Quality of endoscopic screening for colorectal cancer in Italy: A national survey.

BACKGROUND: Colorectal cancer (CRC) is a major healthcare problem all over the world and screening is effective in reducing mortality and increasing survival. Since colonoscopy has a central role in faecal immunochemical test (FIT)-based CRC screening and surveillance, consistent quality measures are essential to ensure quality and outcomes. Nevertheless, screening modalities in clinical practice may differ according to the centers experience and the local availability of instrumentation and devices. AIMS: to assess the quality of endoscopic screening for CRC and adherence to international guidelines across Gastroenterology Departments in Italy. METHODS: All members of the Italian Society of Gastroenterology (SIGE) were invited to answer a web-based survey. RESULTS: Data from 64 hospitals from 17 Italian regions were analyzed. 32/64 (50.0%) were from northern, 12/64 (18.75%) from central and 20/64 (31.25%) from southern Italy. Each center is equipped with a median of 5.0 (3.5-7.0) endoscopists involved in CRC screening, 71.4% of which are gastroenterologists. After a positive FIT, most centers (93.8%) schedule a colonoscopy within 3 months. High-definition video endoscopy is routinely performed in 68.8% and chromoendoscopy in 53.1% of centers. Withdrawal time is ≥6 min in 79.9% and cecal intubation rate is ≥90% in 94.4% of departments. Finally, in 92.7% of centers adenoma detection rate (ADR) overcome the minimum standard of 25%. Analyzing the data by regional areas, a significant higher number of median endoscopic examinations/year (6500 vs 4000 and 3000, respectively, p = 0.024) and of endoscopists per center (6.5 vs 5.0 and 3.5, respectively, p < 0.001) has been registered in the northern compared to central-southern centers. CONCLUSIONS: Data from this survey show adequacy and good quality of endoscopic screening for CRC in Italy, highlighting, at the same time, relevant deficiencies and a discrepancy in procedural attitudes between the different centers. These findings call for a urgent action to overcome the shortcomings, refine and homogenize the behaviour of all screening centers in the national territory and improve the outcomes.

Impact of the COVID-19 pandemic on Gastroenterology Divisions in Italy: A national survey.

BACKGROUND: In Italy, the spread of the COVID-19 pandemic has stressed the entire healthcare system and required a huge re-organization of many Divisions, including those of Gastroenterology. AIMS: to assess the impact of COVID-19 pandemic on Gastroenterology Divisions across Italy. METHODS: All members of the Italian Society of Gastroenterology (SIGE) were invited to answer a web-based survey. RESULTS: Data of 121 hospitals from all 20 Italian regions were analyzed. Overall, 10.7% Gastroenterology Divisions have been converted to Covid Units. Outpatients consultations, endoscopic and ultrasound procedures were limited to urgencies and oncology indications in 85.1%, 96.2% and 72.2% of Units, respectively, and 46.7% of them suspended the screening for colorectal cancer. Moreover, 72.2% of the staff received a training for use of personal protective equipment, although 45.5% did not have sufficient devices for adequate replacement. Overall, 132 healthcare workers in 41 Gastroenterology Divisions were found to be infected. CONCLUSION: This is the first study to evaluate, at a country level, the impact of COVID-19 outbreak on Gastroenterology Divisions. Substantial changes of practice and reduction of procedures have been recorded in the entire country. The long-term impact of such modifications is difficult to estimate but potentially very risky for many digestive diseases.

Membrane human equilibrative nucleoside transporter 1 is associated with a high proliferation rate and worse survival in resected intrahepatic cholangiocarcinoma patients not receiving adjuvant treatments.

