Tumour necrosis factor alpha down-regulation parallels inflammatory regression in ulcerative colitis patients treated with infliximab.

BACKGROUND: Treatment with the anti-tumour necrosis factor alpha monoclonal antibody infliximab has been shown to be effective in moderate-to-severe ulcerative colitis. However, its effect on the mucosal histopathological abnormalities of this disease is largely unknown. This study aimed to assess the immunohistological effect of infliximab in ulcerative colitis. METHODS: Nine patients with moderate-to-severe ulcerative colitis received infliximab (5mg/kg) at weeks 0, 2 and 6, respectively. Colonic biopsies were collected before therapy and at week 10, when the Mayo score (including the endoscopic subscore) was also assessed. Severity of inflammation was evaluated by histologic score and histomorphometry. Immunohistochemical staining with antibody against tumour necrosis factor alpha was performed on all biopsies and expressed as percentage of positive stromal cells/1000 counted (tumour necrosis factor alpha score). RESULTS: A profound down-regulation of mucosal tumour necrosis factor alpha occurred in all the six patients who achieved a clinical response, but not in the three who did not respond. Median tumour necrosis factor alpha score dropped from 44.8 (range 35-58.3) to 12.8 (range 5.3-15.3) in the responders (p=0.03), whilst it remained unchanged in the non-responders. Such effect was related with a dramatic regression of the median histologic score, which dropped from 2.7 (range 2-3) to 0.5 (range 0.0-1.5) in responder patients (p=0.002). This was related to a virtual disappearance of neutrophils in responders (r=0.72; p=0.002; Spearman's test), but not in those who did not improve. Tumour necrosis factor alpha score appeared to be correlated with the histologic, endoscopic and clinical activity. CONCLUSIONS: A profound tumour necrosis factor alpha down-regulation appears to be strictly associated with a dramatic regression of the inflammation in patients with moderate-to-severe ulcerative colitis treated with infliximab. Such immunohistochemical effect seems to be critical for a clinical and endoscopic response to therapy.

Primary clarithromycin resistance in Italy assessed on Helicobacter pylori DNA sequences by TaqMan real-time polymerase chain reaction.

BACKGROUND: Helicobacter pylori clarithromycin resistance is increasing worldwide and different mutations are involved in its mechanisms. Recently, molecular methods have been proposed to assess these mutations. AIM: To assess prevalence of primary clarithromycin resistance in two Italian areas, and the distribution of involved mutations, by using a novel method for real-time polymerase chain reaction. METHODS: Two hundred and thirty-two H. pylori-positive patients undergoing oesophagogastroduodenoscopy in two Italian towns (Rome, centre Italy; Foggia, south Italy) were enrolled. Helicobacter pylori infection was detected by histology, rapid urease and urea breath tests. Clarithromycin resistance was assessed by TaqMan real-time polymerase chain reaction on paraffin-embedded antral biopsies. Results Primary clarithromycin resistance was detected in 62 (26.7%) patients. Its prevalence did not differ between the two areas (31.5%, centre vs. 23.3%, south; P=0.17) and between non-ulcer dyspepsia and peptic ulcer patients (28.4% vs. 20.7%, P=0.2). The A2143G point mutation was detected in 35 (56.4%) patients, A2142G in 14 (22.6%), A2142C in eight (12.9%), whilst a double mutation (A2143G plus A2142C or A2142G) was present in the remaining five (8.1%) cases. CONCLUSIONS: Our study found that primary clarithromycin resistance is highly prevalent in both central and southern Italy, and that A2143G is the most frequent point mutation involved in these areas.

Epithelial cell proliferation of the colonic mucosa in diverticular disease: a case-control study.

