness. The disease may be inherited as an autosomal
dominant, autosomal recessive or X-linked recessive disorder. Multiple genes and loci involved in the retinitis
pigmentosa have been recognized but a large number still remains unknown. The identified genes include
genes encoding for rhodopsin (the first recognized in 1986) on chromosome 3. Further contributes have come
with the discovery of genes RDS/peripherin, ROM1, rod beta-phosphodiesterase (PDE), rod cyclic guanosine
monophosphate (c-GMP)-gated channel, rod PDE-alpha, cellular retinaldehyde-binding protein (CralBP),
ABCR, RPE65. No clear genotype-phenotype relationship has been demonstrated yet.
81 patients with retinitis pigmentosa were examined with the following aims: 1. to estimate the incidence of
the disease in Italy, 2. to analyze the pattern of inheritance, 3. to characterize the ophthalmologic findings, 4. to
create a DNA-bank for molecular analysis, 5. to establish possible genotypic-phenotypic correlations. All patients underwent complete ophthalmological examination, including Goldmann visual field testing and ERG
evaluation. Among our patients we found 32 subjects with sporadic RP, 21 subjects with ADRP, 26 subjects
with ARRP, 5 patients with Usher syndrome, 1 patient with retinitis pigmentosa sine pigmento, 2 patients with
retinitis pigmentosa albescens and 2 patients with RP and neurological disease (Charcot- Marie-Tooth). Peripheral blood extraction for DNA analysis was performed and extracted DNA has been sent to Telethon Institute
of Genetics and Medicine (Naples) for molecular analysis. These data will be part of a multicentric study that
ultimately will lead to define the molecular defects and the frequency of the genetic types of RP in Italy.
Demyelinating optic neuritis: prospective longitudinal study by quantitative magnetic resonance imaging
S. Bianchi Marzoli, A. Falini, A. Ghezzi, L. Melzi, M. A. Rocca, P. Vezzulli, G. Comi, G. Scotti, R. Brancato,
M. Filippi
The aim of our study is to determine conventional Magnetic Resonance Imaging (MRI) and Magnetization
Transfer Imaging (MTI) optic nerve data and their prognostic role in visual outcome in patients with probable
or definite MS with optic neuritis and in patients with idiopathic optic neuritis. We have included 11 patients, 4
with definite MS and 7 with idiopathic optic neuritis (8 women and 3 men, mean age 31 years old, range 20-49).
All patients have a first episode of acute unilateral optic neuritis confirmed by a complete and standardized
neuro-ophtalmologic assessment (including automated visual field testing); they also have a neurologic evaluation and Visual Evoked Potentials (VEPs). All patients have been evaluated at baseline, within 10 days from
symptoms onset, after 3 and 12 months. MRI and MTI from optic nerve (ON) and MRI from the brain are obtained in a single session, by means of standard head coil and scanner operating at 1,5 Tesla. Analysis of the image consists of measuring ON volumes on T1-weighted images, calculating the areas of the ON coronal section
and multiplying the sum of these areas for the slice thickness.
From the corresponding 2 gradient echo images, before and after saturation pulse, we obtained the MTR value, calculated for the same tract considered for ON volume measurement. Results at one year follow-up show
that: (1) no statistically significant difference in volume was detected between affected and unaffected optic
nerve at baseline and during follow-up; (2) MTR in affected ON at baseline was statistically significant higher
than in healthy ones; (3) MTR in affected ON during follow-up was significant lower than in healthy ones; (4)
mean volumes and MRT in healthy ON remained stable over time; (5) no statistically significant difference was
found at baseline and during follow-up for mean volumes and MRT values of affected optic nerve in patients
with MS and patients with idiopathic optic neuritis. These results suggest that in our patients acute optic neuritis probably did not cause loss of tissue, but changes of fibres microstructure.
Reduction in the occurrence of center-threatening diabetic macular Edema
R. Lattanzio, E. Bruschi, F. Legorini, G. Tremolada, F. Scotti, A. Ramoni, S. Donati, L. Bonisolli, R. Brancato
Diabetic Macular Edema (DME) occurs in patients with diabetic retinopathy. DME involving the central part
of the macula, the fovea, can reduce the central visual acuity necessary for reading and for fine visual discrimination. DME is classified by the Early Treatment of Diabetic Retinopathy Study (ETDRS) criteria into two
groups: Non Clinically-Significant Macular Edema (NCSME) and Clinically-Significant Macular Edema
(CSME). The current standard of treatment for DME is the application of focal or grid laser, which can reduce
further vision loss. No medical treatment is currently approved for DME. At the present there are new clinical
trials on medical treatment with possible therapeutical effect in diabetic patients affected by DME. Ruboxistaurin (LY333531) is a bisindolylmaleimide which displays a high degree of specificity in inhibiting PKC ß isoform.
Ruboxistaurin’s ability to inhibit the hyperglicemia-induced increase in retinal vasculature PCK activity, to normalize the diabetes-associated decrease in Na/K-ATPase activity, and to inhibit VEGF-stimulated increases in
retinal permeability, suggests that it may exert a beneficial effect on patients with DME. The primary objective
of this multicenter, randomized, double-masked, parallel, placebo-controlled trial, is to test the hypothesis that
oral administration of 32 mg per day of Ruboxistaurin mesylate for approximately 36 months will reduce, compared to placebo, the occurence of center-threatening diabetic macular edema as assessed by fundus photogra-
phy in patients with non clinically-significant macular edema and non proliferative diabetic retinopathy at baseline. In 2004 we have enrolled 5 consecutive patients. Patients’ enrolment is still ongoing.
Gamma Knife radiosurgery for cavernous sinus and pituitary benign tumors. Results and complications on
afferent and efferent visual pathways
S. Bianchi Marzoli, M. Forti, A. Franzin, P. Picozzi, M. Losa, M. Giovanelli, R. Brancato
The aim of this prospective study was to evaluate morbility on visual system correlated neurological structures of Gamma Knife radiosurgery for cavernous sinus and pituitary benign tumors. From November 2000
and December 2003, 211 consecutive patients (132 with pituitary adenomas and 79 with cavernous sinus
meningiomas) were studied. All patients underwent complete neuro-ophthalmological evaluation included automated perimetry before treatment and every six months after radiosurgery. The mean follow up time was 22
months. Our results show that visual function did not worsen in all subjects. Improvement of ocular motility
was observed in 21.4% of patients with oculomotor cranial nerve palsy. These data show that gamma Knife radiosurgery is a safe treatment for tumors impinging upon afferent and efferent visual sistems. Moreover oculomotor cranial nerve palsy due to cavernous sinus tumors can improve after treatment.
Direct and indirect measurement of human articular cartilage permeability
F. Boschetti, G. M. Peretti, M. Colombo, S. Cattaneo, R. Pietrabissa, F. Gervaso, G. Fraschini
In collaboration with the Laboratory of Biological Structure Mechanics, Politecnico di Milano, our group has
started a series of studies on the analysis of the biomechanical properties of articular cartilage, including the
permeability, which is a crucial property for the functionality of this tissue. It has been so far extrapolated from
indentation-creep mechanical tests (indirect measure). We have developed a method for measuring the permeability of human articular cartilage through its depth by determining in vitro the fluid flow induced by an applied pressure gradient (direct measure). We have also compared the values obtained to those extrapolated
from creep tests (indirect measure). We have demonstrated that the values of permeability decreased with increased applied pressure, and also moving from the superficial to the deep layer. The values of permeability obtained from the creep tests can be up to six times different from those obtained from the permeability direct
tests. We believe that the values obtained from permeability tests could be considered more reliable, because
they give a measured quantity rather than an extrapolated one. More tests are being performed.
Mesangioblasts as possible cell source for articular cartilage repair
G. M. Peretti, S. Biressi, M. Buragas, G. Fraschini
The new methods for repairing articular cartilage lesions require the biopsy of healthy cartilage from a low
weight bearing area of the knee joint as cell source. This implies a surgical access to the knee and a possible
damage to the donor site. The alternative cell sources for chondrocytes harvesting represent a major issue for
several research group. We have recently begun to analyze the potential of mesangioblasts as possible alternative source for chondrocytes. Mesangioblasts are stem cells of vascular origin, with the capacity of differentiating into different phenotypes, as muscular cells. We believe that under appropriate stimuli they can turn their
phenotype toward chondrogeneic lineage. For this reason we have recently started a series of in vitro experiments, where murine mesangioblasts are co-cultured with swine chondrocytes, in monolayer culture, in pellet
culture or in the presence of cartilage explants. The ability of the mesangioblasts to produce the components of
the cartilaginous matrix will be tested by PCR analysis and immunohistochemistry.
Effect of blood on the morphological, biochemical and biomechanical properties of neocartilage,
synthetized by isolated chondrocytes pre-seeded onto a biological scaffold
G. M. Peretti, C. Sosio, F. Boschetti, A. Gigante, C. Bevilaqua, L. Mangiavini, S. Biressi, G. Fraschini
In the past years the use of autologous chondrocytes, transplanted onto a biological scaffold, has become a
clinical reality for the repair of cartilage lesions. However, the effect of the contact of blood on engineered car-
tilage is still unknown. Our group has investigated the effect of blood contact on the morphological, biochemical and biomechanical properties of our model of engineered cartilage. We have isolated articular swine chondrocytes, expanded and seeded onto biological scaffolds in vitro for two weeks. Then we have obtained peripheral blood from other pigs and we have placed our samples in contact with blood for 3 days, mimicking a normal haemarthro following surgery. We have analyzed our samples at different time points after the blood contact and we have compared the results to controls, which did not experience this contact. We have found that
blood contact seems to produce a reduction in physical, biochemical and biomechanical properties, but chondrocytes try to recover in the following standard culture period. We also had the confirmation that this model
could be utilized to test the effects of different conditions on the engineered cartilage.
Study on a tissue engineered cartilage implant model: morphological, physical and biomechanical
G. M. Peretti, C. Sosio, F. Boschetti, A. Gigante, A. Passi, C. Bevilaqua, L. Mangiavini, C. Scotti, S. Biressi,
G. Fraschini
In the attempt of regenerating the articular cartilage tissue, the use of autologous chondrocytes has been introduced in clinical practice. Cells are grown in culture and seeded onto a biological membrane for the transplant to lesion site. The analysis of the effects on this cartilaginous composite of different conditions, such as,
for example, the contact with blood or drugs, the mechanical stresses and others, results difficult after in vivo
transplantation. For this reason, our group has decided to create an in vitro model for engineered cartilage, in
order to be able to test different factors on the development of the cartilaginous tissue. We have first identified
the more appropriate conditions for seeding the biological scaffolds with chondrocytes. Secondary, we have analyzed the morphological, physical, biochemical, and biomechanical properties of the composite overtime in
vitro (3, 4, and 6 weeks), finding an increment of the values overtime, except for the biomechanical test. This
tissue engineered cartilage structure is easily reproducible and it could represent a valuable model for studying
the behaviour of different variables on the newly formed cartilage.
Study on a tissue engineered tendon
G. M. Peretti, C. Sosio, F. Boschetti, L. Mangiavini, C. Scotti, C. Domeneghini, M. Buragas, S. Biressi,
G. Fraschini
Our group has recently started a series of experiments with the goal of creating a tissue engineered tendon. In
fact tendons do not repair spontaneously in the acute or chronic ruptures. Sometimes the gap between the lesion margins requires the transplantation of autologous tissue, with obvious damage at the donor site. Moreover, in the degenerative rupture, as in the rotator cuff, the end-to-end sutures do not guarantee mechanical stability and clinical functionality. We believe that a tendon engineered in vitro with a biological scaffold seeded
with autologous fibroblasts could represent the solution for these problems. We are defining the more appropriate method for isolating fibroblasts. As possible cell source we are testing tendon, ligament, and derma. We
have identified a potential scaffold, a combination of type I and type III collagen sponge. We are studying the
most appropriate cell concentration for seeding the collagen scaffold and the most adequate period of culture
of the composite in vitro, before being transplanted in vivo. Morphological and biomechanical tests are being
performed and the protocol has been submitted to IACUC for transplantation in rat tendon lesion.
Endocavitary hyperthermia
R. Colombo, P. Rigatti
Transurethral hyperthermia in combination with intravescical chemotherapy has proved to be feasible, safe
and effective for the treatment of patients suffering from superficial highly recurrent bladder cancer mainly
when refractory to any standard conservative approach. The technique has been recently standardized on outpatient basis with local anaesthesia. A multicentric study documented that this procedure is more effective than
standard intravescical chemotherapy for prophylaxis of recurrences of superficial bladder cancer after
transurethral resection. A European multicentric study is ongoing in order to verify safety and efficacy of this
novel technique when compared to immunoprophylaxis with BCG as adjuvant treatment for intermediate and
high risk superficial bladder cancer.
Laparoscopy in urology
A. Cestari, G. Guazzoni, P. Rigatti
Laparoscopy is gaining popularity among the urological community. Our experience with this surgical technique started in 1991. We are currently able to perform nearly all the major surgical urological procedures with
this technique employing both the transperitoneal and the retroperitoneal approach. Our experience of laparoscopic treatment of benign adrenal tumors has become the first in Europe. Cases of conservative adrenal surgery have also been performed. Laparoscopic renal surgery has also been successfully accomplished. Laparoscopic nephrectomy is becoming the standard of care when the complete ablation of the kidney is required.
Conservative surgery has also been successfully performed. Our series for laparoscopic renal cryoablation in
case of small renal tumors is the first in Europe and the second in the world. Laparoscopic radical prostatectomy is also routinely performed and a prospective study on laparoscopic prostatectomy versus open prostatectomy is”on going” and several cases of radical cystectomy have also been performed. Finally, reconstructive surgery, such as laparoscopic pyeloplasty and colposacropexy are routinely performed.
Pediatric urology
A. Bocciardi, A. Lesma, P. Rigatti
Surgical experience with Passerini feminizing genitoplasty in case of ambiguous genitalia has been increasingly expanded with positive long-term results. Laparoscopy was applied in children to remove non-functional
kidneys, benign or malignant lesions of adrenal glands. Endoscopic correction of vescico-ureteral reflux has
been successfully performed in the paediatric population with good long term results. A long term study on hypospadia repair has shown that the early age at intervention is related to a correct psychosexual development in
young adults. A regional screening program for adolescent urology is going to start.
Benign Prostatic Hyperplasia (BPH)
F. Montorsi, R. Naspro, P. Rigatti
Our experience with holmium laser enucleation of the prostate (HoLEP) with morcellation for the treatment
of symptomatic benign prostatic hyperplasia (BPH) has certainly broadened and is now standard practice at
our institution. We have continued to conduct trials to compare HoLEP with standard trans-urethral resection
of the prostate (TURP) for prostates <70 gr and with open standard prostatectomy for larger prostates, (up to
130 grs) demonstrating the superiority of the laser technique in terms of blood loss, time of catheterization and
hospital stay, guaranteeing equal effectiveness in relieving obstruction and lower urinary tract symptoms. At the
one year follow-up, complications and functional results were similar in all groups. Our data also show that
HoLEP promotes a significant hospital net cost saving (i.e. 9.6%) over transvescical open prostatectomy. Additional cost reduction might be achieved by using HoLEP with a same-day discharge policy even for those patients with large BPH otherwise candidates to a longer hospital stay with the standard open surgery.