Human equilibrative nucleoside transporter 1 (hENT-1) is a membrane nucleoside transporter mediating the intracellular uptake of nucleosides and their analogues. hENT-1 was recently reported to have a predictive role in intrahepatic cholangiocarcinoma (iCC) patients receiving adjuvant gemcitabine-based chemotherapy, but its biological and clinical significance in iCC remains unsettled. This study investigated the role of hENT-1 in regulating tumour growth and predicting the survival of 40 resected iCC patients not receiving adjuvant treatments. hENT-1 expression was found to be significantly higher in iCC than in the matched non-tumoural liver. Patients harbouring hENT-1 localised on the tumour cell membrane had a worse overall survival than membrane hENT-1-negative patients (median 21.2 months vs 30.3 months, p = 0.031), with an adjusted hazard ratio of 2.8 (95% confidence interval 1.01-7.76). Moreover, membrane hENT-1-positive patients had a higher percentage of Ki67-positive cells in tumour tissue than membrane hENT-1-negative patients (median 23% vs 5%, p < 0.0001). Functional analyses in iCC cell lines revealed that hENT-1 silencing inhibited cell proliferation and induced apoptosis in HUH-28 cells expressing hENT-1 on the cell membrane, but not in SNU-1079 cells expressing the transporter only in the cytoplasm. Overall, these findings suggest that membrane hENT-1 is involved in iCC proliferation and associated with worse survival in resected iCC patients. Further prospective studies on larger cohorts are required to confirm these results and better define the potential prognostic role of membrane hENT-1 in this setting of patients.

Narrow band imaging with magnification endoscopy for celiac disease: results from a prospective, single-center study.

In celiac disease (CD), the intestinal lesions can be patchy and partial villous atrophy may elude detection at standard endoscopy (SE). Narrow Band Imaging (NBI) system in combination with a magnifying endoscope (ME) is a simple tool able to obtain targeted biopsy specimens. The aim of the study was to assess the correlation between NBI-ME and histology in CD diagnosis and to compare diagnostic accuracy between NBI-ME and SE in detecting villous abnormalities in CD. Forty-four consecutive patients with suspected CD undergoing upper gastrointestinal endoscopy have been prospectively evaluated. Utilizing both SE and NBI-ME, observed surface patterns were compared with histological results obtained from biopsy specimens using the k-Cohen agreement coefficient. NBI-ME identified partial villous atrophy in 12 patients in whom SE was normal, with sensitivity, specificity, and accuracy of 100%, 92.6%, and 95%, respectively. The overall agreement between NBI-ME and histology was significantly higher when compared with SE and histology (kappa score: 0.90 versus 0.46; P = 0.001) in diagnosing CD. NBI-ME could help identify partial mucosal atrophy in the routine endoscopic practice, potentially reducing the need for blind biopsies. NBI-ME was superior to SE and can reliably predict in vivo the villous changes of CD.

Van-Gogh-like 2 antagonises the canonical WNT pathway and is methylated in colorectal cancers.

BACKGROUND: Aberrant activation of the canonical WNT signaling is a feature of colorectal cancer (CRC). Van-Gogh-like 2 (VANGL2) belongs to the non-canonical WNT pathway whose activation inhibits canonical WNT signaling. In this study, we investigated the role of VANGL2 and its epigenetic regulation in CRC. METHODS: Van-Gogh-like 2 expression and promoter methylation after 5-aza-2'-deoxycytidine (5-aza) treatment were evaluated in CRC cells. DNA samples from 418 sporadic CRCs were tested for VANGL2 promoter methylation and microsatellite instability (MSI). Proliferation, colony formation and activation of the WNT pathway were tested in cells after VANGL2 overexpression. RESULTS: Van-Gogh-like 2 mRNA was significantly higher in 5-aza-treated RKO, LOVO and SW48, whereas no differences were found in SW480. Van-Gogh-like 2 was fully methylated in RKO, SW48, HCT116, DLD1 and Caco2; partially methylated in LOVO, LS174T and SW837; and unmethylated in SW480, SW620 and HT29. Higher expression of VANGL2 mRNA was found in the unmethylated cell lines. In CRC specimens (8.93% MSI), methylated VANGL2 was associated with MSI, higher grade, proximal colon location and BRAF mutation. Van-Gogh-like 2 overexpression in SW480 significantly decreased proliferation, colony formation and ß-catenin levels. CONCLUSION: Van-Gogh-like 2 is frequently methylated in MSI-CRCs with BRAF mutation and may act as a tumour suppressor gene, counteracting WNT/ß-catenin signaling.

Chronic intestinal pseudo-obstruction related to viral infections.

