Human cytomegalovirus and Epstein-Barr virus infection in inflammatory bowel disease:Need for mucosal viral load measurement

AIM:To evaluate the best diagnostic technique and risk factors of the human Cytomegalovirus(HCMV)and Epstein-Barr virus(EBV)infection in inflammatory bowel disease(IBD).METHODS:A cohort of 40 IBD patients(17 refractory)and 40 controls underwent peripheral blood and endoscopic colonic mucosal sample harvest.Viral infection was assessed by quantitative real-time polymerase chain reaction and immunohistochemistry,and correlations with clinical and endoscopic indexes of activity,and risk factors were investigated.RESULTS:All refractory patients carried detectable levels of HCMV and/or EBV mucosal load as comparedto 13/23(56.5%)non-refractory and 13/40(32.5%)controls.The median DNA value was significantly higher in refractory(HCMV 286 and EBV 5.440 copies/105cells)than in non-refractory(HCMV 0 and EBV 6copies/105 cells;P<0.05 and<0.001)IBD patients and controls(HCMV and EBV 0 copies/105 cells;P<0.001 for both).Refractory patients showed DNA peak values≥103 copies/105 cells in diseased mucosa in comparison to non-diseased mucosa(P<0.0121 for HCMV and<0.0004 for EBV),while non-refractory patients and controls invariably displayed levels below this threshold,thus allowing us to differentiate viral colitis from mucosal infection.Moreover,the mucosal load positively correlated with the values found in the peripheral blood,whilst no correlation with the number of positive cells at immunohistochemistry was found.Steroid use was identified as a significant risk factor for both HCMV(P=0.018)and EBV(P=0.002)colitis.Finally,a course of specific antiviral therapy with ganciclovir was successful in all refractory patients with HCMV colitis,whilst refractory patients with EBV colitis did not show any improvement despite steroid tapering and discontinuation of the other medications.CONCLUSION:Viral colitis appeared to contribute to mucosal lesions in refractory IBD,and its correct diagnosis and management require quantitative real-time polymerase chain reaction assay of mucosal specimens.

Histotype-based prognostic classification of gastric cancer

AIM:To test the efficiency of a recently proposed histotype-based grading system in a consecutive series of gastric cancers.METHODS:Two hundred advanced gastric cancers operated upon in 1980-1987 and followed for a median 159 mo were investigated on hematoxylin-eosinstained sections to identify low-grade [muconodular,well differentiated tubular,diffuse desmoplastic and high lymphoid response (HLR)],high-grade (anaplastic and mucinous invasive) and intermediate-grade (ordinary cohesive,diffuse and mucinous) cancers,in parallel with a previously investigated series of 292 cases.In addition,immunohistochemical analyses for CD8,CD11 and HLA-DR antigens,pancytokeratin and podoplanin,as well as immunohistochemical and molecular tests for microsatellite DNA instability and in situ hybridization for the Epstein-Barr virus (EBV) EBER1 gene were performed.Patient survival was assessed with death rates per 100 person-years and with Kaplan-Meier or Cox model estimates.RESULTS:Collectively,the four low-grade histotypes accounted for 22% and the two high-grade histotypes for 7% of the consecutive cancers investigated,while the remaining 71% of cases were intermediate-grade cancers,with highly significant,stage-independent,survival differences among the three tumor grades (P=0.004 for grade 1 vs 2 and P=0.0019 for grade 2 vs grade 3),thus confirming the results in the original series.A combined analysis of 492 cases showed an improved prognostic value of histotype-based grading compared with the Lauren classification.In addition,it allowed better characterization of rare histotypes,particularly the three subsets of prognostically different mucinous neoplasms,of which 10 ordinary mucinous cancers showed stage-inclusive survival worse than that of 20 muconodular (P=0.037) and better than that of 21 high-grade (P<0.001) cases.Tumors with high-level microsatellite DNA instability (MSI-H) or EBV infection,together with a third subset negative for both conditions,formed the T8 cell-rich HLR group,the largest group among low-grade histotypes.Coexisting HLR proved to be a factor in improved prognosis in tumors with microsatellite instability (P=0.0015 vs HLR-/MSI-H tumors) or DR type human leukocyte antigen expression (P=0.033 vs HLR/HLA-DR+ tumors).CONCLUSION:Identification of lowand high-grade histotypes can improve the prognostic assessment of a substantial proportion of gastric cancers in routine diagnostic practice.

Role of the advanced glycation end products receptor in Crohn's disease inflammation

AIM:To investigate the level of mucosal expression and the involvement of the receptor for the advanced glycation end products(RAGE)in delayed apoptosis and tumor necrosis factor(TNF)-αproduction in Crohn’s disease(CD).METHODS:Surgical and endoscopic specimens from both inflamed and non-inflamed areas of the ileum and/or colon were collected from 20 and 14 adult CD patients,respectively,and used for the assessment of RAGE expression by means of immunohistochemistry and western blotting analysis.Normal tissues from 21 control subjects were used for comparison.The same polyclonal anti-human RAGE antibody(R and D System)was used in all experimental conditions.RAGE staining was quantized by a score including both the amount of positive cells and intensity of immunoreactivity;cellular pattern was also described.The effects of RAGE blocking on apoptotic rate and TNF-αproduction were investigated on immune cells freshly isolated from CD mucosa and incubated both with and without the muramyl dipeptide used as antigenic stimulus.Statistical analysis was performed via the test for trend,with regression models to account for intra-patient correlations.A 2-sided P<0.05 was considered significant.RESULTS:In inflamed areas,RAGE expression in both the epithelial and lamina propria compartments was higher than control tissues(P=0.001 and 0.021,respectively),and a cluster of positive cells were usually found in proximity of ulcerative lesions.Similar results were obtained in the lamina propria compartment of non-inflamed areas(P=0.025).The pattern of staining was membranous and granular cytosolic at the epithelial level,while in the lamina propria it was diffuse cytosolic.When evaluating the amount of protein expression by immunoblotting,a significant increase of both surface area and band intensity(P<0.0001 for both)was observed in CD inflamed areas compared to control tissue,while in non-inflamed areas a significant increase was found only for band intensity(P<0.005).Moreover,a significantly lower expression in noninflamed areas in comparison with inflamed areas was found for both surface area and band intensity(P<0.0006 for both).Finally,RAGE blocking largely affects both the apoptotic rate of mucosal cells(towards an increase in both non-inflamed and inflamed areas of P<0.001 and<0.0001,respectively)and TNF-αsecretion(towards a decrease in both non-inflamed and inflamed areas of P<0.05 and<0.01,respectively),mainly in the presence of antigenic stimulation.CONCLUSION:RAGE is up-regulated in CD,especially in inflamed areas,and it appears to play a role in the mechanisms involved in chronic inflammation.