M. Cretella

Distinct Profiles of Effector Cytokines Mark the Different Phases of Crohn’s Disease

Objective Crohn’s Disease (CD)-associated inflammation is supposed to be driven by T helper (Th)1/Th17 cell-derived cytokines, even though there is evidence that the mucosal profile of cytokine may vary with the evolution of the disease. We aimed at comparing the pattern of effector cytokines in early and established lesions of CD. Design Mucosal samples were taken from the neo-terminal ileum of CD patients undergoing ileocolonic resection, with (early lesions) or without post-operative recurrence, and terminal ileum of CD patients with long-standing disease undergoing intestinal resection (established lesions). Inflammatory cell infiltrate was examined by immunofluorescence and cytokine expression was analysed by real-time PCR, flow-cytometry and ELISA. Results Before the appearance of endoscopic lesions, the mucosa of the neo-terminal ileum contained high number of T cells and macrophages, elevated levels of Th1-related cytokines and TNF-α and slightly increased IL-17A expression. Transition from this stage to endoscopic recurrence was marked by abundance of Th1 cytokines, marked increase in IL-17A, and induction of IL-6 and IL-23, two cytokines involved in the control of Th17 cell responses. In samples with established lesions, there was a mixed Th1/Th17 response with no TNF-α induction. Expression of IL-4 and IL-5 was up-regulated in both early and established lesions even though the fraction of IL-4-producing cells was lower than that of cells producing either interferon-γ or IL-17A. Conclusions Distinct mucosal profiles of cytokines are produced during the different phases of CD. A better understanding of the cytokines temporally regulated in CD tissue could help optimize therapeutic interventions in CD.

IL-15 positively regulates IL-21 production in celiac disease mucosa

Celiac disease (CD)-associated inflammation is characterized by high interleukin- 21 (IL-21), but the mechanisms that control IL-21 production are not fully understood. Here we analyzed IL-21 cell sources and examined how IL-21 production is regulated in CD. Intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs), isolated from CD patients and non-CD controls, were analyzed for cell markers, cytokines, and transcription factors by flow cytometry. IL-21 was highly produced by CD4+ and CD4+/CD8+ IELs and LPLs in active CD. IL-21-producing cells coexpressed interferon-γ (IFN-γ) and to a lesser extent T helper type 17 (Th17) cytokines. Treatment of control LPLs with IL-15, a cytokine overproduced in CD, activated Akt and STAT3 (signal transducer and activator of transcription 3), thus enhancing IL-21 synthesis. Active CD biopsies contained elevated levels of Akt, and blockade of IL-15 in those samples reduced IL-21. Similarly, neutralization of IL-15 in biopsies of inactive CD patients inhibited peptic–tryptic digest of gliadin-induced IL-21 expression. These findings indicate that in CD, IL-15 positively regulates IL-21 production.

A simplified, non-invasive fecal-based DNA integrity assay and iFOBT for colorectal cancer detection.

PurposeNeoplasia cells exfoliated from colorectal epithelium have dysfunctional apoptotic mechanisms, thus it is possible to identify high-molecular weight DNA fragments in feces. This prospective single-center study was performed to evaluate the sensitivity and specificity of fecal-based DNA integrity versus immunological fecal occult blood test (iFOBT) and calprotectin for colorectal cancer (CRC) and adenoma detection.MethodsFeces were collected from 204 subjects and DNA integrity was quantified by quantitative-denaturing high performance liquid chromatography (QdHPLC). Calprotectin and iFOBT were assessed using commercial kits. The diagnostic performance was calculated by receiver operating characteristic (ROC) curves analysis.ResultsA total of 192 fecal specimens were analyzed and 12 samples were excluded due to DNA degradation. We found long DNA (L-DNA) occurrence in feces with a sensitivity of 86% (n = 24/28) and a specificity of 81% for CRC detection. To minimize false-positive cases of the developed test, area under the curve of ROC was evaluated such that the specificity was increased to 92% with decreased sensitivity to 79%, p = 0.0001 for CRC detection. iFOBT was positive in 51% (n = 14/27) while calprotectin was positive in 75% (n = 18/27). The combination of iFOBT and L-DNA identified a greater number of CRC cases with a sensitivity of 89% and a specificity of 95%, p < 0.001. The combination also improved the sensitivity of polyps, particularly high-grade dysplasia and advanced adenoma (33%, p = 0.0015) as opposed to a single evaluation assay (17–21%).ConclusionsThis study illustrates the usefulness of fecal DNA integrity assay by QdHPLC as a non-invasive, easy-to-perform, and reproducible method with a high level of sensitivity in detecting individuals with colorectal neoplasia. Combination of iFOBT and L-DNA improves the sensitivity for CRC and adenoma detection.

Adenoma, advanced adenoma and colorectal cancer prevalence in asymptomatic 40- to 49-year-old subjects with a first-degree family history of colorectal cancer.

First‐degree relatives (FDRs) of patients with colorectal cancer (CRC) have an increased CRC risk. Few studies have addressed if adenoma and advanced adenoma risk is increased among individuals, 40–49 years of age, with a family history of CRC. Therefore, the aim of the study was to define the prevalence and location of adenoma, advanced adenoma and CRC, according to age, in asymptomatic individuals with a family history of CRC.

Quantitative denaturing high performance liquid chromatography (Q-dHPLC) detection of APC long DNA in faeces from patients with colorectal cancer.