Chronic intestinal pseudo-obstruction (CIPO), one of the most severe gastrointestinal motility disorders, is a condition characterized by a clinical picture mimicking small bowel occlusion with related symptoms and signs in the absence of demonstrable mechanical obstruction. Analysis of full-thickness biopsy samples may unravel structural changes of the neuromuscular layer involving the whole gut, although the midgut is usually worst affected. Intestinal pseudo-obstruction can occur in association with systemic neurological, endocrine, and connective tissue diseases or malignancy but, when no recognizable etiology is found, CIPO is referred to as idiopathic (CIIPO). The latter form can be diagnosed early in life due to a genetic etiology or in adulthood when a viral origin may be considered. This review addresses the hypothesis that some systemic neurotrophic viral infections can affect the enteric nervous system thereby altering normal peristaltic activity. Available data are reviewed, focusing specifically on herpesviruses or polyomaviruses (JC virus). These suggest that in comparison to a proportion of CIIPO patients, healthy controls rarely harbor viral DNA in the myenteric plexus, leaving open the possibility that a viral infection might have an etiologic role in the development of CIIPO. The review thus provides some new perspectives in the pathophysiology and perhaps targeted treatment of CIIPO.

JC virus infects the enteric glia of patients with chronic idiopathic intestinal pseudo-obstruction.

BACKGROUND AND AIMS: Chronic idiopathic intestinal pseudo-obstruction (CIIP) is characterised by severe impairment of intestinal propulsive motility that mimics bowel obstruction. JC virus (JCV) is a polyomavirus that can infect brain glial cells causing a fatal disease, but may also be found throughout the normal gastrointestinal tract. The hypothesis that JCV infects the myenteric plexuses of patients with CIIP was tested. METHODS: 10 patients with CIIP and 61 normal specimens (30 ascending colon and 31 ileum) from patients with uncomplicated colon cancer were studied. DNA was extracted from the myenteric plexuses, and JCV T antigen (TAg) DNA and the viral regulatory region were detected by PCR and sequencing. Immunohistochemistry was performed to detect JCV viral protein expression, neuronal and glial markers. Fluorescence in situ hybridisation was performed for cellular localisation of the JCV infection. RESULTS: Clinical studies demonstrated neurogenic impairment, and pathological analyses showed neuropathy in each patient with CIIP. JCV TAg DNA was found in the myenteric plexuses of 8/10 (80%) of the patients with CIIP and 3/31 (9.7%) of the control patients (p<0.001). All samples were JCV Mad-1 strains. Seven of the 10 CIIP specimens expressed both JCV TAg and the JCV viral protein VP1, while none of the controls expressed either. JCV infection co-localised with glial fibrillary acidic protein expression, a marker of enteric glial cells. CONCLUSION: JCV infection occurs in the myenteric plexuses of patients with CIIP. The JCV localisation in enteroglial cells suggests a possible pathological role for this virus in enteric neuropathy.

Cytokine gene polymorphisms in gastric cancer patients from two Italian areas at high and low cancer prevalence.

Polymorphisms of interleukin-1beta (IL-1beta), IL-1 receptor antagonist (IL1-RN), and tumor necrosis factor-alpha (TNF-alpha) genes are supposed to be key determinants of gastric cancer risk. Our aim was to study the association between these polymorphisms and gastric cancer in two areas characterized by high (Pavia/Bologna, North Italy) and low (San Giovanni Rotondo, South Italy) gastric cancer prevalence. Genomic DNA was obtained from 216 healthy donors and 98 gastric cancer patients from Pavia and Bologna, and 146 healthy donors and 86 gastric cancer patients from San Giovanni Rotondo. Two SNP in IL-1beta (-511 C/T) and TNF-alpha (-308 G/A) as well as the VNTR polymorphism of IL-1RN locus were studied. A significant linkage disequilibrium was found between IL-1beta -511 and IL-1RN. Genotype and allele frequencies at the IL-1beta, IL-1RN, and TNF-alpha loci in gastric cancer cases were not significantly different from controls. An epistatic effect between IL-1beta -511 and IL-1RN was found with the IL-1beta -511C/IL-1RN*2 haplotype conferring a significant protection against the intestinal-type of gastric cancer in the Southern population. In conclusion, IL-1beta, IL1-RN, and TNF-alpha genotypes are not associated with gastric cancer in Italian patients. An epistatic interrelationship between IL-1beta -511 and IL-1RN confers protection against gastric cancer in low-risk Italian population.