Surgical tecnique – Radical nephrocapsulectomy in extracorporeal circulation and deep hypothermic
arrested circulation in RCC with thrombosis of the inferior vena cava and right atrium
R. Bertini, P. Rigatti
In the last years the surgical technique for the treatment of renal cell carcinoma with neoplastic thrombus of
the Inferior Vena Cava (IVC) and right atrium has become feasible and radical from an oncological point of
view thanks to the new developments in the area of general anaesthesia and cardiac surgery. We defined the
best therapeutical strategies according to the upper limits of the neoplastic thrombus, since our technique is
personalized for each different case. Furthermore, we developed an en bloc nephro-capsulectomy, during ECC
and deep hypothermic arrested circulation; this approach allows an adequate mini-invasive approach and the
application of available technologies to guarantee an operation in complete safety even in such a dramatic scenario as renal cell carcinoma with thrombus of the supra-hepatic IVC. To reduce intra-operative blood loss, this
strategy requires embolization of the renal artery to be performed the day before the operation. In our experience, median sternotomy is replaced by an anterior right mini-thoracotomy that offers a reduction in operative
time, in post-operative pain with a good aesthetic scarring.
Peyronie’s disease
F. Montorsi, A. Salonia, A. Briganti, F. Dehò, P. Rigatti
Clinical presentation of Peyronie’s disease in patients younger than 40 years old showed a more acute onset
and a lower incidence of associated erectile dysfunction. The long term impact of a new surgical procedure
(namely, multiple relaxing incisions of the tunica albuginea and subsequent placement of a 3-piece inflatable
penile implant) on penile deformity caused by severe cavernous fibrosis was evaluated. Patients with severe penile curvature, shortening and impaired penile rigidity due to penile fibrosis may be offered this surgical alternative, which proved to be effective and safe in long term assessment. The 5-year follow-up outcome of plaque
incision and vein grafting in selected patients with Peyronie’s disease was assessed by extensive pre- and postoperative subjective and objective analysis. Plaque incision and vein grafting is associated with a significant patient dissatisfaction rate and organic morbidity when the 5-year follow-up is achieved.
Erectile dysfunction
F. Montorsi, A. Salonia, A. Briganti, R. Colombo, F. Dehò, P. Rigatti
A large, multicenter study suggested that a significant proportion of patients with angiographically-documented coronary artery disease have erectile dysfunction and that this latter condition may become evident prior to angina symptoms in almost 70% of cases. A prospective study including a control group of patients with
coronary artery disease and normal erectile function was initiated in order to verify whether erectile dysfunction may be considered a real predictor of ischemic heart disease. The role of subsequent early postoperative
administration of alprostadil and sildenafil in the prevention of iatrogenic erectile dysfunction after nerve sparing radical prostatectomy has been demonstrated. Nerve and seminal-sparing cystectomy demonstrated to offer
satisfactory clinical and functional outcomes both in terms of urinary continence and overall sexual function for
young, fully potent and socially active patients suffering from organ-confined bladder cancer.
Prostate cancer
F. Montorsi, L. Da Pozzo, G. Zanni, B. Mazzoccoli, P. Rigatti
Radical prostatectomy (RRP): during the last year a tremendous effort has been produced to set-up an institutional data base especially designed to store clinical information for patients submitted to RRP at our Department. With the help of a new data manager, clinical data concerning this procedure are now continuously updated and available for statistical analysis. Not only oncological information is investigated and stored, but also
data concerning quality of life with particular regard to continence and potency. One major technical aspect of
RRP has already been investigated and was the object of several publications during the last year: general versus
spinal anaesthesia for RRP. Special effort is now being produced to ameliorate the technique of nerve sparing
RRP. The new technology TRIMprob (Tissue Resonance Interaction Method) in the early diagnosis of prostate
cancer is under investigation at our Department. The primary objective of the investigation is to determine the
ability of TRIMprob to diagnose (accuracy) the presence of prostate cancer in terms of agreement with the diagnosis obtained from biopsy.
General Anaesthesia and respiratory function
A. Casati, A. Albertin, G. Torri
The introduction of new intravenous and inhalational agents has been investigated by determining the effects
of two different plasma concentrations of remifentanil (1 and 3 ng/ml) on the minimum alveolar concentration
of sevoflurane blunting cardiovascular responses to skin incision in 50% of patients (MAC-BAR): interestingly,
1 ng/ml remifentanil reduces the MAC-BAR of sevoflurane by nearly 60%, while increasing the plasma concentrations of remifentanil up to 3 ng/ml resulted in a further 30% reduction of MAC-BAR of sevoflurane. We
also determined the plasma concentration of remifentanil blunting cardiovascular responses to tracheal intubation and skin incision in patients receiving a BIS-guided infusion of propofol. The negative effects of mild in-
traoperative hypothermia on patient outcome are well-known; in this light we also evaluated if the use of laparoscopic rather than laparotomic techniques for major abdominal surgery could result in less effects on thermic homeostasis during surgery, but we demonstrated that heat loss is a severe problem irrespectively of the
surgical technique, while active patient warming must be considered. A study is in progress for desflurane.
Anaesthesia in orthopaedic and outpatient surgery
A. Casati, G. Cappelleri, G. Aldegheri, A. Albertin
Among different approaches to central blocks, unilateral spinal anaesthesia and single shot epidural blockade
have been demonstrated to allow less hemodynamic effects than conventional bilateral spinal anaesthesia. Concerning new local anaesthetic molecules introduced in clinical practice, our research group evaluated intra-and
postoperative clinical properties of ropivacaine and levobupivacaine, two new long acting local anaesthetics,
used for both epidural anaesthesia and different peripheral nerve block, such as combined sciatic femoral nerve
block, axillary and interscalene brachial plexus anaesthesia and continuous interscalene, femoral and sciatic
nerve block. The use of different anaesthesia techniques, including central blocks, peripheral nerve blocks or
general anaesthesia with propofol and remifentanil has been evaluated in different outpatient procedures, such
as knee arthroscopy, inguinal hernia repair or gynaecological procedures. Finally a clinical trial on the use of
chirocaine in continuous epidural perfusion for postoperative pain has been performed.
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Murphy, A; Varanese, L. Tadalafil in the treatment of erectile
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[11C]Coline-PET and [18F]FDG-PET in broncoalveolar carcinoma: comparison with histological and
molecular data
C. Messa, M. Picchio, C. Landoni, L. Gianolli, B. Giglioni, G. Arrigoni, F. Sanvito, F. Fazio
BAC is a form of lung cancer frequently negative with [18F]FDG-PET. Preliminary PET data in BAC tumors
with both [18F]FDG and [11C]Choline suggested that the information derived from combining the two tracers, may help distinguish BAC ([18F]FDG negative and [11C]Choline positive) from an inflammatory lesion
([18F]FDG and [11C]Choline positive). In addition, the simultaneous use of two PET tracers evaluating two
different metabolic pathways, together with histopathologic, immunohistochemical and gene expression analysis, may help to improve understanding of tumour in-vivo behaviour. In BAC tumour tissue, the absence of
Glut-1 protein and corresponding mRNA, is a cause for the negative [18F]FDG-PET findings. Immunohistochemical and molecular findings confirmed the biological behaviour of BACs, namely: these are usually well
differentiated carcinomas with lepidic growth and low level of glycolysis, responsible for the absence of
[18F]FDG uptake. Conversely, such behaviour could be detected by using [11C]Choline, whose uptake is regulated by phospholipid metabolism rate.
[11C]Choline-PET sensitivity in re-staging prostate cancer patients: relationship with PSA serum level
M. Picchio, C. Messa, V. Scattoni, C. Landoni, L. Gianolli, M. Matarrese, P. Rigatti, F. Fazio
11C-Choline-PET is an established technique to re-stage prostate cancer patients. However, the relationship
between PSA serum level and the influence of androgen-ablation therapy on [11C]Choline uptake has not yet
been established. To this aim, 188 consecutive prostate cancer patients have been re-staged with [11C]CholinePET/CT in the suspect of recurrence of disease after prostatectomy. [11C]Choline-PET resulted positive
(Chol+) in 47% of patients (mean PSA: 8,62 ng/ml) and negative (Chol-) in 53% (mean PSA: 1,98 ng/ml). In
particular, in patients with PSA<1 ng/ml, 29% of patients resulted Chol+. In patients with PSA<2 ng/ml, 33%
resulted Chol+ and in patients with PSA>2 ng/ml (68% of patients resulted Chol+. At the time of
[11C]Choline-PET study 109 of all 188 patients (58%) were under androgen ablation therapy. Of these, 54 resulted Chol+ and 55 Chol-. As for patients without androgen ablation therapy (n=79), 35 resulted Chol+ and
44 Chol-. [11C]Choline-PET sensitivity improves with increasing PSA serum value, but recurrences can be
found in patients even at very low PSA levels (<2 ng/ml). Androgen ablation therapy seems not to influence
[11C]Choline-PET sensitivity.
Value of PET/CT with [18F]FDG in re-staging ovarian carcinoma
S. Sironi, C. Messa, M. Picchio, B. Zangheri, L. Gianolli, G. Mangili, A. Del Maschio, A. Ferrari, F. Fazio
Ovarian carcinoma is the third most common cancer of the female genital tract. Purpose of our study was to
evaluate the accuracy of [18F]FDG-PET/CT for depicting persistent ovarian carcinoma. Included in the study
were 31 consecutive patients with ovarian cancer scheduled for surgical second-look procedure. Seventeen
(55%) of 31 patients had persistent disease at histopathologic examination after surgical second-look procedure. In these 17 patients, the total number of lesions found positive for malignant tissue was 41 (lymph nodes:
n=16; peritoneal lesions: n=21; pelvic lesions: n=4). PET/CT overall lesion-based sensitivity, specificity, accuracy, positive and negative predictive values for revealing persistent ovarian carcinoma were 78% (32/41), 75%
(12/16), 77% (44/57), 89% (32/36) and 57% (12/21), respectively. The accuracy rate for lesions greater than 1
cm in size was 90% (38/42). The overall patient-based sensitivity, specificity, accuracy, positive, and negative
predictive values of PET/CT were as follows: 53% (9/17), 86% (12/14), 68% (21/31), 82% (9/11), and 60%
(12/20), respectively.
Value of PET/CT with [18F]FDG in pre-operative staging of cervical carcinoma
S. Sironi, C. Messa, M. Picchio, L. Gianolli, G.Mangili, A. Del Maschio, A. Ferrari, F. Fazio
To perspectively determine the accuracy of [18F]FDG-PET/CT for lymph node staging in patients with early stage cervical cancer, 39 patients scheduled for radical hysterectomy with pelvic lymph node dissection were
included in the study. Before surgical treatment, all of them underwent [18F]FDG-PET/CT imaging. Six
(15%) of 39 patients had metastatic lymph nodes at histopathology, and 33 (85%) of 39 had no histologically
confirmed node metastasis. Of the total 975 lymph nodes sampled, 10 (1.02%) were found to be positive for
malignant cells at histopathology. The overall lesion-based sensitivity, specificity, positive predictive value
(PPV), negative predictive value (NPV), and accuracy of PET/CT were 50%, 91%, 62%, 86% and 82%, respectively. The corresponding values calculated for lymph node lesions greater than 0.5 cm were 100%, 91%,
62%, 100%, and 92%. The overall patient-based sensitivity, specificity, PPV, NPV, and accuracy of PET/CT
were 33%, 97%,67% 89% and 87%, respectively. These data proved that PET/CT is a valuable for lymph
node staging in these patients, being 0.5 cm the size threshold for accurate depiction of metastatic node
A thresholding segmentation method for pet fdg lung neoplastic lesions
L. Soma, M. Picchio, M. C. Gilardi, C. Messa, V. Bettinardi, M. Danna, G. Rizzo, I. Dell’Oca, F. Fazio
To implement a thresholding segmentation method for PET FDG lung lesions, phantom and patient studies
were performed by GE PET/CT Discovery ST system. An optimal threshold (THopt) was determined in each
phantom experiment. Curves of THopt versus measured L/B values were created (Thopt-L/B curves). PET
whole body studies, with metabolically active lung lesions, were selected from 12 patients. For each study, the
lesions and their surrounding tissues were recognized in order to calculate L/B ratios. Lesion contours were
generated applying the segmentation thresholding method by deriving the appropriate THopt from the phantom THopt-L/B curves using the measured L/B ratios. As expected THopt-L/B curves fit an inverse behaviour.
The fitting parameters depend however on statistic counts, since statistical noise affects the estimate of L and B.
To achieve an appropriate contouring in patients, different background levels had to be considered, resulting in
different L/B and THopt. In conclusion, these preliminary data suggest that phantom THopt-L/B curves for lesion segmentation could be used in clinical PET, taking into account statistical noise and background heterogeneity
Molecular imaging techniques for the in vivo pet study of neurochemistry in normal and pathological
R. M. Moresco, A. Panzacchi, D. Perani, R. Cavallaro, M. Locatelli, E. Smeraldi, L. Bellodi, E. Turolla, F. Fazio
Part of our research activity is focused on the application of molecular imaging techniques for the in vivo
study of neurochemistry and neuropharmacology of different psychiatric diseases including anxiety and affective disorders. The neurobiological correlates of normal and pathological behaviors is also under investigation.
In particular, the neurochemistry of OCD, major depression and eating disorders together with the neurobiology of personality traits are under investigation using different radiopharmaceuticals. In particular using PET
and selected radioligands we have measured the regional differences in serotonin and dopamine receptors occupancy induced by different neuroleptics and the effect of the serotonin reuptake sites inhibitor Fluvoxamine
on striatal dopamine D2 receptors in a group of drug naïve OCD patients.
fMRI studies of language functions
J. Abutalebi, S. Brambati, F. Fazio, C. Saccuman, M. Tettamanti
Neuroimaging studies provided the in vivo evidence for neural substrates of language processes in normal
and impaired conditions. To this purpose, we used fMRI functional studies to measure the neural correlates of
reading processes in a large sample of developmental familial dyslexia. Reduction of functional activity was ob-
served in the left hemisphere, in the inferior frontal gyrus and in the inferior lateral temporal cortex. This suggests that the underlying functional abnormalities may be responsible for defective written language acquisition
in these subjects. fMRI and SPM analysis in anomic aphasics during naming tasks before and after rehabilitative
training provided evidence for functional recovery of specific neural systems in the left hemisphere and for the
contribute of homologue regions in the right.
fMRI studies in motor imagery
D. Anchisi, F. Fazio, C. Saccuman, M. Tettamanti
We studied, using fMRI, the neural correlates of motor imagery in 4 amputee patients, before and after hand
allograft. We observed activations in sensorimotor circuits as well as in frontal regions controlling for motor
plans. This was evident also in the presurgical phase, suggesting the maintenance of hand representation after a
long lasting deafferentation.
rcbf SPECT studies in Mild Cognitive Impairment (MCI)
D. Anchisi, F. Fazio, M. Franceschi, P. Ortelli, V. Garibotto, B. Borroni
We have evaluated the potential role of 99mTc-ECD Single Photon Emission Computed Tomography
(SPECT) and memory scores in predicting conversion to AD in MCI subjects. The pattern of hypoperfusion
99mTc-ECD SPECT and the severity of memory deficits predict the risk of progression to probable AD dementia in MCI subjects. Platelet Amyloid Precursor Protein forms ratio and 99mTc-ECD SPECT perfusion
analysis were evaluated in Mild Cognitive Impairment (MCI) subjects who progressed to Alzheimer Disease
(AD) and in stable MCI. We reported that their combined evaluation increases the discriminative power of the
analysis in identifying pre-symptomatic AD.
