0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

ISS.17 – Biomarkers and diagnosis
U.O.12 – To investigate whether EBV might be the trigger of an
immunopathologic response involving pDC the following experiments will be
performed through the analysis of IFN signature in MS patients with different
disease courses versus HD by quantitative real time PCR. In particular, to
understand whether EBV reactivation may stimulate the release of type I
IFNs from pDC we will investigate the ex-vivo expression of the IFN-signature
in PBMC obtained from MS patients with different MS courses and at different
states of the disease, and in PBMC from HD. FACS analysis will be performed
by studying the expression of maturation markers on BDCA-
2 + /CD123 + population.
The comparative analysis of pDC response to TLR triggering or EBV
infection in PBMC from MS patients and HD will be performed in PBMC
from MS patients vs HD following in vitro stimulation with TLR-7 and TLR-9
agonists or upon EBV infection. The expression of type I IFN will be evaluated
by real time PCR, ELISA (PBL Biomedical laboratories) or intracellular
staining (Miltenyi Biotec).
The analysis of oxidative status in MS patients will be evaluated by analyzing
the level of anti-oxidant capacity (AOC) in serum from MS patients and
HD while levels of F2-isoprostane will be quantified in the CSF from MS
patients as well as in supernatants from PBMC cultures stimulated in vitro
with TLR agonists or infected by EBV.
U.O.13 – In order to understand whether MS patients display an altered
gene expression repertoire in peripheral blood compared to the healthy population
a comparative analysis of RNA obtained from PBMC and whole blood
will be performed by using high-throughput screening techniques. Hybridization
on Illumina GeneChip arrays containing probes for several thousand
human genes may be performed by a service provider. In addition, potential
diagnostic or treatment-related biomarkers will be validated by quantitative
PCR-based approaches, by Elisa tests on serum samples from patients and
controls for the molecular biomarkers that transcribe for soluble factors,
while Flow cytometry assays will be planned for surface markers. Finally,
commercially available agonists, antagonists, inhibitory molecules, blocking
reagents for some of the identified MS related gene products will be searched
and used in ad hoc designed in vitro studies where modulation of immune cell
reactivity (for example in terms of proliferation and cytokine production) will
be assessed.
U.O.14 – In the attempt to identify soluble molecules and immunoglobulins
in the CSF as correlates of the pathological processes that take place at
lesional sites in MS, autopsy CSF samples from 20 patients with MS and 10
patients with other non-neurological and neurological diseases (UK MS Tissue
Bank), and paired CSF and serum samples from 50 patients with MS, 50
patients with other inflammatory and non-inflammatory neurological diseases,
and 10 patients with benign endocranic hypertension (Neurological
Institute C. Mondino CSF Bank) will be analyzed. The study design addresses
the characterization of molecules and mechanisms that in MS could be
involved in the interplay between EBV-infected B-cell infiltration of the cen-
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