Measuring dyspepsia: a new severity index validated in Bologna.

BACKGROUND: Measurement of the severity of dyspepsia symptoms before and after treatment and determining what is a significant change is a major problem in designing dyspepsia treatment studies. OBJECTIVES: To assess the reproducibility, validity and responsiveness to treatment of a dyspepsia questionnaire to be used in clinical and population-based studies. METHODS: Seventy-three dyspeptic patients (35 male, 38 female; mean age 52 years) and 75 healthy volunteers (32 male, 43 female; mean age 52 years) were included. Subjects were interviewed for the presence/absence and severity/frequency of 19 gastrointestinal symptoms. Severity was measured on a 5-point scale. Frequency was also recorded on a 5-point scale. A global symptom index (severity x frequency) was calculated for the eight most severe symptoms; a mean global symptom index (8-MGSI) was considered for the evaluation of the instrument. To evaluate intra-observer variation, one author interviewed subjects (T0) and then repeated the interview 1 week later (T1). For inter-observer variation, two authors interviewed patients. VALIDITY was measured by comparing 8-MGSI of the dyspepsia patients to those of healthy volunteers. Responsiveness was assessed by comparing mean global symptom index before and 1 month after appropriate therapy. RESULTS: Reproducibility: The mean 8-MGSI was 4.5 at T0 and 3.7 at T1 with a correlation coefficient of 0.62. As for inter-observer variation, the average 8-MGSI was 4.8 by the first author and 3.9 by the second with a correlation coefficient of 0.60. VALIDITY: The mean 8-MGSI was, respectively, 1.4 in healthy volunteers and 4.8 in dyspeptic patients (p = 0.001). Responsiveness: After treatment, a significant improvement in 8-MGSI was detected (p = 0.001). CONCLUSIONS: This questionnaire is a reliable, valid and responsive instrument for measuring the presence, severity and frequency of dyspepsia.

The VEPSY UPDATED Project: clinical rationale and technical approach.

More than 10 years ago, Tart (1990) described virtual reality (VR) as a technological model of consciousness offering intriguing possibilities for developing diagnostic, inductive, psychotherapeutic, and training techniques that can extend and supplement current ones. To exploit and understand this potential is the overall goal of the "Telemedicine and Portable Virtual Environment in Clinical Psychology"--VEPSY UPDATED--a European Community-funded research project (IST-2000-25323, www.cybertherapy.info). Particularly, its specific goal is the development of different PC-based virtual reality modules to be used in clinical assessment and treatment of social phobia, panic disorders, male sexual disorders, obesity, and eating disorders. The paper describes the clinical and technical rationale behind the clinical applications developed by the project. Moreover, the paper focuses its analysis on the possible role of VR in clinical psychology and how it can be used for therapeutic change.

Helicobacter pylori and functional dyspepsia: review of previous studies and commentary on new data.

Many studies have attempted to prove a link between Helicobacter pylori infection and functional dyspepsia but the results have been conflicting. Several mechanisms have been postulated for how H pylori associated inflammation disturbs antral and duodenal function but no pathophysiological explanation of how H pylori may cause dyspeptic symptoms is presently available.

Low-dose lansoprazole and clarithromycin plus metronidazole vs. full-dose lansoprazole and clarithromycin plus amoxicillin for eradication of Helicobacter pylori infection.

AIM: To compare, in a randomized controlled trial, the efficacy and tolerability of two 1-week triple therapies for Helicobacter pylori eradication. METHODS: One hundred and thirty-four consecutive patients with non-ulcer dyspepsia and H. pylori infection were randomized to receive lansoprazole 30 mg once daily, clarithromycin 250 mg twice daily, and metronidazole 500 mg twice daily (LCM group), or lansoprazole 30 mg twice daily, clarithromycin 500 mg twice daily, and amoxicillin 1000 mg twice daily (LCA group). H. pylori status was assessed by rapid urease test, histology and 13C-urea breath test before and after therapy. RESULTS: At 3 months, H. pylori eradication (intention- to-treat/per protocol analysis) was 92.4%/93.8% in the LCM group and 83.1%/85.7% in the LCA group (P=N.S.). Side-effects were more frequently reported in the LCA group (37.9%) than in the LCM group (19.7%) (P < 0.05). CONCLUSIONS: In this open, randomized controlled trial, eradication of H. pylori by low-dose lansoprazole and clarithromycin plus metronidazole was higher with significantly less side-effects than by full-dose lansoprazole and clarithromycin plus amoxicillin. This finding may be related to the stronger synergism of clarithromycin plus metronidazole, even at lower doses, than of clarithromycin plus amoxicillin. Considering the lower cost as well, LCM should be preferred to LCA in the eradication of H. pylori.