BACKGROUND: A higher risk of both advanced adenoma and carcinoma occurs in the sigmoid colon of patients with diverticular disease, for which bacterial carcinogens have been claimed to play a role. AIM: To assess epithelial cell proliferation in colonic mucosa of diverticular disease patients before and after rifaximin treatment. METHODS: Twelve consecutive patients with a new endoscopic diagnosis of left-sided diverticular disease and 12 matched controls were enrolled. Epithelial cell proliferation in the sigmoid mucosa was assessed by using proliferating cell nuclear antigen. The proliferating cell nuclear antigen index of the whole crypt and of the upper third was separately evaluated before and after 10-day rifaximin (400 mg b.d.) therapy. RESULTS: Proliferating cell nuclear antigen index in the upper third of the crypt was significantly higher in the diverticular patients (median: 25, range: 14-32) as compared with controls (median: 15, range: 5-20) (P = 0.038), and it was not reverted by rifaximin therapy. No difference of the proliferating cell nuclear antigen index of the whole crypt was detected between cases (median: 27, range: 23-44) and controls (median: 25, range: 18-42) (P = 0.6). CONCLUSIONS: Our data showed an upward shifting of cellular proliferation in the sigmoid mucosa of patients with diverticular disease. Because of rifaximin failure in reversing this alteration, factors other than the bacterial load should probably be investigated.

Sequential treatment for Helicobacter pylori does not share the risk factors of triple therapy failure.

BACKGROUND: Predicting factors for the outcome of conventional Helicobacter pylori triple therapy have been identified. Of these, the presence of the CagA gene is a strong predictor of successful treatment. Our preliminary data show that this factor becomes irrelevant when sequential therapy is used. AIM: To identify predicting factors for the outcome of H. pylori eradication using two therapeutic schemes (triple and sequential) of equal duration (10 days). METHODS: Ninety-six patients with H. pylori infection were randomly assigned to receive one of the following therapeutic schemes: group A: rabeprazole (20 mg b.d.) plus amoxicillin (1 g b.d.) for 5 days, followed by rabeprazole (20 mg b.d.) plus tinidazole (500 mg b.d.) and clarithromycin (500 mg b.d.) for a further 5 days; group B: rabeprazole (20 mg b.d.) plus amoxicillin (1 g b.d.) and clarithromycin (500 mg b.d.) for 10 days. Age, sex, smoking, endoscopic and histological findings, and CagA and VacA status were considered as candidates for a model of multivariate analysis which used therapeutic outcome as the dependent variable. CagA and VacA status were assessed by polymerase chain reaction on DNA isolated from gastric antral specimens. RESULTS: The sequential scheme was significantly more effective than prolonged triple therapy (P < 0.05). Smoking (P < 0.001) and the absence of the CagA gene (P < 0.05) were significantly associated with the failure of triple therapy, but the effectiveness of sequential treatment was not predicted by these factors. CONCLUSION: Our data suggest that sequential therapy is not affected by bacterial and host factors which have, until now, predicted the outcome of conventional eradication treatments.

Tumor necrosis factor alpha and apoptosis in Helicobacter pylori related progressive gastric damage: a possible mechanism of immune system involvement in epithelial turnover regulation.

Helicobacter pylori (HP) related inflammation is mediated by tumour necrosis factor alpha (TNFalpha), which "in vitro" increases epithelial apoptosis in response to infection. In the early stages of HP gastritis, a raised epithelial apoptosis occurs; this phenomenon becomes less evident with progression towards intestinal metaplasia. Aim of our study was to analyze "in vivo" mucosal TNFalpha in relation to epithelial apoptosis in the progression of HP related histological damage. Antral biopsies from 20 HP positive patients were retrospectively studied: 10 with and 10 without intestinal metaplasia (IM and CG group respectively); samples of 10 dyspeptics with normal HP negative stomach (N) were used as control. The following parameters were evaluated by immunohistochemistry: 85 kDa caspase-cleaved fragment (p85) of human poly (ADP-ribose) polymerase (PARP) labelling index (LI) as marker of apoptosis and TNFalpha LI in stromal cells as marker of inflammatory response. Both epithelial apoptosis and mucosal TNFalpha expression were higher in chronic active gastritis compared to intestinal metaplasia and controls (PARP and TNFalpha LI: CG > IM > N; ANOVA & Student-Neumann-Keuls; p < 0.05 and p < 0.01, respectively). Pearson's coefficient showed a significant correlation between PARP and TNFalpha LI in IM and CG groups. Our data show that mucosal TNFalpha, similarly to what suggested "in vitro", may be related "in vivo" to epithelial apoptosis thus suggesting a possible mechanism for immune system involvement in the control of gastric epithelial turnover.

