L. Ricevuti

Defective mucosal T cell death is sustainably reverted by infliximab in a caspase dependent pathway in Crohn’s disease

Background and aims: To verify whether targeting defective mucosal T cell death underlies the sustained therapeutic benefit of infliximab in Crohn’s disease, we explored its in vivo proapoptotic effect after 10 weeks of treatment, and its in vitro killing activity on lamina propria T cells (LPT) and peripheral blood T cells (PBT), both isolated from Crohn’s disease patients. Methods: Endoscopic intestinal biopsies were collected from 10 Crohn’s disease patients (six steroid refractory and four fistulising) before and after three consecutive infusions of infliximab, administered at week 0, 2, and 6 in a single intravenous dose (5 mg/kg), and from 10 subjects who proved to have functional diarrhoea. Apoptosis was determined in vivo by TUNEL assay, and in vitro by fluorescein isothiocyanate-annexin V/propidium iodide staining on LPT and PBT from Crohn’s disease patients cultured with infliximab. The effect of the broad caspase inhibitor Z-VAD-FMK and the neutralising anti-Fas antibody ZB4 was tested in vitro on LPT and PBT treated with infliximab. Caspase-3 activity was determined by immunoblotting. Results: In Crohn’s disease patients, infliximab treatment induced a sustained LPT apoptosis, still evident four weeks after the last infusion. In vitro infliximab induced death of LPT from Crohn’s disease patients occurred via apoptosis rather than necrosis. LPT showed a higher susceptibility to infliximab induced apoptosis than PBT in Crohn’s disease patients. The signalling pathway underlying the restoration of infliximab induced LPT apoptosis occurred via the caspase pathway but not Fas-Fas ligand interaction in Crohn’s disease. Conclusions: These findings demonstrate that apoptosis is the major mechanism by which infliximab exerts its killing activity on LPT in Crohn’s disease. The sustained LPT proapoptotic action of infliximab, which extends far beyond its circulating half life, may be responsible for the sustained remission induced in Crohn’s disease patients by infliximab retreatment.

Serum bFGF and VEGF correlate respectively with bowel wall thickness and intramural blood flow in Crohn's disease

Serum levels of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)—two factors known to promote tissue repair, fibroblast proliferation, and angiogenesis—were measured in Crohn’s disease patients and correlated with bowel wall thickness (BWT), measured by conventional grey scale ultrasonography, and with the ileal intramural vessel flow, measured by contrast-enhanced color Doppler imaging. Serum samples were obtained from 25 patients with active Crohn’s disease and 22 healthy volunteers, all sex- and age-matched. Serum bFGF and VEGF levels were measured by ELISA assay. All the patients were examined with conventional transabdominal bowel sonography. Color Doppler of the intramural enteric vessels was then performed after the intravenous injection of Levovist, a galactose-based sonographic contrast agent. In Crohn’s disease patients, serum bFGF and VEGF were significantly higher compared with healthy volunteers. A positive correlation between serum bFGF and BWT and between serum VEGF and color Doppler signal intensity was found. The raised serum bFGF levels in Crohn’s disease patients with intestinal strictures compared with patients with other phenotypes (fistulizing, inflammatory), together with the correlation observed between serum bFGF and BWT, suggests a possible involvement of bFGF in the process of transmural fibrogenesis in Crohn’s disease. The higher levels of VEGF in those patients with increased intramural blood flow suggests that VEGF may be considered a marker of angiogenesis in this condition.

Doppler enhancement after intravenous levovist injection in Crohn's disease

Although transabdominal bowel sonography (TABS) has been proposed as a reliable tool to assess increased bowel wall thickness (BWT), the most common sonographic pattern in patients with Crohns disease (CD), its accuracy is limited in the diagnosis of CD. We therefore tried to assess whether color Doppler enhancement with Levovist, a galactose-based intravenous sonographic contrast agent able to enhance the arterial Doppler signal, increases TABS accuracy. Thirty-one patients with ileal CD, diagnosed by endoscopy and enteroclysis, and 20 healthy volunteers were examined with conventional TABS. Color Doppler of the intramural enteric vessels was then performed before and after intravenous injection of Levovist. Twenty-two CD patients had a BWT >4 mm, and 16 of them presented with active disease. Two of the remaining nine CD patients, all with BWT <4 mm, presented with active disease. By means of color Doppler we identified six patients with inactive disease, normal BWT, and normal basal Doppler signal intensity, who showed an enhanced Doppler signal in intramural vessels after contrast agent bolus. Four of these patients, identified only by color Doppler after Levovist injection, relapsed within 6 months. In our experience, sensitivity and specificity of TABS, integrated with additional stimulated acoustic emission mode, were 96.7% and 100%, respectively. The use of Levovist in color Doppler increases the accuracy of TABS in CD diagnosis and follow-up.