Endoscopic surveillance in Barrett's esophagus.

Barrett's esophagus (BE), a complication of chronic gastroesophageal reflux disease (GORD), is a condition that is premalignant for adenocarcinoma of the esophagus and esophagogastric junction. Esophageal adenocarcinoma, once an uncommon entity, has been growing rapidly in incidence over the last two decades in several parts of the world. Barrett's esophagus is a change in the esophageal epithelium of any length that can be recognized at endoscopy and is confirmed to have intestinal metaplasia by biopsy (American College of Gastroenterology guidelines). Because of its premalignant nature, it is recommended that patients with BE undergo regular endoscopic surveillance. The recommendation for endoscopic surveillance is based on unproved and controversial assumptions including: 1) the assumption that Barrett's esophagus adversely influences survival; 2) the assumption that endoscopic surveillance can reliably detect early, curable neoplasia in the columnar lined esophagus. Moreover, the low incidence of adenocarcinoma (reported cancer incidence rates in prospective studies on BE range between 0.5% and 1.9%) is used to support an approach of not surveying patients with Barrett's esophagus. Despite these not convincing data, endoscopic surveillance is considered ''reasonable'' and ''desirable'' by the gastroenterological associations and consensus meetings. Endoscopic surveillance for cancer in Barrett's esophagus (BE) is performed primarily to seek dysplasia, to prevent the progression to invasive malignancy; however, one of the limitations of using dysplasia is a lack of understanding of its natural history. The efficacy of endoscopic surveillance for Barrett's esophagus is likely to remain unclear for a long time. The American College of Gastroenterology has recommended the following practice guidelines: a) for patients with no dysplasia, surveillance endoscopy is recommended at an interval of every 2 to 3 years; b) for patients with low grade dysplasia, surveillance endoscopy every 6 months for the first year is recommended, followed by yearly endoscopy if the dysplasia has not progressed in severity; c) for patients with high grade dysplasia, two alternatives are proposed after the diagnosis has been confirmed by an expert gastrointestinal pathologist. One alternative is intensive endoscopic surveillance until intramucosal cancer is detected at an interval of every 3-6 months. The other alternative is esophageal resection. In the situation of indeterminate dysplasia, whereas the pathologist can not come to definite diagnosis, control biopsies are proposed after 2 months of adequate acid suppression by means of proton pump inhibition. In all cases, the technique of random, four quadrant biopsies taken every 2 cm in the columnar-lined esophagus for standard histologic evaluation is recommended. Any grossly abnormal areas may be biopsied too. One can expect however that during the next future these protocol will change considering new data on dysplasia detection (biochemical markers, flow cytometry), new techniques to identify dysplasia (chromoendoscopy, endosonography, coherence optical tomography, fluorescence techniques) and development of better ablative techniques. At present a marker other than dysplasia identifying a high risk group for cancer on which to focus endoscopic surveillance has not yet been established.

The Loiano-Monghidoro population-based study of Helicobacter pylori infection: prevalence by 13C-urea breath test and associated factors.

OBJECTIVES: As part of a cross-sectional study on cholelithiasis, 1533 out of 1840 residents in Loiano/Monghidoro, a rural area in Northern Italy (792 men, 741 women, age range 28-80 years), agreed to be further evaluated in relation to their Helicobacter pylori status. METHODS: Each participant performed a 13C-urea breath test (13C-UBT) and provided information on sociodemographic, lifestyle and clinical characteristics. RESULTS: The 13C-UBT was positive in 1041 subjects (67.9%; men: 69%; women: 67%) and was positively associated with increasing age (P < 0.001), alcohol consumption (P < 0.01), a higher number of siblings (P < 0.001) and a personal history of peptic ulcer (P < 0.01), but inversely with a nonmanual occupation (P < 0.001). Overall, H. pylori infection was unrelated to smoking, house pets, and a family history of gastroduodenal diseases. The prevalence of H. pylori infection was 72% in subjects reporting one or more dyspeptic symptoms and 65% among asymptomatic participants (P < 0.001); a multivariate analysis showed that only epigastric pain was significantly, although weakly, associated with 13C-UBT positivity. CONCLUSIONS: This large population-based study showed a prevalence of H. pylori infection higher than that reported by serologic surveys in urban areas. Current H. pylori infection was strongly associated with indicators of lower socioeconomic status, alcohol consumption and increasing age. A role of H. pylori infection in determining epigastric pain was suggested.