Helicobacter heilmannii gastritis: a histological and immunohistochemical trait.

AIM: Biopsies of the gastric antrum were reviewed over a period of 10 years to determine the prevalence of Helicobacter heilmannii in symptomatic subjects from this geographical area and to relate its presence to distinctive histopathological and immunohistochemical features. METHODS: Biopsies from 7926 symptomatic patients were reviewed. Ten serial sections were stained with haematoxylin and eosin for conventional histology. Another 10 sections were stained with the Gram method for spiral bacteria. When H heilmannii was suspected, 10 additional serial sections were stained with methylene blue to obtain homogeneous colouring. An equal number of sections from patients affected by isolated H heilmannii or H pylori gastritis were analysed by immunohistochemistry to evaluate lymphoid aggregate/mucosal lymphocyte clonality (CD20 and CD3) and tumour necrosis factor alpha (TNF-alpha) in stromal cells. RESULTS: The prevalence of H heilmannii was 0.1% (eight of 7926), whereas H pylori was present in 60.7% of patients (4813 of 7926). In two of the eight H heilmannii positive patients both helicobacters were found. In all subjects infected by H heilmannii only, distinctive histology (lymphocyte exudation into gastric foveolae) was seen. Lymphoid aggregates, chronic mucosal inflammation with patchy activity, and the absence of epithelial mucus depletion were regular features of H heilmannii gastritis. Immunohistochemistry did not reveal different lymphocyte clonal patterns between H pylori and H heilmannii gastritis: CD20 positive cells were predominant in the centre of aggregates and mucosal infiltrates, whereas CD3 positive cells were prevalent at the periphery of follicles. Only H pylori gastritis showed a significant increase in TNF-alpha positive stromal cells. CONCLUSION: These data suggest that an unusual lymphocyte reaction, with the tendency to invade the foveolar lumen, is a distinctive histopathological aspect of H heilmannii chronic gastritis, although further studies in a larger series are necessary to confirm this fact. Nevertheless, lymphocyte clones do not differ qualitatively from those found in H pylori infection. Moreover, compared with H heilmannii, H pylori provokes a more intense release of TNF-alpha, suggesting that different inflammatory responses exist to these two organisms.

Epithelial proliferation and ras p21 oncoprotein expression in rectal mucosa of patients with ulcerative colitis.

In ulcerative colitis (UC), epithelial proliferation plays a role in crypt repair and neoplastic evolution. Proliferative status is predominantly connoted in active disease, but not defined in remission. Histologically, remission is characterized by normalization of the picture or development of atrophy. Mutation of the ras oncogene is involved in intestinal carcinogenesis. Aim of this work was to assess the proliferative pattern of rectal epithelium in UC during disease activity and in remission and correlate it with ras oncoprotein p21. The study was performed retrospectively in rectal biopsies from four groups each of 10 patients: active ulcerative colitis (AUC), remission with a normal histology (RUC), remission with rectal atrophy (ARUC), and irritable bowel syndrome (C, control group). In all, immunohistostain was employed to evaluate the proliferation cell nuclear antigen labeling index (PCNA LI) and ras p21. Statistical analysis was performed by ANOVA and Student-Neumann-Keuls tests. PCNA LI was significantly higher in AUC and ARUC than in RUC and C. Positive cells were predominant in the lower zone of crypts in RUC and C, while a significant expression of PCNA was also observed in the upper areas in AUC and ARUC. Oncoprotein p21 was expressed on the apical surface of the epithelium in 3/10 AUC patients, in all 10 ARUC patients and in none of RUC and C. The persistently increased epithelial proliferation associated with ras p21 expression in ARUC may be due to the action of an abnormal, mutated ras gene that could play a role in UC-related tumorigenesis.