Evaluation of new potential radioligands for the in vivo PET imaging of neurodegeneration
R. M. Moresco, S. Belloli, S. Todde, M. Matarrese, P. Simonelli, A. Carpinelli, F. Fazio. M. Galli, P. Popoli
This research line is focused on the evaluation of new ligands for the in vivo imaging of neurodegeneration.
The time course of A2 receptor and D2 dopamine receptor loss has been evaluated in a toxicological model of
striatal degeneration. The expression of D2 and A2 receptors have been measured using [11C]SCH442416 and
[11C]raclopride at 8, 15, 30 and 60 days after the intra-striatal administration of quinolinic acid. Preliminary results of the study, that is still ongoing, show a progressive loss of both receptors subtype indicating that the
process of degeneration of intra-striatal GABAergic neurons is still present two month after a single administration of the neurotoxin. Further researches will correlate in the same animal model the rate of receptor loss with
the presence of activated microglial cells.
In vivo imaging of peripheral benzodiazepine receptors as a marker of microglia activation in patients with
neurodegenerative diseases
R. M. Moresco, C. Messa, A. Panzacchi, L. Pietra, M. Matarrese, C. Cobelli, O. Bugiani, F. Tagliavini, F. Fazio
The use of PET and [11C]PK11195 allows the in vivo assessment of microglia activation the follows neurodegeneration processes. Using this method we investigated that activation of microglial cells which occurs in
CJD following the deposition of the prion protein. An increase in tracer binding was presenti in different brain
regions of CJD patients. Interestingly, the increase in [11C]PK11195 binding was observed in structures which
where also hyperintense in MRI images. This study provides an in vivo evidence of microglial activation in CJD
patients, and supports the view that MRI hyperintensities are related with the presence of activated microglia.
Furthermore, it proposes a role of molecular imaging in the clinical evaluation of CJD patients.
Cerebrovascular diseases and Magnetic Resonance Angiography (MRA)
N. Anzalone, F. Scomazzoni, L. S. Politi, M. Cadioli, G. Scotti
Evaluation of intracranial aneurysms, pre- and post endovascular treatment and intracranial arterial stenosis
with MR angiography at both 1.5T and 3T to demonstrate the advantages of high field strength in studying the
intracranial circulation. Due to the increasing interest in the atherosclerotic plaque, especially at the carotid artery, we began to study plaque morphology and structure with MR at 1.5T using special surface dedicated coils;
we compare in vivo studies with ex-vivo (after endoarterectomy) studies and pathology to correlate different
plaque components. The aim is to compare data with echotomography and to correlate them with patients’
clinical and laboratory data.
Interventional and therapeutic neuroradiology
F. Simionato, F. Scomazzoni, C. Righi, G. Scotti
Evaluation of coated coils, self expanding stents and other new devices in the endovascular treatment of intracranial aneurysms. Endovascular Treatment of Brain Artero-Venous Malformations with a new cyanoacrylic
glue. Angioplasty and Stenting: long term evaluation in the treatment of stenosis of the supra-aortic arteries is
continuing, in particular results are being evaluated for the treatment of carotid stenosis with low profile self
expanding stents. Evaluation of intracranial stenting on atherosclerotic intracranial stenosis. Evaluation of different strategies in the intra-arterial acute treatment of stroke, both with pharmacological and mechanical fibrinolysis. Involvement in italian registry data collection both on aneurysm and stroke endovascular therapies.
In vitro labelling of mouse neural precursor cells with SPIO particles: relevance for in vivo cell tracking
L. S. Politi, M. Bacigaluppi, M. Cadioli, A. Falini, S. Pluchino. G. Martino, G. Scotti
Adult mouse Neural Precursor Cells (aNPCs) were in vitro labelled with increasing concentrations of Fe using three different Super Paramagnetic Iron Oxide (SPIO) contrast media (Endorem®; Resovist®; Sinerem®).
Iron uptake, cell viability, proliferation and differentiation were tested in vitro 72 hours and 8 days after in vitro
labelling. We prepared agarose phantoms containing dispersed unlabelled and labelled cells or free SPIO and
measured at 1.5T (with a 23 mm surface coil) signal change with routinely used sequences and R1 and R2 relaxivities. Increasing doses of SPIOs were used (from 0.01 to 0.8 mg/ml Fe). Uptake of all SPIOs was much higher when aNPCs were incubated in vitro with both polylisine-L and iron nanoparticles. Mild cell toxicity was
measured when cells were incubated with 0.8 mg/ml Fe. All relaxation parameters parallely increased cell concentration of the iron particles. Between contrast media tested, Endorem® showed the highest signal decrease.
aNPCs can be successfully labelled in vitro with novel SPIOs contrast media. This method might allow in vivo
aNPC tracking.
Outcome of HIV-related PML in HAART treated patients: clinical radiological, and viro-immunological
S. Gerevini, P. Cinque, S. Bossolasco, A. Pazzi, G. Scotti
PML is a progressive demyelinating diseases caused by the virus JCV. It is rapidly fatal in approximately 50%
of HIV-infected patients treated with HAART. There is neither effective treatment for this disease nor baseline
indicators of disease progression. This study has the objective of monitoring brain MRI changes in parallel with
clinical neurological examination, quantification of JCV in the CSF and JCV-specific lymphoproliferation assays in peripheral blood, in order to define early markers of disease progression during follow-up. Patients receiving experimental treatment regimens (corticosteroids, various types of HAART) will be evaluated.
HAART-induced immune reconstitution syndrome disease of the CNS
S. Gerevini, P. Cinque, S. Bossolasco, A. Pazzi, G. Scotti
HIV-infected patients starting HAART usually show a response to treatment in terms of increased CD4 and
decrease of HIV-1 RNA load. It has been observed in some patients that these changes are accompanied by the
onset of symptoms related to inflammation at different sites, including visceral organs, skin and CNS. The objective of this study is to use neuroimaging to characterize CNS immune reconstitution diseases, to evaluate
their frequency and describe the natural history.
HAART-induced immune HIV-related CNS abnormalities in HIV-infected patients failing to respond to
S. Gerevini, P. Cinque, S. Bossolasco, A. Pazzi, G. Scotti
HIV-infected patients starting HAART usually show a response to treatment in terms of increased CD4 and
decrease of HIV-1 RNA load. It has been observed in some patients that these changes are accompanied by the
onset of symptoms related to inflammation at different sites, including visceral organs, skin and CNS. A large
proportion of HIV-infected patients on long-term HAART fail to respond to treatment in term of reduced or
lack of CD4 cell increase. Despite the low number of CD4, however, these patients seem less likely to develop
HIV-associated CNS disease, which was common in the pre-HAART. It is possible that, despite failure of immunological reconstitution, treatment maintains a neuroprotective effect. The objective of the study is to assess
the extent of clinical, neuropsychologic and brain MRI abnormalities in HAART-treated or untreated patients
with low CD4 cell numbers.
Delayed gadolinium-enhanced cardiac mr imaging in myocarditis: comparison with biopsy findings
F. De Cobelli, A. Esposito, E. Belloni, R. Mellone, M. Pieroni, C. Chimenti, A. Frustaci, A. Maseri,
A. Del Maschio
Acute Myocarditis (AM) is an inflammatory disease of the myocardium, most frequently caused by viruses.
Apparently the spontaneous recovery of AM is common, nevertheless the disease may progress to fatal arrhytmias, with sudden death or to dilated cardiomyopathy and heart failure. Clinical grounds, EKG, laboratory data, echocardiography, myocardial scintigraphy and coronary angiography are of limited value except for the exclusion of acute coronary syndromes and the diagnosis of AM is often presuntive. The identification of more
accurate diagnostic tools is crucial for the diagnosis of AM. Our purpose was to compare the findings obtained
by the contrast-enhanced Cardiac Magnetic Resonance (CMR) with the histopathological findings obtained by
endomyocardial biopsy (EMB) in 25 consecutive patients to establish the potential role of the non-invasive MR
procedure in the diagnosis of AM. At histopathologic analysis, AM was found in 13 patients. At CMR, areas of
late enhancement (LE) were present in 12 over 13 AM patients (sensitivity 92%) with patchy foci or
midwall/subepicardial striae. Thus, CMR can be considered a valuable tool in the diagnosis of LV active myocarditis.
31Phosphorus Magnetic Resonance Spectroscopy (31P MRS) and delayed gadolinium-enhanced cardiac
magnetic resonance imaging in patients with hypertrophic cardiomyopathy
F. De Cobelli, G. Perseghin, A. Esposito, P. Scifo, E. Belloni, T. Canu, R. Mellone, A. Maseri, A. Del Maschio
Hypertrophic cardiomyopathy (HCM) is a primary disease of the myocardium characterized by abnormal
symmetric or asymmetric thickening of the left ventricular wall. Myocardial disarray, intramyocardial small-vessel disease, interstitial fibrosis and macroscopic scars are the major histological abnormality. Replacement scarring fibrosis has been postulated to represent an important anatomic substrate of ventricular arrhythmias.
Gadolinium Delayed-Enhanced Magnetic Resonance Imaging (DEMRI) allows visualization of myocardial
scarring. In HCM patients had been also described to have an impairment of myocardial energy metabolism by
evaluation of high-energy phosphorus metabolites with 31P Magnetic Resonance Spectroscopy (31P-MRS). We
have studied 17 HCM patients with DEMRI, 31P-MRS and endomyocardial biopsy. The biopsies obtained
from delayed-enhanced areas showed abnormal presence of fibrosis. Comparing the volume of enhanced tissue
visualized at DEMRI and PCr/ATP obtained from MR spectra, a strong negative correlation was found (R=0.76; P<0.005), suggesting that myocardial fibrosis replacement is one of the main factors contributing to impairment of myocardial metabolism in HCM.
MRS of the skeletal muscle and of the liver and impact on the insuline resistance syndrome
G. Perseghin, F. De Cobelli, P. Scifo, A. Esposito, T. Canu, L. Luzi, A. Del Maschio
Ectopic fat accumulation within the skeletal muscle and the liver may contribute to the derangement of glucose and fatty acids metabolism in obesity, diabetes and in the insulin resistance syndrome. Our ongoing research program based on the non-invasive assessment of intracellular substrate concentrations by means of MR
Spectroscopy (MRS) techniques (in opposite to the traditional biopsy technique) has been successful in clarifying the relationships between ectopic fat accumulation within the skeletal muscle and whole body insulin resistance using 1H, 13C and 31P MRS techniques. During the last year we have been focusing our studies on the
metabolic effects induced by abnormal fat accumulation within the liver (liver steatosis) using 1H MRS and on
the intrahepatic metabolism of High Energy Phosphates (HEPs) searching for a primary defects in the hepatic
fatty acids oxidative disposal.
Non-invasive assessment of High Energy Phosphate (HEP) metabolism and function of human heart with
31Phosphorus Magnetic Resonance Spectroscopy (31P MRS) and cardiac magnetic resonance imaging
G. Perseghin, F. De Cobelli, P. Scifo, A. Esposito, T. Canu, L. Luzi, A. Del Maschio
This technique explores the “energetic” homeostasis of the heart in vivo and in association with the MRI determination of morphological and hemodynamic parameters constitutes a unique tool to study heart performance in health and diseases. In collaboration with Dr. Fiorina and the Transplant Unit directed by Prof Secchi,
we have studied the early metabolic and functional heart alterations associated with type 1 diabetes and we
have demonstrated that kidney-pancreas transplantation is successful in reversing these abnormalities vs kidney
transplantation alone. Moreover, in collaboration with Dr Fragasso and Cardiology Department we have studied metabolic, functional and hemodynamics heart alterations in patient with chronic heart failure. In this
group of patients we are assessing the effects of therapy on functional and metabolic heart performance, with
31P MRS and MRI approach. Recently, in collaboration with faculty of exercise sciences of Università degli
Studi di Milano, we have started to study intracardiac metabolism and cardiac function in elite athlete professionals.
IG.1. Antonini, A; Moresco, RM; Gobbo, C; De Notaris, R; Panzacchi, A; Barone, P; Bonifati, V; Pezzoli, G; Fazio, F. Striatal
dopaminergic denervation in early and late onset Parkinson’s
disease assessed by PET and the tracer [C-11]FECIT: preliminary findings in one patient with autosomal recessive parkinsonism (Park2). Neurol.Sci.; 2002; 23 (): S51 S52
IG.2. Benndorf, G; Kroppenstedt, S; Campi, A; Unterberg, A. Selective neck occlusion of a large complex aneurysm of the middle cerebral artery trifurcation with the ultrasoft coil.
Am.J.Neuroradiol.; 2002; 23 (6): 965 969
IG.3. Bettinardi, V; Pagani, E; Gilardi, MC; Alenius, S; Thielemans, K; Teras, M; Fazio, F. Implementation and evaluation of
a 3D one-step late reconstruction algorithm for 3D positron
emission tomography brain studies using median root prior.
Eur.J.Nucl.Med.; 2002; 29 (1): 7 18
IG.4. Buvat, I; Castiglioni, I. Monte Carlo simulations in SPET
and PET. Q.J.Nucl.Med.; 2002; 46 (1): 48 61
IG.5. Castiglioni, I; Cremonesi, O; Gilardi, MC; Savi, A; Bettinardi, V; Rizzo, G; Bellotti, E; Fazio, F. A Monte Carlo model of
noise components in 3-D PET. IEEE Trans.Nucl.Sci.; 2002; 49
(5): 2297 2303
IG.6. Cortesi, L; Canossi, B; De Santis, M; Panizza, P; Rossi, G;
Turchetti, D; Del Maschio, A; Ferrari, S; Romagnoli, R; Federico, M; Silingardi, V. Usefulness of breast MRI in a patient with
genetic risk. J.Exp.Clin.Cancer Res.; 2002; 21 (3): 131 136
IG.7. Del Maschio, A; De Gaspari, A; Panizza, P. Present indications for the use of breast MRI. J.Exp.Clin.Cancer Res.; 2002;
21 (3): 55 58
IG.8. Del Sole, A; Gambini, A; Falini, A; Lecchi, M; Lucignani,
G. In vivo neurochemistry with emission tomography and magnetic resonance spectroscopy: clinical applications. Eur.Radiol.;
2002; 12 (10): 2582 2599
IG.9. Di Domenico, G; Motta, A; Zavattini, G; Del Guerra, A;
Damiani, C; Bettinardi, V; Gilardi, MC. Characterization of the
Ferrara animal PET scanner. Nucl.Instrum.Methods
Phys.Res.Sect.A-Accel.Spectrom.Dect.Assoc.Equip.; 2002; 477
(1-3): 505 508
IG.10. Herholz, K; Salmon, E; Perani, D; Baron, JC; Holthoff, V;
Frolich, L; Schonknecht, P; Ito, K; Mielke, R; Kalbe, E; Zundorf, G; Delbeuck, X; Pelati, O; Anchisi, D; Fazio, F; Kerrouche, N; Desgranges, B; Eustache, F; Beuthien-Baumann, B;
Menzel, C; Schroder, J; Kato, T; Arahata, Y; Henze, M; Heiss,
WD. Discrimination between Alzheimer dementia and controls
by automated analysis of multicenter FDG PET. Neuroimage;
2002; 17 (1): 302 316
IG.11. Jones, ME; Gesu, G; Ortisi, G; Sahm, DF; Critchley, IA;
Goglio, A. Proficiency of Italian clinical laboratories in detecting reduced glycopeptide susceptibility in Enterococcus and
Staphylococcus spp. using routine laboratory methodologies.
