Individuazione di anticorpi nei soggetti a rischio di celiachia
  • Responsabile: dott. Tarcisio Not

La celiachia è una malattia autoimmune, che si manifesta in soggetti geneticamente predisposti all’intolleranza al glutine. La diagnosi di celiachia è ora agevolata per la disponibilità degli anticorpi anti-transglutaminasi che hanno una sensibilità e specificità diagnostica del 95%. In questa malattia si osserva un’ampia variabilità clinica, non ancora compresa, caratterizzata sia da soggetti con severa sintomatologia gastro-intestinale (es. diarrea, perdita di peso) o più spesso extra-intestinale (es. anemia ferro carenziale, osteoporosi, bassa statura isolata in età pediatrica, lesioni dello smalto dentale), che da soggetti con sintomatologia sfumata (lieve anemia) o priva di sintomi come nei soggetti identificati nel corso di screening di gruppi a rischio come i familiari di celiaci o soggetti con malattie autoimmuni (es. diabete insulino dipendente). Queste variabili cliniche si accompagnano a differenti caratteristiche biologiche come il grado di danno intestinale e la concentrazione degli anticorpi anti-transglutaminasi nel sangue. Il presente progetto intende identificare un fattore capace di modulare l’espressione clinica e biologica della celiachia. In particolare lo studio intende valutare la presenza nel sangue periferico di anticorpi in grado di bloccare il legame degli anticorpi anti-transglutaminasi alla propria proteina bersaglio e agire in senso anti-infiammatorio anche sui linfociti che sintetizzano gli anti-transglutaminasi. In altre parole cerchiamo di isolare e misurare la concentrazione dell’anticorpo che blocca l’auto-anticorpo responsabile dell’infiammazione glutine dipendente. Da un punto di vista clinico la possibilità di misurare la concentrazione di questo anticorpo protettivo potrebbe spiegare i diversi quadri clinici con cui si manifesta la malattia celiaca e permetterebbe di individuare precocemente soggetti a rischio di celiachia che stanno evolvendo in forme sintomatiche della malattia a causa dei bassi valori di questo anticorpo protettivo.

Background

Celiac disease (CD) is an immune-mediated disorder elicited by gluten. The common factor for all patients is the presence of variable combination of clinical manifestations, specific serum concentrations of anti-transglutaminase antibodies (anti-ttg), different degrees of enteropathy and the HLA DQ2/8. The anti-ttg often herald the overt CD by years before the onset of intestinal atrophy. This is the case of potential CD defined by fluctuating anti-ttg titres, that in several patients disappear from serum during follow-up, without intestinal lesions. In our institution potential CD patients now account for 18% of biopsy proven CD, and there is no agreement on how to diagnose and to treat these patients. Although the anti-ttg are often considered to be an epiphenomenon resulting from the gluten-dependent intetinal inflammation, recent studies suggest that they play an active role in rearrangement of intestinal epithelium as well as to the increased epithelial permeability. By creating phage antibody libraries against tranglutaminase enzyme from CD patients, we produced a specific human monoclonal antibody (anti-ttg 2.8), that is able to modify the structure of the intestinal villa. To investigate the possible role of anti-ttg in CD pathogenesis, we artificially induced the production of anti-ttg 2.8 in mice. We found that this treatment induced 2.8-specific anti-idiotypic antibodies (anti-Ids) that efficiently blocked the binding of anti-ttg 2.8 to the antigen and was accompanied by a reduction of organ lesions. We hypothesized that the anti-Ids could affect the disease progression by preventing anti-ttg binding to its antigen. We preliminary found that CD patients showed a specific lack of serum anti-Ids to disease associated anti-ttg 2.8, whereas gluten-genetically predisposed subjects with normal mucosa presented higher serum concentrations of the specific anti-Ids.

Aims

The project intends to study the anti-idiotypic network against the anti-ttg and to evaluate the protective role of the anti-Ids in the genesis of the intestinal lesions; and to evaluate the positive and negative predictive value of the anti-Ids in subjects genetically predisposed to gluten intolerance before the onset of CD.

Study Design

We will enroll, biopsy proven CD patients tested positive for anti-ttg . Relatives of CD patients will be eligible for the study if they are HLA DQ2 or DQ8 positive and had tested positive and negative for serum anti-ttg antibodies (fluctuating serum anti-ttg concentrations) at least twice in the previous year (on a normal diet). With the help of a physician, in the course of a routine examination, the subjects will fill in a questionnaire regarding their medical history and clinical condition. They also will undergo intestinal biopsy to determine the presence of anti-Ids in the intestinal mucosa, as well as to ascertain the histological state of the mucosa. All the serum samples will be analyzed for the measurement of specific anti-Ids. The levels of both mucosal and serum anti-Ids will be correlated with the intestinal mucosal condition, the gastro-intestinal or non-gastro-intestinal complaints. We will enroll sick control subjects suffering from gastro-intestinal complaints with no personal or family history of CD. All relatives with high levels of anti-Ids will be monitored along with clinical condition, serum anti-transglutaminase concentrations, and serum anti-Ids. Relatives with decrement of anti-Ids will be invited to undergo to second intestinal biopsy to verify the intestinal mucosa condition and to measure mucosal anti-Ids.

Summary Preliminary Data

We expect to demonstrate the presence of the anti-idiotype network against the anti-ttg both in the intestinal mucosa and the serum of celiac subjects and subjects with potential celiac disease. The main result regards the different concentration of the anti-Ids in the two main groups of the study: celiac patients in acute phase and potential celiacs without intestinal lesions. We expect to find a significantly higher concentration of anti-Ids in potential celiac subjects compared to the celiacs. This result will require confirmation by the sequential analysis of the sera of potential celiac patients prior to diagnosis of CD, in which the concentrations of the anti-Ids will have to be progressively decreasing up to the CD diagnosis. These results might to define genetically predisposed gluten intolerance with no intestinal lesions in the presence of elevated anti-Ids, as opposed to celiac disease with intestinal lesions and low levels of anti-Ids. In the contest of genetically gluten-intolerance, the specific anti-Ids dosing test will identify subjects that are moving towards classic CD, according to a constant decrease in the anti-Ids in the serum and/or in the intestinal mucosa.

Materials and methods

We have select 50 subjects with potential CD characteristics, and we will diagnose about 100 newly biopsy proven CD ( ESPHGAN criteria), and will enrol 50 sick control subjects suffering from gastro-intestinal complaints with no personal or family history of CD. At the enrolment all patients will undergo a clinical and laboratory assessment (CD-related autoantibodies, anti-ttg anti-Ids). We will produce the human anti-ttg antibody (named 2.8 anti-ttg) composed by the variable part of human VH-VL anti-ttg isolated from the intestinal mucosa of CD subjects. The 2.8 anti-ttg will capture the specific anti-Ids, by using an immunoenzimatic assay.

Main Expected Results and Impact

The anti-Ids dosing test might identify the symptomatic and asymptomatic subjects that are moving towards classic CD, for preventing delay of CD diagnosis.

Significance and Relevance for National Health System (SSN)

Improving the diagnostic tools for CD, avoiding restrictive and expensive diet, for patients and health care system, in not necessary patients and preventing CD dependent complications.

Periodo: 
31/12/2015

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