0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

Sezione III: Attività per progetti
METHODS
Patients. The study will be conducted on a total of 170 RR-MS, 100 CIS, 60
PP-MS and 60 SP-MS, 20 pediatric MS patients, 100 inflammatory and non
inflammatory neurological diseases, 10 benign endocranic hypertension
patients and 80 sex- and age-matched healthy subjects with no previous history
of neurological disease. All patients will be recruited in the stable or in
the acute phase of the disease, as judged by clinical assessment, but will be
free from immune-modulating therapy for at least 3 months preceding entry
in the study and their EBV sero-status will be also assessed. Comparisons
between pairs of data-sets will be performed by means of Student’s t-tests for
unrelated samples.
U.O.11 – In the attempt to identify biomarkers that correlate with the risk
or progression of the disease and which may be used as an aid to diagnosis
and to therapeutic efficacy, we will use polychromatic flow cytometry which
enables the precise definition of different subsets of cells, and we will evaluate
the immune response toward EBV antigens. Flow cytometry and cell preparation.
Flow Cytometric analysis will be carried out on a FACSCalibur, on a
FACSCanto (BD Bioscience) or CyAn (Coulter). High Speed Cell sorting will
be performed by a 4 laser MoFlo (Coulter). Human mononuclear cells will be
isolated by Ficoll gradient centrifugation (Pharmacia, Uppsala, Sweden).
Dead cells will be excluded by propidium iodide. Data will be analyzed using
Flowjo software (Treestar Inc., Ashland, OR).
Antibodies and reagents. The following antibodies will be used: Foxp3 from
eBioscience, San Diego, CA; 5-(and-6)-carboxyfluorescein diacetate, succinimidyl
ester (CSFE) will be purchased from Invitrogen (Carlsbad, CA, USA).
CD2, CD3, CD4, CD5, CD8, CD25, CD26, CD27, CD28, CD30, CD38, CD40L,
CD44, CD45RO, CD45RA, CD45RB, CD49d, CD50, CD54, CD56, CD58,
CD62L, CD69, CD70, CD95, CD122, CD134, CD162, CTLA4, HLA-DR, CCR4,
CCR6, CCR7, CXCR2, CXCR3, CXCR5 are from BD Pharmingen; GITR,
CCR1, CCR2, CCR3, CCR5, CCR8, CCR9, CXCR4, CXCR6 are from R&D.
These antibodies are conjugated with the following fluorochromes: FITC, PE,
Cy5-PE, APC, PE-Cy7, PE-TexasRed, APC-Cy5.5, APC-Cy7, APC-Alexa 750,
PE-Alexa 610, Cascade Yellow, Pacific Blue. IFN-g, IL-10, IL-4, TNF-a are
from Becton Dickinson; IFN a is from Miltenyi Biotech.
PBLs will be stimulated with EBV pentamers (Pro5 ® MHC Class I Pentamers
Proimmune), or with plate-bound anti-CD3 and soluble anti-CD28
(1 mg/ml, both from Pharmingen) as a positive control. Production of inflammatory
cytokines will then be measured via intracellular staining or ELISA.
Moreover, the frequency of cells which bind the FITC-conjugated pentamers
will also be measured, and polychromatic flow cytometry will be used to characterize
these cells. Finally, pentamer + cells will be sorted and expanded
in vitro to be further characterized (cytotoxicity assays using B-EBV targets).
The frequency of Treg cells will also be measured, and their phenotype will be
studied in detail. Functional assays will evaluate their suppressive abilities
and their role in the control of EBV-specific T cells.
640 2009