Clin.Microbiol.Infect.; 2002; 8 (2): 101 111
IG.12. Kroencke, TJ; Wasser, MN; Pattynama, PMT; Barentsz,
JO; Grabbe, E; Marchal, G; Knopp, MV; Schneider, G;
Bonomo, L; Pennell, DJ; Del Maschio, A; Hentrich, HR;
Dapra, M; Kirchin, MA; Spinazzi, A; Taupitz, M; Hamm, B.
Gadobenate dimeglumine-enhanced MR angiography of the
abdominal aorta and renal arteries. Am.J.Roentgenol.; 2002;
179 (6): 1573 1582
IG.13. Lucignani, G; Gobbo, C; Moresco, RM; Antonini, A; Panzacchi, A; Bonaldi, L; Carpinelli, A; Caraceni, T; Fazio, F. The
feasibility of statistical parametric mapping for the analysis of
positron emission tomography studies using 11C-2-b-carbometaxy-3-b-(4-fluorophenyl)-tropane in patients, movement
disorders. Nuc.Med.Commun.; 2002; 23 (11): 1047 1055
IG.14. Mainardi, LT; Origgi, D; Lucia, P; Scotti, G; Cerutti, S. A
wavelet packets decomposition algorithm for quantification of
in vivo H-1-MRS parameters. Med.Eng.Phys.; 2002; 24 (3): 201
IG.15. Marin, C; Seoane, JM; Sanchez, M; Ruiz, Y; Garcia, JA.
Magnetic resonance imaging of primary lymphoma of the
cervix. Eur.Radiol.; 2002; 12 (6): 1541 1545
IG.16. Matarrese, M; Moresco, RM; Romeo, G; Turolla, EA; Simonelli, P; Todde, S; Belloli, S; Carpinelli, A; Magni, F; Russo,
F; Kienle, MG; Fazio, F. [C-11]RN5: A new agent for the in vivo imaging of myocardial alpha(1)-adrenoceptors. Eur.J.Pharmacol.; 2002; 453 (2-3): 231 238
IG.17. Matarrese, M; Sudati, F; Soloviev, D; Todde, S; Turolla,
EA; Kienle, MG; Fazio, F. Automation of [C-11]acyl chloride
syntheses using commercially available C-11-modules. Appl.Radiat.Isot.; 2002; 57 (5): 675 679
IG.18. Montagna, P; Provini, F; Plazzi, G; Vetrugno, R; Gallassi,
R; Pierangeli, G; Ragno, M; Cortelli, P; Perani, D. Bilateral
paramedian thalamic syndrome: abnormal circadian wake-sleep
and autonomic functions. J.Neurol.Neurosurg.Psychiatry;
2002; 73 (6): 772 774
IG.19. Moresco, RM; Dieci, M; Vita, A; Messa, C; Gobbo, C; Galli, L; Rizzo, G; Panzacchi, A; De Peri, L; Invernizzi, G; Fazio, F.
In vivo serotonin 5HT(2A) receptor binding and personality
traits in healthy subjects: A positron emission tomography
study. Neuroimage; 2002; 17 (3): 1470 1478
IG.20. Moresco, RM; Volonte, MA; Messa, C; Gobbo, C; Galli, L;
Carpinelli, A; Rizzo, G; Panzacchi, A; Franceschi, M; Fazio, F.
New perspectives on neurochemical effects of amantadine in
the brain of parkinsonian patients: a PET [C-11]raclopride
study. J.Neural Transm.; 2002; 109 (10): 1265 1274
IG.21. Parazzini, C; Baldoli, C; Scotti, G; Triulzi, F. Terminal
zones of myelination: MR evaluation of children aged 20-40
months. Am.J.Neuroradiol.; 2002; 23 (10): 1669 1673
IG.22. Picchio, M; Landoni, C; Messa, C; Gianolli, L; Matarrese,
M; De Cobelli, F; Del Maschio, A; Fazio, F. Positive [C11]choline and negative [F-18]FDG with positron emission tomography in recurrence of prostate cancer. Am.J.Roentgenol.;
2002; 179 (2): 482 484
IG.23. Pirola, R; Mundo, E; Bellodi, L; Bareggi, SR. Simultaneous
determination of clomipramine and its desmethyl and hydroxy
metabolites in plasma of patients by high- performance liquid
chromatography after solid-phase extraction. J.Chromatogr.B;
2002; 772 (2): 205 210
IG.24. Rabiner, EA; Messa, C; Sargent, PA; Husted-Kjaer, K;
Montgomery, A; Lawrence, AD; Bench, CJ; Gunn, RN; Cowen,
P; Grasby, PM. A database of [C-11]WAY-100635 binding to 5HT1A receptors in normal male volunteers: Normative data
and relationship to methodological, demographic, physiological, and behavioral variables. Neuroimage; 2002; 15 (3): 620632
IG.25. Redaelli, A; Rizzo, G; Arrigoni, S; Di Martino, E; Origgi,
D; Fazio, F; Montevecchi, F. An assisted automated procedure
for vessel geometry reconstruction and hemodynamic simulations from clinical imaging. Comput.Med.Imag.Grap.; 2002;
(2): 143-152
IG.26. Riddell, C; Buvat, I; Savi, A; Gilardi, MC; Fazio, F. Iterative reconstruction of SPECT data with adaptive regularization.
IEEE Trans.Nucl.Sci.; 2002; 49 (5): 2350 2354
IG.27. Sironi, S; Bellomi, M; Villa, G; Rossi, S; Del Maschio, A.
Clinical stage I carcinoma of the uterine cervix: Value of preoperative magnetic resonance imaging in assessing parametrial invasion. Tumori; 2002; 88 (4): 291 295
IG.28. Tortorano, AM; Biraghi, E; Astolfi, A; Ossi, C; Tejada, M;
Farina, C; Perin, S; Bonaccorso, C; Cavanna, C; Raballo, A;
Grossi, A. European Confederation of Medical Mycology
(ECMM) prospective survey of candidaemia: report from one
Italian region. J.Hosp.Infect.; 2002; 51 (4): 297 304
IG.29. Veronesi, G; Landoni, C; Pelosi, G; Picchio, M; Sonzogni,
A; Leon, ME; Solli, PG; Leo, F; Spaggiari, L; Bellomi, M;
Fazio, F; Pastorino, U. Fluoro-deoxi-glucose uptake and angiogenesis are independent biological features in lung metastases.
Br.J.Cancer; 2002; 86 (9): 1391 1395
IG.30. Viviani, MA; Antinori, S; Cogliati, M; Esposto, MC; Pinsi,
G; Casari, S; Bergamasco, A; De Santis, ML; Ghirga, P; Bonaccorso, C; Jacchetti, G; Niero, F; Ammassari, A; Morace, G;
Foppa, CU; Ossi, C; Montagna, MT; Angarano, G; Farina, C;
Maggiolo, F; Moroni, M; Pierdomenico, S; Michelone, G; Cavanna, C; Viti, F; Carli, T; Barchiesi, F; Agrappi, C; Mena, M;
Giola, M; Lombardi, G; Tinelli, M; Ceraminiello, A; Codeluppi, M; Casolari, C; Foresti, S; Bramati, S; Ruggieri, A; Caggese,
L; Astolfi, A; Bonora, S; Scotton, PG; Rigoli, R; Angioni, G;
Meneghetti, F; Di Perri, G. European Confederation of Medical Mycology (ECMM) prospective survey of cryptococcosis:
Report from Italy. Med.Mycol.; 2002; 40 (5): 507 517
IG.31. Zetterberg, H; Coppola, A; D’Angelo, A; Rymo, L; Spandidos, DA; Blennow, K. MTHFR C677T and A1298C polymorphisms and mutated sequences occurring in cis.
Eur.J.Hum.Genet.; 2002; 10 (10): 579 582
IG.32. Bettinardi, V; Alenius, S; Numminen, P; Teras, M; Gilardi,
MC; Fazio, F; Ruotsalainen, U. Implementation and evaluation
of an ordered subsets reconstruction algorithm for transmission
PET studies using median root prior and inter-update median
filtering. Eur.J.Nucl.Med.Mol.Imaging; 2003; 30 (2): 222-231
IG.33. Bettinardi, V; Gilardi, MC; Lecchi, M; Savi, A; Castiglioni,
I; Rizzo, G; Fazio, F. Integrated CT/PET systems. Nucl.Phys.BProc.Suppl; 2003; 125 (): 139-144
IG.34. Binetti, G; Signorini, S; Squitti, R; Alberici, A; Benussi, L;
Cassetta, E; Frisoni, GB; Barbiero, L; Feudatari, E; Nicosia, F;
Testa, C; Zanetti, O; Gennarelli, M; Perani, D; Anchisi, D; Ghidoni, R; Rossini, PM. Atypical dementia associated with a novel
presenilin-2 mutation. Ann.Neurol; 2003; 54 (6): 832-836
IG.35. Deblieck, C; Pesenti, G; Scifo, P; Fazio, F; Bricolo, E; Lo,
Russo, G; Scialfa, G; Cossu, M; Bottini, G; Paulesu, E. Preserved functional competence of perilesional areas in drug-resistant epilepsy with lesion in supplementary motor cortex: fMRI and neuropsychological observations. Neuroimage; 20 (4):
IG.36. Goerendt, IK; Messa, C; Lawrence, AD; Grasby, PM; Piccini, P; Brooks, DJ. Dopamine release during sequential finger
movements in health and Parkinson’s disease: a PET study.
Brain; 2003 126 ()Part 2:312-325
IG.37. Matarrese, M; Soloviev, D; Todde, S; Neutro, F; Petta, P;
Carpinelli, A; Brussermann, M; Kienle, MG; Fazio, F. Preparation of [C-11] radioligands with high specific radioactivity on a
commercial PET tracer synthesizer. Nucl.Med.Biol; 2003; 30
(1): 79-83
IG.38. Messa, C; Colombo, C; Moresco, RM; Gobbo, C; Galli, L;
Lucignani, G; Gilardi, MC; Rizzo, G; Smeraldi, E; Zanardi, R;
Artigas, F; Fazio, F. 5-HT2A receptor binding is reduced in
drug-naive and unchanged in SSRI-responder depressed patients compared to healthy controls: a PET study. Psychopharmacology (Berl.): 2003; 167 (1): 72-78
IG.39. Parazzini, C; Mammi, S; Comola, M; Scotti, G. Magnetic
resonance diffusion-weighted images in Creutzfeldt-Jakob disease: case report. Neuroradiology; 2003; 45 (1): 50-52
IG.40. Pastorino, U; Bellomi, M; Landoni, C; De Fiori, E; Arnaldi, P; Picchio, M; Pelosi, G; Boyle, P; Fazio, F. Early lung-cancer detection with spiral CT and positron emission tomography
in heavy smokers: 2-year results. Lancet; 2003; 362 (9384): 593597
IG.41. Paulesu, E; Perani, D; Blasi, V; Silani, G; Borghese, NA;
De Giovanni, U; Sensolo, S; Fazio, F. A functional-anatomical
model for lipreading. J.Neurophysiol; 2003; 90 (3): 2005-2013
IG.42. Perani, D; Abutalebi, J; Paulesu, E; Brambati, S; Scifo, P;
Cappa, SF; Fazio, F. The role of age of acquisition and language
usage in early, high-proficient bilinguals: An fMRI study during
verbal fluency. Hum.Brain Mapp; 2003; 19 (3): 170-182
IG.43. Perani, D; Cappa, SF; Tettamanti, M; Rosa, M; Scifo, P;
Miozzo, A; Basso, A; Fazio, F. A fMRI study of word retrieval in
aphasia. Brain Lang; 2003; 85 (3): 357-368
IG.44. Picchio, M; Messa, C; Landoni, C; Gianolli, L; Sironi, S;
Brioschi, M; Matarrese, M; Matei, DV; De Cobelli, F; Del Maschio, A; Rocco, F; Rigatti, P; Fazio, F. Value of [C-11]cholinepositron emission tomography for re-staging prostate cancer: A
comparison with [F-18]fluorodeoxyglucose-positron emission
tomography. J.Urol; 2003; 169 (4): 1337-1340
IG.45. Picchio, M; Savi, A; Lecchi, M; Landoni, C; Gianolli, L;
Brioschi, M; Rossetti, C; Gilardi, MC; Fazio, F. Evaluation of
the clinical performances of a large NaI(Tl) crystal 3D PET
scanner. Q.J.Nucl.Med; 2003; 47 (2): 90-100
IG.46. Picchio, M; Sironi, S; Messa, C; Mangili, G; Landoni, C;
Gianolli, L; Zangheri, B; Vigano, R; Aletti, G; De Marzi, P; De
Cobelli, F; Del Maschio, A; Ferrari, A; Fazio, F. Advanced ovarian carcinoma: usefulness of [F-18]FDG-PET in combination
with CT for lesion detection after primary treatment. Q.J.Nucl.Med; 2003; 47 (2): 77-84
IG.47. Righini, A; Bianchini, E; Parazzini, C; Gementi, P; Ramenghi, L; Baldoli, C; Nicolini, U; Mosca, F; Triulzi, F. Apparent diffusion coefficient determination in normal fetal brain: A
prenatal MR imaging study. Am.J.Neuroradiol; 2003; 24 (5):
IG.48. Salmon, E; Garraux, G; Delbeuck, X; Collette, F; Kalbe,
E; Zuendorf, G; Perani, D; Fazio, F; Herholz, K. Predominant
ventromedial frontopolar metabolic impairment in frontotemporal dementia. Neuroimage; 2003; 20 (1): 435-440
IG.49. Simionato, F; Scomazzoni, F; Righi, C. GDC embolization
of three intracranial wide-neck aneurysms using a novel self-expandable nitinol microstent. Riv.Neuroradiol; 2003; 16 (1): 175177
IG.50. Sironi, S; Picchio, M; Mangili, G; Garavaglia, E; Zangheri,
B; Messa, C; Voci, C; Taccagni, GL; Del Maschio, A; Fazio, F.