Incidence and recurrence rates of colorectal adenomas in first-degree asymptomatic relatives of patients with colon cancer.

OBJECTIVES: Subjects with one first-degree relative affected with colorectal cancer are considered to be at increased risk of colorectal adenomas. We compared the recurrence and incidence rates of colorectal adenomas among subjects with one first-degree relative with colorectal cancer and those without family history. METHODS: A series of consecutive asymptomatic subjects successfully underwent a colonoscopy, were found to have either normal results or at least one adenoma, provided a detailed family history, and were offered a second colonoscopy 3 yr later; 190 out of 436 subjects accepted, 134/172 with one or more adenomas and 56/264 with no abnormalities at the initial examination. A first-degree family history was reported by 43/134 and 26/56, respectively. RESULTS: By multivariate analysis, the presence of adenomas at follow-up examination was significantly associated with a positive family history of colorectal cancer in both subgroups, those with a previously resected adenoma (odds ratio = 2.23, 95% CI = 1.04-4.79) and those without (odds ratio = 8.95, CI = 1.29-62.22). CONCLUSION: A history of one first-degree relative with colorectal cancer is associated with a significant increase in 3-yr cumulative incidence and recurrence rates of adenomas.

Mad-1 is the exclusive JC virus strain present in the human colon, and its transcriptional control region has a deleted 98-base-pair sequence in colon cancer tissues.

JC virus (JCV), along with other members of the polyomavirus family, encodes a class of highly conserved proteins, T antigens, that are capable of inducing aneuploidy in cultured cells. We have previously isolated T-antigen DNA variants of JCV from both colon cancer tissues and the corresponding nonneoplastic gastrointestinal tissues, raising new questions about the role of JCV in the development of chromosomal instability of the colon. Based on the sequence of the transcriptional control region (TCR), JCV can be classified as archetype or tandem repeat variants. Among the latter, Mad-1, the prototype virus first isolated from a patient with progressive multifocal leukoencephalopathy, is characterized by lacking the 23- and 66-bp sequences that are present in the archetype and by duplication of a 98-bp sequence. In this study, we evaluated differences in the TCR of JCV isolated from colon cancer tissues and nonneoplastic epithelium. To characterize JCV variants, we first treated eight pairs of DNA samples from colon cancers and noncancerous tissue with topoisomerase I and then amplified and cloned the JCV TCR. We obtained 285 recombinant clones from the JCV TCR, 157 from nonneoplastic samples, and 128 from colon cancer tissues. Of these clones, 262 spanned the length of the JCV Mad-1 TCR: 99.3% from nonneoplastic samples and 82.8% from colon cancer tissues. In sequencing 54 clones in both directions, we did not find archetype JCV either in the nonneoplastic tissue or in the cancer samples. From all colon cancer tissues, 18 clones had a deletion of one 98-bp tandem repeat. This deleted strain was not detected in any of the nonneoplastic tissues (14 versus 0% [chi(2) = 23.6; P < 0.001]). Our study demonstrates that the only JCV strain present in the human colon is Mad-1, and the variant with a single 98-bp sequence is found exclusively in the cancer tissues. This strain may be involved in the development of chromosomal instability.

JC virus DNA sequences are frequently present in the human upper and lower gastrointestinal tract.

