RICERCA ITALIANA     

Research program

STUDIES ON GENETIC AND IMMUNOLOGIC FACTORS MODULATING THE TISSUE DAMAGE AND THE CLINICAL COURSE IN RHEUMATOID ARTHRITIS PATIENTS.

University Co-ordinator

Università degli Studi di ROMA "La Sapienza" - CLINICA E TERAPIA MEDICA APPLICATA - ROMA(RM)

Research Unit Leader

Guido VALESINI

Description

It is now four years that our Reumatology unit is involved in a clinical study devoted to the follow up of Rheumatoid Arthritis (RA) patients under treatment with biological anti-TNF products. Such an observational study (ANTARES) has been approved by the Ministry of Health (Decreto 24.05.01, Gazzetta Ufficiale della Repubblica Italiana n.127, 04.06.2001).
Using the ANTARES inclusion criteria and measures of outcome we have identified a large cohort of RA patients and we have collected a huge number of clinical data and paired serum samples.
Aim of this research project is to evaluate a few genetic markers and their correlation with the clinical and serological characteristics at the moment of inclusion in the ANTARES protocol and then during the follow-up, in order to identify some prognostic markers able to steer the choose of the best treatment in terms of efficacy and safety.
The genetic markers (Class II HLA, PTPN22 gene, genes involved in the citrullination process, and osteopontin) will be evaluated in Novara on the DNA samples we will collect in our department in Rome.
The serological markers we will check on the serum samples ( collected and stored at -70°C during the previous step of the ANTARES protocol) are the anti-CCP and the Rheumatoid Factors (RF) of IgG, IgM, IgA isotype.
The clinical variables evaluated prospectively in the course of routine clinical care include joint counts, patient and physician global assessment and patient pain assessment by visual analogue scale (VAS), as well as acute phase reactants.
Therapeutic response has been evaluated using the EULAR response criteria, which employ the 28 joint disease activity score (DAS28).
The DAS has been determined prospectively at the initiation of treatment and then serially. A DAS28 of more than 5.1 indicates high disease activity; between 3.2 and 5.1, moderate disease activity; and below 3.2, low disease activity.
A decrease in DAS28 of >1.2 represents a good EULAR response (unless DAS is in the high disease activity range at the end of observation); a decrease of >0.6 but <=1.2 is considered a moderate response; a change of ≤ 0.6 (or high disease activity whatever the change in the DAS) is regarded as lack of clinical response.
The ACR 20% (ACR20) response was also evaluated, that is, a 20% decrease in the number of swollen and painful joints and in at least three of the following: patient pain assessment, patient and physician global assessment, erythrocyte sedimentation rate (ESR) or C reactive protein, and health assessment questionnaire (HAQ) score.

Aim of the study
1.serum determination of anti-CCP and RF antibodies (IgG, IgA, IgM) before and after anti-TNF treatment ( infliximab, enbrel, adalimumab);
2.Synovial fluid determination of anti-CCP and RF antibodies;
3.Synovial fluid determination of total amount of IgG, IgA, IgM.

Serum and synovial fluid determination of anti-CCP, RF, and IgG, IgA, IgM .

Anti-CCP antibodies will be evaluated using a commercial ELISA kits (Axis Shield; Dundee, Scotland) using the manufacturer's instructions.

Total amount of IgG, IgM, and IgA will ne assayed using a quantitative immunonephelometry test ( Behring, Germany).

The serum levels of IgM-RF, IgA-RF and IgG-RF will be measured by an enzyme-linked immunosorbent assay (ELISA) as previously described. Polystirene microplate wells will be coated overnight at 4°C with normal rabbit IgG diluted in carbonate/bicarbonate buffer (pH 9.6). After incubation for 2 hours (r/t) serum samples, diluted 1 :40 in PBS-Tween 20, will be placed in IgG coated wells. After 2 hours (r/t) the microplates will be incubated with goat-F(ab)2 anti-human IgM (  chain specific) and IgA (chain specific) each labelled with alkalyne phosphatase. Finally, p-nitrophenyl phosphate (1mg/ml), diluted in Tris-HCl 0.1M (pH 8.6) will be added.
After 30 minutes the reaction will be stopped and the optical density (OD) of wells will be measured on a microplate reader at 405 nm wavelenght.
For the determination of IgG-RF, all sera will be pretreated with pepsine and rabbit anti-human IgG ( chain specific) conjugated with peroxidase used. The substrate will be o-phenylendiamine (1mg/ml) diluted in citric/phosphate buffer containing H2O2, and the plates will be read at 492 nm wave length. The results will be expressed as a binding index (B.I.)