[F-18]fluorodeoxyglucose positron emission tomography as a
useful indicator of metastatic gestational trophoblastic tumor:
preliminary results in three patients. Gynecol.Oncol; 2003; 91
(1): 226-230
IG.51. Wikstrom, J; Wasser, MN; Pattynama, PMT; Bonomo, L;
Hamm, B; Del Maschio, A; Knopp, MV; Marchal, G; Barentsz,
JO; Oudkerk, M; Hentrich, HR; Dapra, M; Kirchin, MA; Shen,
NY; Spinazzi, A; Ahlstrom, H. Gadobenate dimeglumine-enhanced magnetic resonance angiography of the pelvic arteries.
Invest.Radiol; 2003; 38 (8): 504-515
IG.52. Anzalone, N; Scotti, R; Riva, R. Neuroradiologic differential diagnosis of cerebral intraparenchymal hemorrhage. Neurol. Sci.: 2004; 25(Suppl. 1): S3-S5
IG.53. Belloli, S; Moresco, RM; Matarrese, M; Biella, G; Sanvito,
F; Simonelli, P; Turolla, E; Olivieri, S; Cappelli, A; Vomero, S;
Galli-Kienle, M; Fazio, F. Evaluation of three quinoline-carboxamide derivatives as potential radioligands for the in vivo pet
imaging of neurodegeneration. Neurochem. Int.: 2004; 44(6):
IG.54. Bettinardi, V; Danna, M; Savi, A; Lecchi, M; Castiglioni, I;
Gilardi, MC; Bammer, H; Lucignani, G; Fazio, F. Performance
evaluation of the new whole-body PET/CT scanner: Discovery
ST. Eur. J. Nucl. Med. Mol. Imaging: 2004; 31(6): 867-881
IG.55. Brasca, LE; Zanello, A; De Cobelli, F; Zerbi, A; Fazio, F;
Del Maschio, A. Intrapancreatic accessory spleen mimicking a
neuroendocrine tumor: magnetic resonance findings and possible diagnostic role of different nuclear medicine tests. Eur. Radiol.: 2004; 14(7): 1322-1323
IG.56. Cappelli, A; Giuliani, G; Pericot Mohr, G; Gallelli, A;
Anzini, M; Vomero, S; Cupello, A; Scarrone, S; Matarrese, M;
Moresco, RM; Fazio, F; Finetti, F; Morbidelli, L; Ziche, M. A
non-peptide NK1 receptor agonist showing subpicomolar affinity. J. Med Chem. 2004 Mar 11;47(6):1315-8
IG.57. De Cobelli, F; Fiorina, P; Perseghin, G; Magnone, M; Venturini, M; Zerbini, G; Zanello, A; Mazzolari, G; Monti, L; Di
Carlo, V; Secchi, A; Del Maschio, A. L-arginine-induced vasodilation of the renal vasculature is preserved in uremic type 1
diabetic patients after kidney and pancreas but not after kidney-alone transplantation. Diabetes Care: 2004; 27(4): 947-954
IG.58. Di Serio, C; Biffi, S. Graphical chain models and mental
disorders: An application to pathological gambling. Biom. J.:
2004; 46(2): 273-283
IG.59. Fazio, F; Picchio, M; Messa, C. Is C-11-choline the most
appropriate tracer for prostate cancer? Eur. J. Nucl. Med. Mol.
Imaging: 2004; 31(5): 753-756
IG.60. Gugiatti, A; Grimaldi, A; Rossetti, C; Lucignani, G; De
Marchis, D; Borgonovi, E; Fazio, F. Economic analyses on the
use of positron emission tomography for the work-up of solitary
pulmonary nodules and for staging patients with non-small-celllung-cancer in Italy. Q. J. Nucl. Med. Mol. Imaging: 2004;
48(1): 49-61
IG.61. Lanzetta, M; Perani, D; Anchisi, D; Rosen, B; Danna, M;
Scifo, P; Fazio, F; Lundborg, G. Early use of artificial sensibility in hand transplantation. Scand. J. Plast. Reconstr. Surg.
Hand Surg.: 2004; 38(2): 106-111
IG.62. Lucignani, G; Panzacchi, A; Bosio, L; Moresco, RM;
Ravasi, L; Coppa, I; Chiumello, G; Frey, K; Koeppe, R; Fazio, F.
GABA(A) receptor abnormalities in Prader-Willi syndrome assessed with positron emission tomography and [C-11]flumazenil. Neuroimage: 2004; 22(1): 22-28
IG.63. Mangin, JF; Riviere, D; Cachia, A; Duchesnay, E; Cointepas, Y; Papadopoulos-Orfanos, D; Scifo, P; Ochiai, T;
Brunelle, F; Regis, J. A framework to study the cortical folding
patterns. Neuroimage: 2004; 23(Suppl. 1): S129-S138
IG.64. Matarrese, M; Salimbeni, A; Turolla, EA; Turozzi, D;
Moresco, RM; Poma, D; Magni, F; Todde, S; Rossetti, C; Sciarrone, MT; Bianchi, G; Kienle, MG; Fazio, F. C-11-Radiosynthesis and preliminary human evaluation of the disposition of the
ACE inhibitor [C-11]zofenoprilat. Bioorg. Med. Chem.: 2004;
12(3): 603-611
IG.65. Moresco, RM; Cavallaro, R; Messa, C; Bravi, D; Gobbo, C;
Galli, L; Lucignani, G; Colombo, C; Rizzo, G; Velonà, I;
Smeraldi, E; Fazio, F. Cerebral D2 and 5-HT2 receptor occupancy in schizophrenic patients treated with olanzapine or
clozapine. J. Psychopharmacol.: 2004; 18 (3):355-365
IG.66. Moresco, RM; Todde, S; Belloli, S; Simonelli, P; Panzacchi,
A; Rigamonti, M; Galli-Kienle, M; Fazio, F. In vivo imaging of
adenosine A2a receptors in rat and primate brain using
[11C]SCH442416. Eur. J. Nucl. Med. Mol. Imaging: 2004; 11
IG.67. Pelosi, E; Messa, C; Sironi, S; Picchio, M; Landoni, C; Bettinardi, V; Gianolli, L; Del Maschio, A; Gilardi, MC; Fazio, F.
Value of integrated PET/CT for lesion localisation in cancer patients: a comparative study. Eur. J. Nucl. Med. Mol. Imaging:
2004; 31(7): 932-939
IG.68. Rizzo, G; Cattaneo, M; Castellone, P; Castiglioni, I;
Ceresoli, GL; Messa, C; Landoni, C; Gilardi, MC; Arienti, R;
Cerutti, S; Fazio, F. Multi-modal medical image integration to
optimize radiotherapy planning in lung cancer treatment. Ann.
Biomed. Eng.: 2004; 32: 1399-1408.
IG.69. Sardanelli, F; Giuseppetti, GM; Panizza, P; Bazzocchi, M;
Fausto, A; Simonetti, G; Lattanzio, V; Del Maschio, A. Sensitivity of MRI versus mammography for detecting foci of multifocal, multicentric breast cancer in fatty and dense breasts using
the whole-breast pathologic examination as a gold standard.
Am. J. Roentgenol.: 2004; 183(4): 1149- 1157
IG.70. Sironi, S; Messa, C; Cistaro, A; Landoni, C; Provenzi, M;
Giraldi, E; Sonzogni, A; Fazio, F. Recurrent hepatoblastoma in
orthotopic transplanted liver: Detection with FDG positron
emission tomography. Am. J. Roentgenol.: 2004; 182(5): 12141216
IG.71. Sironi, S; Messa, C; Mangili, G; Zangheri, B; Aletti, G;
Garavaglia, E; Vigano, R; Picchio, M; Taccagni, G; Del Maschio, A; Fazio, F. Integrated FDG PET/CT in patients with
persistent ovarian cancer: Correlation with histologic findings.
Radiology: 2004; 233(2): 433-440
IG.72. Venturini, M; Angeli, E; Salvioni, M; De Cobelli, F; Ronzoni, M; Aldrighetti, L; Stella, M; Carlucci, M; Staudacher, C;
Di Carlo, V; Ferla, G; Villa, E; Del Maschio, A. Complications
after percutaneous transaxillary implantation of a catheter for
intraarterial chemotherapy of liver tumors: Clinical relevance
and management in 204 patients. Am. J. Roentgenol.: 2004;
182(6): 1417-1426
IG.73. Zangheri, B; Messa, C; Picchio, M; Gianolli, L; Landoni,
C; Fazio, F. PET/CT and breast cancer. Eur. J. Nucl. Med. Mol.
Imaging: 2004; 31(Suppl 1):S135-42
ALEMBIC: an innovative microscopy facility
D. Dunlap, M. C. Panzeri, C. Covino, F. Grohovaz
The Advanced Light and Electron Microscopy BioImaging Center (ALEMBIC) is a DIBIT-HSR resource intended to expedite or perform biological experiments requiring electronic or optical imaging through the ready
access to state-of-the-art instrumentation. In the past year the staff has (1) performed or coordinated maintenance as required and has performed numerous experiments as a service. (2) To promote independent research,
three courses covering the theory and practice of modern microscopy were presented. (3) A meeting of a pilot
group was held to establish priorities and discuss operational procedures. As a result, the staff identified two recently developed microscopes that might be appropriate for the facility. A one-week trial of one of these was
held in December. (4) An electron microscope with spectral analysis capabilities was purchased. (5) A microscope with digital color camera for histological work was installed. (6) Preliminary work was carried out for future implementation of single molecule microscopy setups. (7) Numerous meetings were held to oversee the realization of new laboratories for the microscopy facility.
CFCM: the San Raffaele-Telethon Core Facility for Conditional Mutagenesis
M. E. Bianchi, L. Ronfani, T. Pusterla, I. Benzoni, P. Scarfone, E. Allievi, J. Gonzalez
The Core Facility for Conditional Mutagenesis (CFCM) was born in 1999 to support research using mouse
models. Transgenic and knock-out mice are widely used because of their high impact on the understanding of
protein function, and as models for human diseases. CFCM staff routinely microinjects DNA and recombinant
ES cells, creating transgenic and gene targeted mice, respectively. Further, the Facility rederives mice making
them compatible with the sanitary standards of the HSR Animal House. CFCM now also performs ES cell engineering. Researchers who start work for the first time with mice receive help and suggestions by the CFCM
staff: how to choose the proper animal model, how to plan experiments and breeding, how to manipulate and
genotype mice, etc. More information can be found at the site http://www.sanraffaele.org/research/cfcm/ under Research>Services and Open Labs. Up to now CFCM has completed about 150 jobs with a success rate of
higher than 95%.
PROtein MIcrocharacterization FAcility
M. Alessio, A. Bachi, A. Cattaneo
The Protein Microsequencing Facility was established in 2000 at the Scientific Institute San Raffaele-DiBit, in
collaboration with IFOM (FIRC Institute of Molecular Oncology), under the sponsorship of FIRC (Federazione Italiana Ricerca sul Cancro). The ProMiFa provides technological innovations, training of expert staff in
2DE gel electrophoresis, biological mass spectrometry and protein micro-characterization. The facility is
equipped with all the tools required for proteomics studies: state of the art mass spectrometers, N-terminal se-
quencer, 2D gel electrophoresis and bioinformatics. Access to mass spectrometry and bioinformatics is a crucial
key in the emerging field of proteomics, the large scale study of proteins and of their interactions, which is giving a major contribution to the description and understanding of biological processes at the molecular level.
Several collaborations have been established with a number of Italian and foreign scientists to develop new
methods and technologies fulfilling the requirements of biological research.
Development of 2 dimensional PAGE facility for the analysis, isolation and characterization of
differentially expressed proteins
V. Corti, A. Conti, M. Alessio
In the post-genomic era, large scale analytical tools are necessary for high throughput protein characterization. Proteomic analysis of biological samples is a rapidly expanding field encompassing both broad-based
screening and strategies focused on analysis of discrete subproteomes. Proteomics design the analysis and characterization of cellular proteins by using a combination of sophisticated techniques including two-dimensional
gel electrophoresis (2DE), image analysis, mass spectrometry, and bioinformatics. To date, the 2DE technique
remains one of the central separation methods for the global analysis of complex mixtures of proteins.We setup
high resolution 2DE systems used for the identification of proteins differentially expressed in different pathological and physiological conditions, as well as for the identification of some protein post-translational modifications. The system is widely used in the Institute within several collaborative projects.
YAC Screening Centre
C. Sala, M. Goegan, D. Toniolo
The YAC Screening Centre provides genomic libraries in PAC and BAC vectors. Clones from the human library RPCI-11 in BAC were fully sequenced by different sequencing projects. The YSC distributes clones from
segment 1, made of 288 microtiter plates. The YSC also provides two PAC libraries. The human RPCI-5 and
the mouse RPCI-21 that have been only partly sequenced. The RPCI-21 library was constructed from DNA of
the mouse strain 129/SvevTACfBr and clones can be used to generate mouse mutants by homologous recombination in ES cells. The Centre provides DNA pools for screening and clones from all libraries.
Bioinformatics Service
G. Verde
The new Bioinformatic service mission is to support the biological research with information technology.
This goal has to be achieved through the following means:
Computing facilities
Computing power and storage space
Installation and maintenance of public bioinformatics analysis software
Development of parallel software and databases for bioinformatics
Centralization of experimental data management
Increased data security
Improved privacy management
Improved data availability
Add value to experimental data
Generate new data and new software
Statistical analysis
Biological system modeling
Web-based data publishing
San Raffaele plans to apply extensively parallel computing techniques to biomedical problems. The parallel
supercomputer DeepBio, capable of 400 billion operations per second, has recently been donated by IBM and
is currently being configured and tested. Several collaborations with major national institutions are being established to support the effort of creating new bioinformatics software for massively parallel computers.
During the past year, several research and development activities have been performed in internal collaborations: image analysis and mathematical modelling for DNA binding visualization; annotation of protein database for mass-spectrometry and customization of the proteomics software Mascot; data management for analysis of genetic isolates; molecular dynamics using the GROMACS software in parallel computing.
See: Genomics for human disease diagnosis in Molecular Biology & Functional Genomics
External Quality Assessment Schemes (EQAS)
F. Ceriotti, C. A. Ferrero, A. Carobene, E. Guerra
The activities in the field of External Quality Assessment (EQA) continued successfully in 2004. The participation in the program for Clinical Chemistry involved 407 laboratories. This program remains the only Italian
EQAS that can provide a real accuracy target. In fact the control materials distributed to the participants are
tested for commutability and have a target value assigned by a reference method for 11 components (glucose,
creatinine, cholesterol, uric acid, sodium, potassium, AST, ALT, GGT, CK, LDH and Amylase). The two EQA
programs on coagulation and haematology involved 360 participants each. The EQA scheme on Hormones and
Tumour markers, initiated in 2003, now involves more than 200 participants.
Cholesterol network activities
F. Ceriotti, C. A. Ferrero, A. Carobene, E. Guerra, E. Platania
During the year 2004 our unit continued its activity of certification of the cholesterol measurement of several
Italian laboratories. The project of standardisation and control of lipid measurements in 12 lipid clinics involved in the clinical trial “MINDIT” is still ongoing and two standardisation exercises have been performed in
2004. The results obtained in the first ring trial organised by the Joint Committee on Traceability in Laboratory
Medicine (JCTLM) were satisfactory for cholesterol and our unit will probably receive a provisional accreditation as Reference Laboratory.