BACKGROUND & AIMS: JC virus (JCV), a human polyomavirus, has been found in a limited number of normal human tissues and cancers. The oncogenic potential of this virus is mediated by a transforming protein, the T antigen (TAg). We have previously demonstrated the presence of JCV-TAg in colorectal cancers, in adjacent normal colonic mucosa from these patients, and in the human colon cancer cell line SW480. The mode of transmission of this virus is unclear, and we hypothesized that the gastrointestinal (GI) tract may be a reservoir for the virus. METHODS: DNA was extracted from 129 normal GI tissue samples collected from 33 patients. Topoisomerase I-assisted polymerase chain reaction (PCR) was used to detect the virus using exact and degenerate primers. Nested PCR and Southern blot analysis confirmed the identity of the PCR products. Single-stranded conformation polymorphism (SSCP) analysis and sequencing were used to evaluate the presence of viral quasispecies. RESULTS: JCV sequences were found in 75.8% of patients (70.6% of upper GI and 81.2% of colonic samples); no significant differences in rates of infection were found by site. The use of degenerate primers combined with topoisomerase I treatment led to viral detection in 58.9% of samples, compared with 27.9% of samples using exact primers and topoisomerase I (P < 0.01). SSCP and sequencing analysis confirmed the amplification of viral quasispecies and the authenticity of TAg sequences. CONCLUSIONS: The results show that JCV DNA sequences are highly prevalent in the human upper and lower gastrointestinal tract of immunocompetent individuals.

Steady-state regulation of the human DNA mismatch repair system.

Steady-state levels of human DNA mismatch repair (MMR) transcripts and proteins were measured in MMR-proficient and -deficient cell lines by the newly developed competitive quantitative reverse transcription- polymerase chain reaction and Western analysis normalized with purified proteins. In MMR-proficient cells, hMSH2 is the most abundant MMR protein and is expressed 3 to 5 times more than hMLH1. The hMLH1 protein was expressed 1.5 to 2.5 times more than hPMS2. Steady-state levels of mRNA expression correlated well with protein expression. hMSH2-mutated LoVo cells did not express detectable hMSH3 or hMSH6 proteins. Similarly, hMLH1-mutated HCT116 cells did not express detectable hMLH1 or hPMS2 protein, whereas in hMLH1-restored HCT116+ch3 cells, hPMS2 protein was re-expressed. In hMSH6-mutated HCT15 cells, both hMSH3 protein and mRNA were increased. In SV40-transformed lung fibroblasts, all MMR mRNAs and proteins examined were expressed at levels 1.5-5-fold higher than in their nontransformed counterpart. The steady-state levels of MMR proteins indicate that substantially more hMutS proteins, which are involved in DNA mismatch recognition, are present in comparison with the hMutL proteins. Stability of hMSH3 and hMSH6 proteins appears to depend upon the presence of the hMSH2 protein, and, similarly, the stability of the hPMS2 protein depends upon hMLH1. When the hMSH6 is mutationally inactivated, hMSH3 increases by both transcriptional up-regulation and enhanced protein stability. A balanced up-regulation of all of the components was seen after viral transformation in a fibroblast model. Quantitative changes of the MMR components are a potential mechanism to modify the DNA MMR capabilities of a cell.

Validation of the 13C-urea breath test for the diagnosis of Helicobacter pylori infection in children: a multicenter study.

OBJECTIVE: The 13C-urea breath test (13C-UBT) is a safe, noninvasive, and accurate test for the detection of Helicobacter pylori (H. pylori) infection in adults. The aim of this study was to evaluate sensitivity and specificity of 13C-UBT in children using different types of test meal, doses of 13C-urea and breath sampling intervals. As yet, a validated, standardized 13C-UBT protocol for children has not been formulated. METHODS: 13C-UBT was performed in 115 children and repeated within 3 days, modifying the test meal or the dose of 13C-urea. H. pylori status was assessed by histology and rapid urease test. 13C-UBT was performed using 100 mg or 50 mg of 13C-urea and a fatty test meal (100 FA; 50 FA), 50 mg of 13C-urea, and a carbohydrate test meal (50 CA). Breath samples were collected every 10 min for 60 min. RESULTS: The 13C-UBT in children was highly sensitive and specific with all three protocols used. The best combination of sensitivity (97.92%) and specificity (97.96%) was obtained with Protocol 50 FA at 30 min with a cut-off of 3.5 per mil. CONCLUSIONS: The 13C-UBT is an accurate test for the detection of H. pylori infection also in children. Administration of 50 mg of 13C-urea, a fatty test meal, and breath sampling at 30 min appears to be the most convenient protocol.