Group leader: FRANCESCO PALLONE
Phase 1. Patient recruitment
· 120 patients with active CD (CDAI>200) with any disease extent and
duration.
Phase 2. Patient assessment
All patients will be tested for:
1. DNA-sequence variations of the human IL-12 p40 promoter within human chromosome
5q31-
5q33 assessed as described in the Genetics and genomics workpackages;
2. hTM5, ASCA and p-ANCA serum IgG tested by ELISA, Western Blotting and immunofluorescence
( );
3. Serum levels of soluble IL-2R, TNFRI, TNFRII by ELISA assays.
4. "In vitro" release and mRNA expression of p40/IL-12, IL-18, IFN-g,
TNF-a by lamina propria mononuclear cells (LPMC) isolated from endoscopical
biopsies by DTT-EDTA-Collagenase and Percoll.
mRNA expression measured by semiquantitative PCR ( ).
Phase 3. Efficacy of biologic therapies tested patients.
1. 100 CD patients will be treated with anti-TNF-a monoclonal antibody (MoAb)
(5 mg/Kg e.v., with 3 infusions at 0,2,6 weeks).
2. In a pilot study, 20 out of the 120 active CD patients will be treated with
anti-IL-12 MoAb.
3. Anti-TNF-a efficacy assessed at 6 weeks according to the CDAI (remission
CDAI<150).
Phase 4. Relation between responsiveness to biological therapies and subclinical
markers
1. At the end of treatment it will be assessed whether responsiveness to anti-TNF-a
and anti-IL12 is
observed in the subgroup of active CD patients showing:
a. DNA-sequence variations of the human IL-12 p40 promoter within chromosome
5q31-5q33;
b. ASCA+/p-ANCA-/hTM5- phenotype;
c. Higher IL-2R, TNFRI, TNFRII serum levels;
d. Increased "in vitro" release and mRNA expression of p40/IL-12,
IL-18, IFN-g,TNFa.
Phase 5. Preparation of an ADV bearing a specific IL-18 antisense oligonucleotide
and examination
of its effects on CD T cell activation markers.
The IL-18 antisense DNA prepared to block the first ATG of the IL-18 mRNA. The
oligonucleotide efficiency verified by semiquantitative RT-PCR and Western Blotting.
The IL-18 antisense cDNA cloned in a transfer vector and the transfer vector
co-transfected in cells with a portion of Ad5 genome. Recombinants purified
by plaque assays and characterized. Adenovirus (ADV) effects examined in mucosal
CD biopsies. CD3, CD4, CD8, CD25, CD45RA, CD45RO expression in mucosal samples
tested by immunohistochemistry. Transcripts for IFN-g, TNF-a, IL-10, TGF-b,
IL-18. IL-12Rb2 and NF-KB subunits expression tested by Southern Blotting. The
amount of protein for each gene product by Western Blotting or ELISA.
Screening studies - recruitment of a prediabetic population
The subjects will be characterized with;
· an Oral GlucoseTolerance Test (OGTT) according to WHO
· a modified and validated euglycemic hyperinsulinemic clamp allowing
examination of insulin action;
· indirect calorimetry to determine basal metabolic rate and carbohydrate
oxidation rate;
· intravenous glucose tolerance test to determine the first phase insulin
secretion capacity;
· body composition (fat and lean body mass) using bioimpedance and/or
a whole body counter;
· assessment of endothelial dysfunction by an non-invasive ultrasound
technique;
· assessment of intima/media thickness of carotid arteries;
· presence of autoantibodies, including GAD and ICA, to exclude LADA
subjects
· lipid profiles
· adipose and muscle biopsies for gene expression assays.
Feasibility studies of these goals have been performed and most methodologies
have been established.
At least forty patients with definite MS will be enrolled in the study.
Peripheral blood samples will be obtained from the Centre for Multiple Sclerosis
of the Department of Neuroscience, University of Tor Vergata, Rome, under the
supervision of Professor Giorgio Bernardi and Dr. Caramia. Dr. Caramia will
perform the diagnosis and provide coded samples obtained at various stages of
the disease process. At the time of peripheral blood cells (PBL) collection,
patients will be clinically evaluated and informations regarding disease duration,
relapse rate, previous treatments will be collected. The expanded disability
status scale (EDSS) score will be also obtained for each patient to evaluate
their neurological disability. Blood samples will be obtained following informed
consent procedures approved by the experimentation committees of the University.
Peripheral blood samples will also be obtained from normal healthy volunteers
and will be matched for age and sex with the MS blood samples.
Patients affected by the following types of emphysema will be recruited: heterogeneous,
bullous and non-bullous emphysema with hyperinflation, which presents a marked
restriction in daily activities despite the most aggressive medical therapy.
The following clinical criteria will be considered:
- Severe obstructive ventilatory defect (FEV1<35%)
- Age<75 years old;
- Willingness to undertake risk of morbidity and mortality associated with reduction
pneumoplasty
- Pulmonary artery systolic pressure< 55mmHg
- PaCO2<55mmHg
- ASA score < 3
- No oral corticosteroids or less than 15 mg prednisolone equivalent
- Nutritional status within 70-130% of ideal body weight
- Ability to participate in a vigorous pulmonary rehabilitation program
- No coexisting medical problems that would significantly increase operative
risk
- No neoplastic disease with life expectancy of less than 2 years
- Abstinence from cigarette smoking for at least 4 months
- No previous thoracotomy or pleurodesis on side of proposed operation
- Absence of overt oedema and signs of respiratory tract infection.
Surgery:
The operation will be performed through videothoracoscopic access. Thoracotomy
is preferred in the case of tenacious pleural adherence. One or two side operation
will be performed according to the presence of target areas and the distribution
of hyperinflation.
The patients will undergo routine preoperative evaluation, which includes:
Pulmonary evaluation; Lung volumes will be measured according to standard criteria
using plethysmographic techniques, timed spirometry, and single-breath diffusing
capacity for carbon monoxide (DLCO). All values will be compared with predictions
and will be expressed as the mean of three consecutive tests. All patients will
be considered to have fixed airflow limitation since the FEV1 following two
inhalations of aereosolized salbutamol improved by <20%.
Physical and psycological evaluation. Exercise tolerance will be assessed with
a standard 6-minutes walk test (6MWT). Dyspnea will be rated according to the
American Thoracic Society's Medical Research Council score. Quality of life
will be scored by the Short-Form 36 Health Survery.
Microbiologic evaluation: All patients, preoperatively, will undergo fiberoptic
bronchoscopy and bronchoalveolar lavage for culture and cytology examination.
Radiologic evaluation: inspiratory and expiratory chest radiographs will be
performed to evaluate the degree of thoracic inflation, and high-resolution
CT scan of the chest to evaluate emphysema morphology. Single photon emission
computed tomography (SPECT) of the lung for more accurate recognition of the
target areas to be resected.
Cardiac evaluation: Routine tests and echocardio-color Doppler and right-heart
catheterization will be performed in all patients for the assessment of pulmonary
artery pressures and right-heart function.
Nutritional Status evaluation: Anthropometric measure consisting in weight,
height, arm circumference, triceps bicipital subscapular and iliac fold thickness
measurement will be assessed.
Energy expenditure evaluation: Measurements of energy expenditure will be performed
by indirect calorimetry by whole body calorimetry.