Standardisation in enzymology
F. Ceriotti, C. Ferrero, A. Carobene, E. Guerra, E. Platania
In the framework of an international joint project between IFCC (International Federation of Clinical Chemistry) and IRMM (Institute for Reference Materials and Measurements of the European Community) our unit
collaborated in the verification of the amylase reference method. We are waiting for the certification campaign
for ALP and AST reference materials. The results obtained in the first ring trial organised by the Joint Committee on Traceability in Laboratory Medicine (JCTLM) were satisfactory for AST and our unit will probably receive a provisional accreditation as Reference Laboratory for enzymatic analyses.
Separative Tecniques applied to Clinical & Basic Research
I. Fermo, L. Zagato, G. Cighetti, L. Cremonesi, A. D’Angelo, P. Fiorina, R. Paroni, P. Piatti
Our activity concerns the use of HPLC and Capillary Electrophoresis (CE) to develop original analytical
methods. Markers of endothelial dysfunction: we are always involved in the Hcy analysis to investigate the influence of genetics defects in patients with hyperhomocysteinemia or with Down’s syndrome. Using an HPLC
procedure set-up in our laboratory, methylated arginines ADMA and SDMA were studied in subjects with renal failure and with metabolic syndrome. Enzymatic Activity: thanks to a fast and simple HPLC method, the
current year we have monitored ACE activity in Human Embrionic Kidney (HEK293) cells transfected with
genes of interest and primary rat mesangial cells from parental and congenic strains. Proteins: in the field of
proteins we set up an original CE procedure for monitoring Carbohydrate Deficient Transferrine, one of the
most reliable markers of chronic alcoholic abuse. This method will allow to identify eventual genetic variants
and will be used as a confirmatory technique of the current immunoassay used for routine screening. Identification of DNA mutations: using D-HPLC we set-up conditions to screen different gene involved in several diseases.
E-services for life and health
A. Sanna, P. Bonini
E-Services for Life and Health is an interdisciplinary Information Technology and Business Modeling team of
15. The Team Mission is to design-develop-deploy highly innovative & effective, safe & trusted healthcare delivery systems, in cooperation with leading international industrial, academic and research partners. Core competencies focus on information and communication technology that enables systematic and timely provision of
tailored services to support patients-citizens decisions (e.g., life style behaviors, drug compliance, nutrition):
e.g., European R&D Projects: · DRIVE (Drug in Virtual Enterprise – IST 12040: drug compliance and patient
safety for hospitalized patients and drug supply chain services); · PIPS (personalized information platform for
health and life services – IST 507019 innovative knowledge-based services to the citizen in his/her daily life).
Services are personalized and based on preventive/predictive medicine, ranging from drug compliance to continuity of care and impact on life styles. A related area of research is Citizen Privacy, PRIME (privacy and identity management in Europe – IST 507591: an integrated project in the area of trust and security, aiming at the realization of a multi-sector and large scale-sustainable approach to privacy and identity management.
Haemostasis, fibrinolysis and inflammation in cardiac surgery
A. D’Angelo, P. Della Valle
In collaboration with the Division of Cardiac Surgery and the Department of Anesthesia, we evaluated the
blood-sparing effects of intra-operative moderate acute normovolemic hemodilution combined with preoperative tranexamic acid treatment and “on demand” shed blood re-infusion in patients undergoing off-pump coronary surgery (OPCAB), showing a potential for reduced allogeneic transfusion requirements. We also evaluated
the influence of tranexamic acid infusion on bleeding and laboratory parameters of coagulation, fibrinolysis
and inflammation in patients scheduled for on-pump (CABG) or OPCAB surgery. Patients were double-blind
randomized to treatment with tranexamic acid or saline. Tranexamic acid reduced total postoperative bleeding
in both OPCAB and CABG patients, with 80% reduction in bleeding exceeding 600 ml and no apparent effect
on thrombotic complications or outcome. This was associated to a reduction in plasma D-dimer and interleukin-6 levels. By affecting fibrinolysis and inflammation tranexamic acid reduces bleeding both in OPCAB
and CABG surgery, without an apparent increased risk of thrombotic complications.
The CD40/CD40 ligand signalling pathway in the antiphospholipid antibody syndrome
A. D’Angelo, L. Crippa, A. Fattorini
Patients with antiphospholipid antibodies (aPL) may suffer arterial and venous thrombosis. The CD40/CD40
ligand (CD40L) signalling pathway promotes atherosclerosis and exerts prothrombotic activity. In collaboration with colleagues from the University La Sapienza, Rome, we explored sCD40L (the soluble form of CD40L)
plasma levels in 30 consecutive SLE outpatients with (aPL+) and without (aPL-) antiphospholipid antibodies.
Compared to healthy controls, SLE patients had higher significantly sCD40L and prothrombin fragment 1.2
(F1+2) levels. 8 of 14 aPL+ patients and 2 of 16 aPL- patients had suffered thrombotic events. SLE aPL- patients had sCD40L levels higher than healthy controls, but significantly lower than SLE aPL+ patients. SLE
aPL+ patients with history of thrombosis, but not subjects with previous thrombosis free of SLE and aPL, had
higher circulating sCD40L levels than SLE aPL+ patients with no history of thrombosis. Enhanced circulating
levels of sCD40L may account for a novel mechanism of thrombosis in the aPL syndrome.
Intrasurgical autologous plasmin in diabetic macular edema
A. D’Angelo, P. Della Valle
In collaboration with the Department of Opthalmology and Visual Sciences, we evaluated in a prospective
study the efficacy of intrasurgical autologous plasmin in producing posterior vitreous detachment in patients
with diabetic macular edema secondary to posterior hyaloidal traction. Blood (30 ml) was drawn from patients
in 0.129 M trisodium citrate 5-15 days before surgery. After obtainment of platelet poor plasma, all following
procedures were carried out under laminar flow. Plasminogen was isolated by chromatography on lysinesepharose and eluted with 15 mM e-aminocaproic acid. After concentration in a centrifugal filter unit, the eACA was removed from the samples by repeated washing and centrifugation. The plasminogen preparation (1
mg/ml) was activated by incubation with streptokinase (50,000 U/mg plasminogen) for 10 min at RT. The plasmin preparation was sterilized and stored in 0.25 ml aliquots at –80° C until use. All preparations were free of
bacteria or endotoxin contamination. Plasmin activity ranged from 20 to 30 IU/ml, was stable for at least two
months at –80°C, and declined to 77% of baseline 24 hours after thawing at RT.
Fondaparinux or enoxaparin for the initial treatment of symptomatic deep venous thrombosis a
randomized trial
A. D’Angelo, A. Fattorini, S. Viganò D’Angelo, P. Della Valle, L. Crippa
The current standard initial therapies for deep venous thrombosis (DVT) are low-molecular-weight heparin
and unfractionated heparin. To evaluate whether fondaparinux has efficacy and safety similar to those of enoxaparin in patients with deep venous thrombosis we took part into a randomized, double-blind study involving
154 centers worldwide and enrolling 2205 patients with acute symptomatic DVT treated with either fondaparinux, 7.5 mg s.c. once daily, or enoxaparin, 1 mg/kg of body weight, s.c. twice daily for at least 5 days and
until vitamin K antagonists induced an INR greater than 2.0. The primary efficacy outcome was the 3-month incidence of symptomatic recurrent venous thromboembolic complications. The main safety outcomes were major bleeding during initial treatment and death. Recurrent thromboembolism (3.9% vs. 4.1%), major bleeding
(1.1% vs 1.2%) and mortaliy rates (3.8% vs 3.0%) were similar in patients receiving fondaparinux or enoxaparin respectively. Once-daily subcutaneous fondaparinux was at least as effective (not inferior) and safe as
twice-daily, body weight–adjusted enoxaparin in the initial treatment of patients with symptomatic DVT.
Coagulation variables in diabetes mellitus
A. D’Angelo, P. Della Valle
In collaboration with the Departments of Internal Medicine and Surgery we evaluated in a cross-sectional
study hemostatic abnormalities of uremic type 1 diabetic patients (U+T1DM), type 1 diabetic patients
(T1DM), uremic type 1 diabetic kidney-pancreas transplanted patients (KP), uremic type 1 diabetic kidneyalone transplanted diabetic patients (KD), and healthy controls (C). The U+T1DM group showed the higher
degree of abnormalities in platelet properties. The KP group, and to a lesser extent the KD group, had normal
platelet receptor expression and normal levels of hypercoagulability markers and natural anticoagulants. In collaboration with the Unit of Immunology of Diabetes, we evaluated coagulation and inflammatory markers in
islet culture media and in type 1 diabetic patients transplanted with the same islet preparations. Plasma Ddimer levels increased early in transplanted patients. Tissue factor (TF) and MCP-1 were always detected in supernatants of islet preparations. D-dimer, ALT and AST peak values correlated with TF and MCP-1 levels in
islet supernatants. Reducing the islet pro-inflammatory state may avoid early post-transplant complications.
Modeling late toxicity in 3D conformal radiotherapy by correlating clinical data with dose-volume
parameters and biological models
C. Fiorino, G. M. Cattaneo, T. Rancati, C. Cozzarini, G. Sanguineti, V. Vavassori, G. Fellin, R. Valdagni
3D Conformal Radiotherapy (3DCRT) using modern treatment planning systems (TPS) opened new possibilities in the modelling of dose-volume effects of irradiated normal tissues. Individual 3D dose-volume information may be used to model the clinically reported effects in order to define quantitative estimators of the risk of
late toxicity after 3DCRT. In this way, during planning optimisation, the calculated dose-volume histogram
(DVH) of the considered organs at risk may be used to predict the risk of toxicity and orient the planner in selecting the best treatment. A number of investigations have been conducted on patient populations treated at
our and other Institutions. Important results were found for late rectal bleeding after 3DCRT for prostate cancer and lung toxicity after lung cancer 3DCRT. Rectal bleeding was found to be strongly correlated with the
fraction of rectum irradiated at certain levels of dose; optimal parameters of the radiobiological model were also found to fit clinical data. A new multi-centric prospective investigation on rectal toxicity coordinated by our
group completed patients enrollement during 2004 and final results are expected within 2007.
Treatment planning optimization by means of the use of fused imaging modalities and IMRT delivery
S. Broggi, C. Fiorino, P. Mangili, G. M. Cattaneo, R. Calandrino, I. Dell’Oca, C. Cozzarini, N. Di Muzio,
F. Fazio
Intensity Modulated Radiation Therapy (IMRT) aims to concentrate the dose in the Planning Target Volume
(PTV) while sparing the surrounding organs at risk (OAR). A new imaging technique combining state-of-theart PET and CT (PET/CT) has been recently introduced in clinical use. The availability of multi-modal images
allows both a more accurate definition of the real extension of neoplastic disease and, in certain cases, the definition of “radiobiological” maps of the tumor. Moreover, for thoracic/abdominal diseases, gated PET/CT acquisition reduces the effects of breathing and consequently the impact of tumor mobility, reducing the irradiated volumes through gated RT. Helical tomotherapy is an innovative solution for IMRT: the continuous gantry
and couch motion combined with a fan “modulated” beam, offer advantages with respect to 3D-conformal RT
and to other IMRT modalities, in creating deep dose gradients between PTV and OAR and to “boost” biological targets inside the PTV. The “dose sculpting” offered by Tomotherapy may significantly reduce the dose to
OARs permitting to explore new fractionation regimens with the hope of increasing the therapeutic ratio of
many cancers.
Rodent histopathology unit
F. Sanvito, S. Olivieri, M. Ponzoni, C. Doglioni
The principal aim of the Rodent Histopathology Unit is to support, complement and favour the advancement
of scientific projects, by providing conventional morphological analysis and immunophenotyping. The facility
is located in the Department of Pathology and offers the technical and the interpretative experience for the
analysis of tissues from animal models, evaluating the best histological or immunocytochemical procedures,
based on the expected results. To date, multiple collaborations within our Institute have been settled in order
to analyze the morphological and immunophenotipical patterns of murine models of diseases, treated with different therapeutical approaches.
Chlamydia infection and B-cell lymphomas: from molecular genetics to clinical treatment
M. Ponzoni, A. J. M. Ferreri, R. Dolcetti, A. G. Resti, M. Freschi, P. Ghia, L. Pecciarini, M. G. Cangi,
C. Doglioni
Our group recently reported for the first time an association between Chlamydia psittaci infection and Ocular Adnexal Lymphomas (OAL), including both MALT and non-MALT histotypes. We documented also an objective clinical response to the antibiotic doxycycline in a group of patients with Ocular Adnexal Lymphomas
(OAL) of MALT-type. We are actively working on extending this pilot therapeutic study and we are planning to
organize a multicenter phase II trial for the evaluation of antibiotic therapy as the first-line treatment of
Chlamydia psittaci-positive OALs. We are characterizing OAL genetic abnormalities to verify if MALT type
translocations could be predictive marker of antibiotic response; we are also analyzing IgHV status of B lymphoma cells in order to gain new insight on the pathogenesis of these disorders. The search for Chlamydia
psittaci and other Chlamydiaceae DNA in a larger group of nodal and extranodal, extra-orbital lymphomas,
will provide an assessment of the distribution of this pathogen in NHLs. The identification of this bacterium in
other NHL could support a more widespread role of Chlamydia species in human lymphomagenesis.
Primary Central Nervous System Lymphoma (PCNSL): from biology knowledge toward the optimization
of treatment
M. Ponzoni, A. J. M. Ferreri, F. Bertoni, M. G. Uguccioni, M. R. Terreni, C. Doglioni
Primary Central Nervous System Lymphoma (PCNSL) is an uncommon extranodal lymphoma, representing
more than 4% of brain tumors. Its incidence is increasing both in immunosuppressed and in immunocompetent individuals. PCNSL is a very aggressive tumour with poor outcome. A collaborative study with the IOSI
(Istituto Oncologico Svizzera Italiana) is under way with the aims to improve the knowledge of PCNSL pathogenesis and to optimize the treatment.
The role of the interaction between NM23 and PRUNE in correlation to worse prognosis in cancers.
G. Arrigoni, M. Zollo, A. D’Angelo, A. Andrè, V. Aglio, P. Carotenuto, L. Garzia, A. Cossu, G. Palmieri
The gene PRUNE was identified and fully characterised (Reymond et al 1999). The protein is a phosphoesterase ubiquitously expressed in human adult tissue. Prune amplification was found in 166 (90%) cases of
sarcoma. We have performed IHC in order to correlate tumour grading and staging with the level of nm23H1,H2 and prune (Forus et al 2001). A correlation with the metastastic potential and tumors aggressiveness, was
found in high grade sarcoma.We have analyzed the nm23-H1 and prune TMA technology, FISH and IHC in
breast cancer and the data show a correlation between Prune amplification, protein overexpression and reduced nm23-H1 expression in M+ breast cancer. PRUNE amplification leads to an increased prune phosphodiesterase activity that is due to a physical interaction with the nm23-H1 protein. The complex formation results in a substantial decrease of the level of free nm23-H1, influencing cell proliferation, cellular motility and
metastatic process (D’angelo et al 2004,Zollo et al 2005). In addition, PRUNE interaction with NM23S120G
impairs gain of function mutation, frequently observed in advanced stage neuroblastoma. Our goal is the prune
analysis of neuroblastoma.