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer-related deaths. However, prevention is possible by early detection. In the present work, we have demonstrated and validated a novel quantitative method based on a DNA integrity assay and mutation in faeces of CRC patients using denaturing high performance liquid chromatography (dHPLC). Methods: Faecal DNA (fDNA) was isolated from 28 CRC, 96 healthy and 61 patients with adenomas. Adenomatosis polyposis coli (APC)-Long-DNA and its mutations were analysed using dHPLC and the Sanger sequencing method. The diagnostic performance was assessed using receiver operating characteristic curve analysis. Results: We detected APC-Long-DNA in 21/28 CRC subjects with a sensitivity of 75% and specificity of 91.7%. A cut-off ratio of 0.2317 was used for APC/β-actin. The Q-dHPLC detection limit was 0.02 ng/injection. The average initial fDNA presence based on a single gene of β-actin was 26.12±13.39 ng/mL for healthy, and 49.61±46.28 ng/mL for CRC subjects, with a sensitivity of 71.4% and a specificity of 84.4% at a cut-off value >29 ng/mL. We also detected a novel mutation at codon 1576 Lys/Glu using dHPLC. Conclusions: This study highlights a novel application of Q-dHPLC in the DNA integrity assay, which demonstrates high performance, good reproducibility, and low cost for the CRC detection using faeces. Further studies in a larger population are needed to confirm these results. Clin Chem Lab Med 2010;48:1303–11.

P.108 EUS ACCURACY IN RESTAGING RECTAL CANCER AFTER NEOADJUVANT CHEMORADIOTHERAPY: A PRELIMINARY REPORT

Colorectal cancer (CRC) is the second most frequent cause of cancer-related death in Western countries: one third of CRC is represented by rectal cancer (RC). Accurate staging is recommended to provide an optimal treatment strategy. Neoadjuvant chemoradiotherapy (NCRT) is increasingly used in the treatment of advanced RCwith the intention of downsizing and downstaging the tumour and to reduce tumour recurrence. Accuracy of EUS staging is reported to be 75-94% for tumour penetration and 7283% for nodal metastases. However after NCRT the EUS accuracy for T staging may decrease (50%) because marked fibrosis, peritumoral infiltration of inflammatory cell necrosis. AIMS: the aims of this study was to verify the accuracy of EUS in restaging RC after neoadjuvant CRT and the efficacy of the therapy in the downstaging and downsizing the tumour. METHODS: we prospectively enrolled consecutive patients affected by RC in stage II-b and III who underwent EUS and MRI or CT or PET before and after neoadjuvant CRT. EUS scanning was performed by one endosonographer using an electronic radial ecoendoscope (Olympus GF UE 160-AL5) with imaging at 7,5-10 MHz. Patients underwent surgical resection after reassessment staging. The reduction of diameter of RC was evaluated using RECIST criteria. Pathological staging was undertaken according to the TNM classification. RESULTS: 14 patients with locally advanced RC were assessed: 10 male, median age 64 yrs (range 56-73). The median distance from the anus was 5 cm. Seven patients were in in stage II-b and 7 in stage III. All patients underwent radiotherapy with no complication associated with chemotherapy (10 pts with CDDP, 2 pts with FOLFOX and 2 pts with FUFA). The downstaging after neadjuvant therapy was observed in 7/14 patients, and significant reduction of tumor diameter in 13/14 patients. There were 13 anterior resections and 1 abdominoperineal resections. EUS post-NCRT T stage was correct in 11/14 pts (3 T0, 5 T3, 3 T2), while 3 pts were over staged (2 T2 and 1 T1). Overall accuracy of EUS post-RCT for pathologic T stage was 93% for T0-T1 stage and 86% for T3 stage. EUS accuracy for N-stage was 79%. CONCLUSION: in our preliminary study neoadjuvant CRT was useful to downstage and downsize locally advanced RC. EUS seems to be an accurate tool to restage RC after neoadjuvant CRT. However our results needed to be confirmed in a large cohort of patients with locally advanced RC.

Local injection of infliximab in the postoperative recurrence of Crohn's Disease during endoscopy: A prospective longitudinal study

BACKGROUND Local injection of infliximab in Crohns disease (CD) lesions may reduce the risk of rare side effects, reduce the dose, and increase the efficacy of the drug. The objective was to prospectively assess the feasibility and the safety of local injection of infliximab for the postoperative recurrence of patients with CD who were followed for at least 1 year. METHODS In a pilot, open-label study, 8 patients with CD (3 men; median age 48 years, range 35-82 years) undergoing ileocolonoscopy were prospectively enrolled. Inclusion criteria included the following: (1) localized (<5 cm) recurrence, (2) inflammatory pattern, and (3) clinically inactive CD. At the first endoscopy, lesions were injected with infliximab (median, 30 mg; range, 8-60 mg); a control endoscopy was performed at 2 weeks in 4 patients (3 received a second injection followed by a control endoscopy at 6 weeks) and at 4 weeks in 4 patients (2 received a second injection followed by a control endoscopy at 8 weeks). OBSERVATIONS No patients showed side effects or clinical relapse in the short term and the long term (median follow-up, 20 months; range, 14-21 months). Endoscopic score improved in 3/8 patients. The histologic scores were reduced in 4 patients, worsened in 3, and were unchanged in one patient with CD. CONCLUSIONS Local injection of infliximab into patients with CD recurrence is feasible and safe, requiring a low dose. Present findings suggest the need of placebo-controlled trials to assess the efficacy of this new and safe procedure in subgroups of patients with CD.