See: Neurosciences
See: Imaging
See: Imaging
See: Imaging
CF.1. Bonini, P; Plebani, M; Ceriotti, F; Rubboli, F. Errors in laboratory medicine. Clin.Chem.; 2002; 48 (5): 691 698
CF.2. Broggi, S; Fiorino, C; Calandrino, R. In vivo estimation of
midline dose maps by transit dosimetry in head and neck radiotherapy. Br.J.Radiol.; 2002; 75 (900): 974 981
CF.3. Capitanio, M; Romano, G; Ballerini, R; Giuntini, M; Pavone,
FS; Dunlap, D; Finzi, L. Calibration of optical tweezers with differential interference contrast signals. Rev.Sci.Instrum.; 2002; 73
(4): 1687-1696
CF.4. Ceriotti, F; Del Buono, L; Bonini, P; Germagnoli, L; Ferrero,
CA; Pezzo, M; Marocchi, A. A two-center evaluation of the
blood gas Immediate Response Mobile Analyzer (IRMA).
Clin.Chem.Lab.Med.; 2002; 40 (2): 182 191
CF.5. Cighetti, G; Allevi, P; Anastasia, L; Bortone, L; Paroni, R.
Use of methyl malondialdehyde as an internal standard for malondialdehyde detection: Validation by isotope-dilution gas chromatography-mass spectrometry. Clin.Chem.; 2002; 48 (12): 2266
CF.6. D’Angelo, A; Piovella, F. Optimal duration of oral anticoagulant therapy after a first episode of venous thromboembolism:
where to go? Haematologica; 2002; 87 (10): 1009-1013
CF.7. D’Angelo, A; Valle, PD; Crippa, L; Fattorini, A; Pattarini, E;
D’Angelo, SV. Relationship between international normalized ratio values, vitamin K-dependent clotting factor levels and in vivo
prothrombin activation during the early and steady phases of
oral anticoagulant treatment. Haematologica; 2002; 87 (10):
CF.8. Dominici, R; Cabrini, E; Cattozzo, G; Ceriotti, F; Grazioli, V;
Scapellato, L; Franzini, C. Intermethod variation in serum carcinoembryonic antigen (CEA) measurement. Fresh serum pools
and control materials compared. Clin.Chem.Lab.Med.; 2002; 40
(2): 167 173
CF.9. Fattorini, A; Crippa, L; D’Angelo, SV; Pattarini, E; D’Angelo,
A. Risk of deep vein thrombosis recurrence: High negative predictive value of D-dimer performed during oral anticoagulation.
Thromb.Haemost.; 2002; 88 (1): 162-163
CF.10. Fermo, I; Arcelloni, C; Paroni, R. High-performance liquid
chromatographic method to quantify total cysteine excretion in
urine. Anal.Biochem.; 2002; 307 (1): 181 183
CF.11. Fratelli, M; Demol, H; Puype, M; Casagrande, S; Eberini, I;
Salmona, M; Bonetto, V; Mengozzi, M; Duffieux, F; Miclet, E;
Bachi, A; Vandekerckhove, J; Gianazza, E; Ghezzi, P. Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes. Proc.Natl.Acad.Sci.U.S.A.;
2002; 99 (6): 3505-3510
CF.12. Galardi, S; Fatica, A; Bachi, A; Scaloni, A; Presutti, C; Bozzoni, I. Purified box C/D snoRNPs are able to reproduce sitespecific 2’-O-methylation of target RNA in vitro. Mol.Cell.Biol.;
2002; 22 (19): 6663-6668
CF.13. Jeppsson, JO; Kobold, U; Barr, J; Finke, A; Hoelzel, W;
Hoshino, T; Miedema, K; Mosca, A; Mauri, P; Paroni, R; Thienpont, L; Umemoto, M; Weykamp, C. Approved IFCC reference
method for the measurement of HbA(1c) in human blood.
Clin.Chem.Lab.Med.; 2002; 40 (1): 78 89
CF.14. Levy, F; Burri, L; Morel, S; Peitrequin, AL; Levy, N; Bachi,
A; Hellman, U; Van den Eynde, BJ; Servis, C. The final N-terminal trimming of a subaminoterminal proline- containing HLA
class I-restricted antigenic peptide in the cytosol is mediated by
two peptidases. J.Immunol.; 2002; 169 (8): 4161-4171
CF.15. Magni, F; Galbusera, C; Tremolada, L; Ferrarese, C; Kienle,
MG. Characterisation of adducts of the lipid peroxidation product 4-hydroxy-2-nonenal and amyloid beta-peptides by liquid
chromatography/electrospray ionisation mass spectrometry.
Rapid Commun.Mass Spectrom.; 2002; 16 (15): 1485-1493
CF.16. Panteghini, M; Ceriotti, F; Pagani, F; Secchiero, S; Zaninot-
to, M; Franzini, C. Recommendations for the routine use of pancreatic amylase measurement instead of total amylase for the diagnosis and monitoring of pancreatic pathology.
Clin.Chem.Lab.Med.; 2002; 40 (2): 97 100
CF.17. Paroni, R; Fermo, I; Cighetti, G. Validation of methyl malondialdehyde as internal standard for malondialdehyde detection by capillary electrophoresis. Anal.Biochem.; 2002; 307 (1):
92 98
CF.18. Piovella, F; Crippa, L; Barone, M; D’Angelo, SV; Serafini, S;
Galli, L; Beltrametti, C; D’Angelo, A. Normalization rates of
compression ultrasonography in patients with a first episode of
deep vein thrombosis of the lower limbs: association with DVT
recurrence and new thrombosis. Haematologica; 2002; 87 (5):
CF.19. Plebani, M; Bonini, P. Wrong biochemistry results Interdepartmental cooperation may help avoid errors in medical laboratories. Br.Med.J.; 2002; 324 (7334): 423 424
CF.20. Rastaldi, MP; Ferrario, F; Giardino, L; Dell’Antonio, G;
Grillo, C; Grillo, P; Strutz, F; Muller, GA; Colasanti, G; D’Amico, G. Epithelial-mesenchymal transition of tubular epithelial
cells in human renal biopsies. Kidney Int.; 2002; 62 (1): 137 146
CF.21. Schumann, G; Bonora, R; Ceriotti, F; Clerc-Renaud, P; Ferrero, CA; Ferard, G; Franck, PFH; Gella, FJ; Hoelzel, W; Jorgensen, PJ; Kanno, T; Kessner, A; Klauke, R; Kristiansen, N;
Lessinger, JM; Linsinger, TPJ; Misaki, H; Panteghini, M;
Pauwels, J; Schimmel, HG; Vialle, A; Weidemann, G; Siekmann,
L. IFCC primary reference procedures for the measurement of
catalytic activity concentrations of enzymes at 37 degrees C. Part
2. Reference procedure for the measurement of catalytic concentration of creatine kinase. Clin.Chem.Lab.Med.; 2002; 40 (6):
635 642
CF.22. Schumann, G; Bonora, R; Ceriotti, F; Clerc-Renaud, P; Ferrero, CA; Ferard, G; Franck, PFH; Gella, FJ; Hoelzel, W; Jorgensen, PJ; Kanno, T; Kessner, A; Klauke, R; Kristiansen, N;
Lessinger, JM; Linsinger, TPJ; Misaki, H; Panteghini, M;
Pauwels, J; Schimmel, HG; Vialle, A; Weidemann, G; Siekmann,
L. IFCC primary reference procedures for the measurement of
catalytic activity concentrations of enzymes at 37 degrees C. Part
3. Reference procedure for the measurement of catalytic concentration of lactate dehydrogenase. Clin.Chem.Lab.Med.; 2002; 40
(6): 643 648
CF.23. Schumann, G; Bonora, R; Ceriotti, F; Ferard, G; Ferrero,
CA; Franck, PFH; Gella, FJ; Hoelzel, W; Jorgensen, PJ; Kanno,
T; Kessner, A; Klauke, R; Kristiansen, N; Lessinger, JM;
Linsinger, TPJ; Misaki, H; Panteghini, M; Pauwels, J; Schiele, F;
Schimmel, HG; Weidemann, G; Siekmann, L. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. Part 4. Reference procedure for the measurement of catalytic concentration of alanine
aminotransferase. Clin.Chem.Lab.Med.; 2002; 40 (7): 718 724
CF.24. Schumann, G; Bonora, R; Ceriotti, F; Ferard, G; Ferrero,
CA; Franck, PFH; Gella, FJ; Hoelzel, W; Jorgensen, PJ; Kanno,
T; Kessner, A; Klauke, R; Kristiansen, N; Lessinger, JM;
Linsinger, TPJ; Misaki, H; Panteghini, M; Pauwels, J; Schiele, F;
Schimmel, HG; Weidemann, G; Siekmann, L. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. Part 5. Reference procedure for the measurement of catalytic concentration of aspartate
aminotransferase. Clin.Chem.Lab.Med.; 2002; 40 (7): 725 733
CF.25. Schumann, G; Bonora, R; Ceriotti, F; Ferard, G; Ferrero,
CA; Franck, PFH; Gella, FJ; Hoelzel, W; Jorgensen, PJ; Kanno,
T; Kessner, A; Klauke, R; Kristiansen, N; Lessinger, JM;
Linsinger, TPJ; Misaki, H; Panteghini, M; Pauwels, J; Schiele, F;
Schimmel, HG; Weidemann, G; SiekmaSchumann, G; Bonora,
R; Ceriotti, F; Ferard, G; Ferrero, CA; Franck, PFH; Gella, FJ;
Hoelzel, W; Jorgensen, PJ; Kanno, T; Kessner, A; Klauke, R;
Kristiansen, N; Lessinger, JM; Linsinger, TPJ; Misaki, H; Panteghini, M; Pauwels, J; Schiele, F; Schimmel, HG; Weidemann,
G; Siekmann, L. IFCC primary reference procedures for the
measurement of catalytic activity concentrations of enzymes at 37
degrees C. Part 6. Reference procedure for the measurement of
catalytic concentration of gamma-glutamyltransferase.
Clin.Chem.Lab.Med.; 2002; 40 (7): 734 738
CF.26. Siekmann, L; Bonora, R; Burtis, CA; Ceriotti, F; Clerc-Renaud, P; Ferard, G; Ferrero, CA; Forest, JC; Franck, PFH; Gella, FJ; Hoelzel, W; Jorgensen, PJ; Kanno, T; Kessner, A; Klauke,
R; Kristiansen, N; Lessinger, JM; Linsinger, TPJ; Misaki, H.
IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. Part 7.
Certification of four reference materials for the determination of
enzymatic activity of gamma- glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase according to IFCC reference procedures at 37 degrees C.
Clin.Chem.Lab.Med.; 2002; 40 (7): 739 745
CF.27. Siekmann, L; Bonora, R; Burtis, CA; Ceriotti, F; Clerc-Renaud, P; Ferard, G; Ferrero, CA; Forest, JC; Franck, PFH; Gella, FJ; Hoelzel, W; Jorgensen, PJ; Kanno, T; Kessner, A; Klauke,
R; Kristiansen, N; Lessinger, JM; Linsinger, TPJ; Misaki, H;
Mueller, MM; Panteghini, M; Pauwels, J; Schiele, F; Schimmel,
HG; Vialle, A; Weidemann, G; Schumann, G. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. Part 1. The concept of
reference procedures for the measurement of catalytic activity
concentrations of enzymes. Clin.Chem.Lab.Med.; 2002; 40 (6):
631 634
CF.28. Stearns-Kurosawa, DJ; Swindle, K; D’Angelo, A; Della
Valle, P; Fattorini, A; Caron, N; Grimaux, M; Woodhams, B;
Kurosawa, S. Plasma levels of endothelial protein C receptor respond to anticoagulant treatment. Blood; 2002; 99 (2): 526-530
CF.29. Testa, S; Morstabilini, G; Fattorini, A; Galli, L; Denti, N;
D’Angelo, A. Discrepant sensitivity of thromboplastin reagents
to clotting factor levels explored by the prothrombin time in patients on stable oral anticoagulant treatment: impact on the international normalized ratio system. Haematologica; 2002; 87 (12):
CF.30. Vytopil, M; Ricci, E; Dello, Russo, A; Hanisch, F; Neudecker, S; Zierz, S; Ricotti, R; Demay, L; Richard, P; Wehnert, M;
Bonne, G; Merlini, L; Toniolo, D. Frequent low penetrance mutations in the Lamin A/C gene, causing Emery Dreifuss muscular
dystrophy. Neuromusc.Disord.; 2002; 12 (10): 958-963
CF.31. Cappuzzo, F; Gregorc, V; Rossi, E; Cancellieri, A; Magrini,
E; Paties, CT; Ceresoli, G; Lombardo, L; Bartolini, S; Calandri,
C; De Rosa, M; Villa, E; Crino, L. Gefitinib in pretreated nonsmall-cell lung cancer (NSCLC): Analysis of efficacy and correlation with HER2 and epidermal growth factor receptor expression in locally advanced or metastatic NSCLC. J.Clin.Oncol;
2003; 21 (14): 2658-2663
CF.32. Conconi, A; Martinelli, G; Thieblemont, C; Ferreri, AJM;
Devizzi, L; Peccatori, F; Ponzoni, M; Pedrinis, E; Dell’Oro, S;
Pruneri, G; Filipazzi, V; Dietrich, PY; Gianni, AM; Coiffier, B;
Cavalli, F; Zucca, E. Clinical activity of rituximab in extranodal
marginal zone B-cell lymphoma of MALT type. Blood; 2003; 102
(8): 2741-2745
CF.33. Corletto, D; Iori, M; Paiusco, M; Brait, L; Broggi, S;
Ceresoli, G; Iotti, C; Calandrino, R; Fiorino, C. Inverse and forward optimization of one- and two-dimensional intensity-modulated radiation therapy-based treatment of concave-shaped planning target volumes: the case of prostate cancer. Radiother.Oncol; 2003; 66 (2): 185-195
CF.34. Cozzarini, C; Fiorino, C; Ceresoli, GL; Cattaneo, GM;
Bolognesi, A; Calandrino, R; Villa, E. Significant correlation between rectal DVH and late bleeding in patients treated after rad-
ical prostatectomy with conformal or conventional radiotherapy
(66.6-70.2 GY). Int.J.Radiat.Oncol.Biol.Phys; 2003; 55 (3): 688694
CF.35. Ferrari, M; Stenirri, S; Bonini, P; Cremonesi, L. Molecular
diagnostics by microelectronic microchips. Clin.Chem.Lab.Med;
2003; 41 (4): 462-467
CF.36. Fiorino, C; Gianolini, S; Nahum, AE. A cylindrical model of
the rectum: comparing dose-volume, dose- surface and dose-wall
histograms in the radiotherapy of prostate cancer. Phys.Med.Biol; 2003; 48 (16): 2603-2616
CF.37. Fiorino, C; Sanguineti, G; Cozzarini, C; Fellin, G; Foppiano,
F; Menegotti, L; Piazzolla, A; Vavassori, V; Valdagni, R. Rectal
dose-volume constraints in high-dose radiotherapy of localized
prostate cancer. Int.J.Radiat.Oncol.Biol.Phys; 2003; 57 (4): 953962
CF.38. Foppiano, F; Fiorino, C; Frezza, G; Greco, C; Valdagni, R.
The impact of contouring uncertainty on rectal 3D dose-volume
data: Results of a dummy run in a multicenter trial (AIROPROS01-02). Int.J.Radiat.Oncol.Biol.Phys; 2003; 57 (2): 573-579
CF.39. Gesu, GP; Marchetti, F; Piccoli, L; Cavallero, A. Levofloxacin and ciprofloxacin in vitro activities against 4,003 clinical bacterial isolates collected in 24 Italian laboratories. Antimicrob.Agents Chemother; 2003; 47 (2): 816-819
CF.40. Gianelli, U; Ponzoni, M; Moro, A; Alfano, RM; Pellegrini,
C; Giardini, R; Patriarca, C; Armiraglio, E; Bosari, S; Parafioriti,
A; Coggi, G. Mutations of the 5’ noncoding region of the BCL-6
gene in primary bone lymphomas. Ann.Hematol; 2003; 82 (11):
CF.41. Lia, G; Bensimon, D; Croquette, V; Allemand, JF; Dunlap,
D; Lewis, DEA; Adhya, SC; Finzi, L. Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping. Proc.Natl.Acad.Sci.U.S.A; 2003; 100 (20):
CF.42. Mancini, N; Canducci, F; Carletti, S; Berardinelli, E; Serafini, G; Grieco, A; Perotti, M; Malcangi, G; Danieli, MG; Varaldo,
PE; Clementi, M; Burioni, R. Heterogeneity of the humoral antiHCV/E2 response in persistently infected patients as demonstrated by divergent patterns of inhibition of the binding of antiHCV/E2 human monoclonal antibodies. J.Biol.Regul.Homeost.Agents; 2003; 17 (2): 183-187
CF.43. Mosca, A; Bonora, R; Ceriotti, F; Franzini, C; Lando, G; Patross, MC; Zaninotto, M; Panteghini, M. Assay using succinyldithiocholine as substrate: The method of choice for the
measurement of cholinesterase catalytic activity in serum to diagnose succinyldicholine sensitivity. Clin.Chem.Lab.Med; 2003; 41
(3): 317-322
CF.44. Mosca, A; Paleari, R; Ceriotti, F; Lapolla, A. Biological variability of albumin excretion rate and albumin-to-creatinine ratio
in hypertensive type 2 diabetic patients. Clin.Chem.Lab.Med;
2003; 41 (9): 1229-1233
CF.45. Pelosi, G; Pasini, F; Fraggetta, F; Pastorino, U; Iannucci, A;
Maisonneuve, P; Arrigoni, G; De Manzoni, G; Bresaola, E; Viale,
G. Independent value of fascin immunoreactivity for predicting
lymph node metastases in typical and atypical pulmonary carcinoids. Lung Cancer; 2003; 42 (2): 203-213
CF.46. Russo, A; Viotti, PL; Vitali, M; Clementi, M. Antimicrobial
activity of a new intact skin antisepsis formulation. Am.J.Infect.Control; 2003; 31 (2): 117-123
CF.47. Arrigoni, G; Doglioni, C. Atypical lipomatous tumor: molecular characterization. Curr. Opin. Oncol.: 2004; 16(4): 355358
CF.48. Barbareschi, M; Roldo, C; Zamboni, G; Capelli, P; Cavazza,
A; Macri, E; Cangi, MG; Chilosi, M; Doglioni, C. CDX-2 homeobox gene product expression in neuroendocrine tumors - Its
role as a marker of intestinal neuroendocrine tumors. Am. J.
Surg. Pathol.: 2004; 28(9): 1169-1176
CF.49. Benedetti, S; Merlini, L. Laminopathies: from the heart of
the cell to the clinics. Curr. Opin. Neurol.: 2004; 17(5): 553-560
CF.50. Bertoni, F; Conconi, A; Cogliatti, SB; Schmitz, SFH;
Ghielmini, M; Cerny, T; Fey, M; Pichert, G; Bertolini, F; Ponzoni, M; Baldini, L; Jones, C; Auer, R; Zucca, E; Cavalli, F; Cotter, FE. Immunoglobulin heavy chain genes somatic hypermutations and chromosome 11q22-23 deletion in classic mantle cell
lymphoma: a study of the Swiss Group for Clinical Cancer Research. Br. J. Haematol.: 2004; 124(3): 289-298
CF.51. Boeri, E; Canducci, F; Grasso, MA; Presi, S; Carrera, P;
Racca, S; Clementi, M. Phylogenetic internal control for HIV-1
genotypic antiviral testing. New microbiol.: 2004; 27(Suppl. 1):
CF.52. Buller, HR; Davidson, BL; Decousus, H; Gallus, A; Gent,
M; Piovella, F; Prins, MH; Raskob, G; Segers, AEM; Cariou R;
Leeuwenkamp, O; Lensing, AL; for the Matisse Investigators (A.
D’Angelo). Fondaparinux or Enoxaparin for the initial treatment
of symptomatic deep venous thrombosis. A randomized trial.
Ann. Intern. Med.: 2004; 140(11):867-73
CF.53. Burioni, R; Mancini, N; Carletti, S; Perotti, M; Grieco, A;
Canducci, F; Varaldo, PE; Clementi, M. Cross-reactive
pseudovirus-neutralizing anti-envelope antibodies coexist with
antibodies devoid of such activity in persistent hepatitis C virus
infection. Virology: 2004; 327(2): 242-248
CF.54. Casati, V; Benussi, S; Sandrelli, L; Grasso, MA; Spagnolo, S;
D’Angelo, A. Intraoperative moderate acute normovolemic hemodilution associated with a comprehensive blood-sparing protocol in off-pump coronary surgery. Anesth. Analg.: 2004; 98(5):
CF.55. Casati, V; Della Valle, P; Benussi, S; Franco, A; Gerli, C;
Baili, P; Alfieri, O; D’Angelo, A. Effects of tranexamic acid on
postoperative bleeding and related hematochemical variables in
coronary surgery: Comparison between on-pump and off-pump
techniques. J. Thorac. Cardiovasc. Surg.: 2004; 128(1): 83-91
CF.56. Clementi, M. Can modulation of viral fitness represent a target for anti-HIV-1 strategies? Microbiologica: 2004; 27(3): 207214
CF.57. Clementi, M; Canducci, F; Bagnarelli, P; Menzo, S. Intrahost evolution of human immunodeficiency virus type 1 and viral
fitness. New microbiol.: 2004; 27( Suppl. 1): 41-44
CF.58. D’Angelo, A; Mazzola, G; Fermo, I;. Gene-gene and geneenvironment interactions in mild hyperhomocysteinemia. Pathophysiol. Haemost. Thromb.: 2004: 33(5-6):337-41
CF.59. D’Angelo, A; Garzia, L; Andre, A; Carotenuto, P; Aglio, V;
Guardiola, O; Arrigoni, G; Cossu, A; Palmieri, G; Aravind, L;
Zollo, M. Prune cAMP phosphodiesterase binds nm23-H1 and
promotes cancer metastasis. Cancer Cell: 2004; 5(2): 137-149
CF.60. Fermo, I; Germagnoli, L; Soldarini, A; Dorigatti, F; Paroni,
R. Comments on “Capillary zone electrophoresis for determination of carbohydrate-deficient transferrin in human serum” - Reply. Electrophoresis: 2004; 25(11-ott): 1724-1725
CF.61. Fermo, I; Germagnoli, L; Soldarini, A; Dorigatti, F; Paroni,
R. Capillary zone electrophoresis for determination of carbohydrate-deficient transferrin in human serum. Electrophoresis:
2004; 25(3): 469-475
CF.62. Ferro, D; Pignatelli, P; Loffredo, L; D’Angelo, A; Varesini,
G; Violi, F. Soluble CD154 plasma levels in patients with systemic lupus erythematosus: modulation by antiphospholipid antibodies. Arthritis Rheum.: 2004:50; 1693-94.
CF.63. Foglieni, B; Cremonesi, L; Travi, M; Ravani, A; Giambona,
A; Rosatelli, MC; Perra, C; Fortina, P; Ferrari, M. beta-thalassemia microelectronic chip: A fast and accurate method for
mutation detection. Clin. Chem.: 2004; 50(1): 73-79
CF.64. Giorda, R; Cerritello, A; Bonaglia, MC; Bova, S; Lanzi, G;
Repetti, E; Giglio, S; Baschirotto, C; Pramparo, T; Avolio, L;
Bragheri, R; Maraschio, P; Zuffardi, O. Selective disruption of
muscle and brain-specific BPAG1 isoforms in a girl with a 6;15
translocation, cognitive and motor delay, and tracheo-oesophageal atresia. J. Med. Genet.: 2004; 41(6): e71
CF.65. Hoelzel, W; Weykamp, C; Jeppsson, JO; Miedema, K; Barr,
JR; Goodall, I; Hoshino, T; John, WG; Kobold, U; Little, R;
Mosca, A; Mauri, P; Paroni, R; Susanto, F; Takei, I; Thienpont,
L; Umemoto, M; Wiedmeyer, HM. IFCC reference system for
measurement of hemoglobin A(lc) in human blood and the National Standardization Schemes in the United States, Japan, and
Sweden: A method-comparison study. Clin. Chem.: 2004; 50(1):
CF.66. Mangili, P; Stea, L; Cattani, F; Lappi, S; Giglioli, F; Calamia,
E; Ziglio, F; Martinelli, R; Longobardi, B. Comparative study of
permanent interstitial prostate brachytherapy post-implant evaluation among seven Italian institutes. Radiother. Oncol.: 2004;
71(1): 13-21
CF.67. Nemni, R; Caniatti, LM; Gironi, M; Bazzigaluppi, E; De
Grandis, D. Stiff person syndrome does not always occur with
maternal passive transfer of GAD65 antibodies. Neurology:
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Mosca, A. Experiences in the measurement of RBC-bound IgG
as markers of cell age. Bioelectrochemistry: 2004; 62(2): 175-179
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Fitting late rectal bleeding data using different NTCP models:
results from an Italian multi-centric study (AIROPROS0101).
Radiother. Oncol.: 2004; 73(1): 21-32
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target volume definition and dose-volume parameters of rectum
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STAFF - 201
Massimo Degano
Paola Tornaghi
Biological mass spectrometry
Angela Bachi
Angela Cattaneo
Biology of myelin
Lawrence Wrabetz
Laura Feltri
Cinzia Ferri, Desirée Zambroni
Biomolecular NMR laboratory
Giovanna Musco
Cell Adhesion
Ivan de Curtis
Simona Paris
Cerebral cortex development
Antonello Mallamaci
Maria Pannese
Luisa Pintonello
Chromatin dynamics
Marco E. Bianchi, Professor of Molecular Biology
Alessandra Agresti
Francesco De Marchis
Cystic fibrosis
Massimo Conese
See: Gene and Stem Cells Therapy
Developmental neurobiology
Manolis Fanto
Silvia Frontini
Dynamic fluorescence spectroscopy in biomedicine
Valeria R. Caiolfa
Genetics of common disorders
Daniela Toniolo
Cinzia Sala
Genomics for human disease diagnosis
Maurizio Ferrari
Paola Carrera, Laura Cremonesi, Vito Lampasona
Nadia Soriani
Human inherited neuropathies
Alessandra Bolino
Leukocyte biology
Ruggero Pardi, Associate Professor of Pathology
Samuele Burastero, Monica Fabbri
Barbara Clissi, Stella Putignano
Molecular basis of Polycystic Kidney Disease (PKD)
Alessandra Boletta
Gianfranco Distefano
Molecular genetics
Francesco Blasi, Professor of Molecular Biology
Massimo Crippa, Roberta Mazzieri, Daniela Talarico, Nicolai Sidenius
Massimo Resnati
Molecular genetics of behavior
Riccardo Brambilla
Molecular genetics of membrane traffic
Maria Vittoria Schiaffino
Molecular histology
Pier Carlo Marchisio, Professor of Anatomy
Stefano Biffo, Associate Professor of Comparative Anatomy
Annarita Miluzio
Molecular oncology
Laura Beguinot
Jeanette Majer, Associate Professor of General Pathology
Monica Riolfo
Protein transport and secretion
Roberto Sitia, Professor of Molecular Biology
Simone Cenci
Claudio Fagioli, Elena Pasqualetto, Elisa Soprana
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Proteome biochemistry
Massimo Alessio
Antonio Conti
Proteomics of iron metabolism
Sonia Levi
Anna Cozzi, Paolo Santambrogio
General Medicine, diabetes, endocrinology and metabolic diseases
Emanuele Bosi, Associate Professor of Internal Medicine
Gabriella Galimberti, Roberto Lanzi, Matteo Rocco Pastore, Piermarco Piatti, Luca Falqui
Alberto Davalli, Marco Manzoni, Sabina Martinenghi, Maria Grazia Perfetti, Alessandro Saibene,
Maurizio Storti
Franco Folli, Lucilla D. Monti
Monica Marchi
Alessandra Caputo, Sabrina Costa, Emilia Sandoli
Clinical transplantation
Antonio Secchi, Associate Professor of Internal Medicine
Ennio La Rocca, Paola Maffi, Rossana Caldara
Paolo Fiorina, Federico Bertuzzi
Telethon/JDRF Center for beta cell replacement and immunology of diabetes
Ezio Bonifacio
Marika Falcone, Lorenzo Piemonti
Cell therapy for type 1 diabetes
Federico Bertuzzi
Rita Nano
Barbara Antonioli, Paola Magistretti, Raffaella Melzi
Metabolic unit
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Alberto Battezzati, Andrea Caumo, Gianluca Perseghin, Ileana Terruzzi, Giampaolo Zerbini
Daniela Gabellini, Francesca Ragogna
Bone metabolism
Alessandro Rubinacci
Isabella Villa
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Paolo Giorgio Arcidiacono, Mario Guslandi, Enzo Masci, Sandro Passaretti
Lorella Fanti, Alberto Mariani, Edi Viale
Silvia Carrara, Maura Corsetti, Simona Curioni, Roberto Frego, Gianni Mezzi
Nephrology and Hypertension
Giuseppe Bianchi, Professor of Nephrology
Chiara Lanzani, Paolo Manunta, Marco Melandri, Rita Quartagno, Maria Teresa Sciarrone
Alibrandi, Giorgio Slaviero, Donatella Spotti, Paola Stella, Giuseppe Vezzoli
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Nadia Lanzi
Giuseppe Chiumello, Professor of Pediatrics
Giovanna Weber, Maria Grazia Roncarolo
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Massimo Bernardi, Fabio Ciceri, Alessandra Pescarollo
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Paolo Servida,
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Medical Oncology
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