Expressions of E2 and E7-HPV16 proteins in pre-malignant and malignant lesions of the uterine cervix.

Continuous production of the E7 protein from different types of high risk human papilloma virus (HPV) is required for progression of malignancy. We developed antibodies against HPV type 16 E7 and E2 proteins to evaluate their utility as markers for diagnosis during early stages of cervical cancer. Forty biopsies from uterine cervices were diagnosed as low grade intraepithelial lesion (LSIL), high grade intraepithelial lesion (HSIL), squamous carcinoma (SC), in situ adenocarcinoma (ISA) and invasive adenocarcinoma (AC), all of which were infected with HPV 16. Immunohistochemistry was used to investigate the expressions of E7 and E2 (both from HPV 16) and p16. P16 was expressed in eight of 12 LSILs, in all HSILs, in 16 of 18 SC and in all ACs. E2 was expressed in six of 12 LSILs. E7 was positive in eight of 12 LSILs and in all HSIL and carcinomas. The expressions of E2 and E7 of HPV16 related to p16 expression confirmed the value of the viral oncogenic proteins as complementary to histology and support the carcinogenic model of the uterine cervix, because HPVDNA integration into cellular DNA implies the destruction of the gene encoding E2, which suppresses the expression of the E6 and E7 oncoproteins. E2 from HPV16 could be marker for LSILs, while E7 could be a marker for progression of LSILs to HSILs in patients infected by HPV16, because viral typing has little positive predictive value.

[Endoscopic submucosal dissection]. / Endoskopische Submukosadissektion.

Endoscopic submucosal dissection (ESD) was developed in Japan but has now also become permanently established in various centers in Europe. ESD is an endoscopic en bloc mucosal resection technique for the treatment of early cancers with a diameter >1 cm and also superficial precancerous lesions, which could only be removed unsatisfactorily in several fragments or with uncertain lateral safety margins using previous loop excision procedures. Using ESD a lesion is excised after circular marking and generous submucosal injection with a safety margin of approximately 5 mm and subsequently resected at the level of the submucosa with a 1-3 mm short diathermic knife. ESD requires high technical skills in interventional endoscopy and is more time-consuming than snare resection techniques. However, numerous studies have shown a clear superiority for ESD with respect to the R0 resection rate and the local recurrence rate. The present article gives a current review of the use of ESD in the upper and lower gastrointestinal tract and demonstrates perspectives of the procedure.

Colon capsule endoscopy: European Society of Gastrointestinal Endoscopy (ESGE) Guideline.

PillCam colon capsule endoscopy (CCE) is an innovative noninvasive, and painless ingestible capsule technique that allows exploration of the colon without the need for sedation and gas insufflation. Although it is already available in European and other countries, the clinical indications for CCE as well as the reporting and work-up of detected findings have not yet been standardized. The aim of this evidence-based and consensus-based guideline, commissioned by the European Society of Gastrointestinal Endoscopy (ESGE) is to furnish healthcare providers with a comprehensive framework for potential implementation of this technique in a clinical setting.

International Conference on Capsule and Double-Balloon Endoscopy (ICCD). Paris, 27-28 August 2010.

This review reports the highlights of the first International Conference on Capsule and Double-Balloon Endoscopy, held in Paris (27 - 28 August 2010), under the chairmanship of Professor G. Gay (Nancy, France). The conference was devoted to the technical issues and clinical experience related to capsule endoscopy and double-balloon endoscopy (DBE), techniques that have been developed over the past decade for the endoscopic investigation of intestinal diseases. The conference focused on the technical advances of new-generation endoscopes and capsules and the clinical experience gained with capsule endoscopy and DBE. Important advances in the knowledge of obscure gastrointestinal bleeding, inflammatory bowel diseases, and celiac disease were also discussed. The second part of the conference was mainly devoted to the investigation of the colon and discussed the technical issues and results of initial controlled studies of the colonic capsule vs. conventional colonoscopy. In addition, the clinical usefulness of double colonoscopy was demonstrated in patients with previously failed colonoscopy. Finally, the role of DBE was discussed with respect to the investigation and treatment of patients with surgically modified anatomy. The conference was attended by 750 delegates and presented a unique opportunity for an in-depth review of the current knowledge in the field and to ascertain/determine the main lines for the future developments of capsule endoscopy and DBE.

Different patterns of regulation for the copper and cadmium metallothioneins of the ectomycorrhizal fungus Hebeloma cylindrosporum.

Metallothioneins (MTs) are small cysteine-rich peptides involved in metal homeostasis and detoxification. We have characterized two MT genes, HcMT1 and HcMT2, from the ectomycorrhizal fungus Hebeloma cylindrosporum in this study. Expression of HcMT1 and HcMT2 in H. cylindrosporum under metal stress conditions was studied by competitive reverse transcription-PCR analysis. The full-length cDNAs were used to perform functional complementation in mutant strains of Saccharomyces cerevisiae. As revealed by heterologous complementation assays in yeast, HcMT1 and HcMT2 each encode a functional polypeptide capable of conferring increased tolerance against Cd and Cu, respectively. The expression levels of HcMT1 were observed to be at their maximum at 24 h, and they increased as a function of Cu concentration. HcMT2 was also induced by Cu, but the expression levels were lower than those for HcMT1. The mRNA accumulation of HcMT1 was not influenced by Cd, whereas Cd induced the transcription of HcMT2. Zn, Pb, and Ni did not affect the transcription of HcMT1 or of HcMT2. Southern blot analysis revealed that both of these genes are present as a single copy in H. cylindrosporum. While the promoters of both HcMT1 and HcMT2 contained the standard stress response elements implicated in the metal response, the numbers and varieties of potential regulatory elements were different in these promoters. These results show that ectomycorrhizal fungi encode different MTs and that each of them has a particular pattern of expression, suggesting that they play critical specific roles in improving the survival and growth of ectomycorrhizal trees in ecosystems contaminated by heavy metals.

Esophageal capsule endoscopy in patients with suspected esophageal disease: double blinded comparison with esophagogastroduodenoscopy and assessment of interobserver variability.

BACKGROUND AND STUDY AIMS: The study aim was to compare the diagnostic yield of capsule endoscopy and esophagogastroduodenoscopy (EGD) in patients with suspected esophageal disease. Secondary aims were to assess interobserver variability of capsule endoscopy readings and safety. PATIENTS AND METHODS: In total, 98 patients (53 men, 53 +/- 13 years) with an indication for EGD were included. The patient population was artificially enriched to include two thirds of patients with abnormal esophageal findings at EGD, which was followed by capsule endoscopy. Capsule recordings were blindly read by three endoscopists, one from the center that recruited the patient and two from the other center. Study outcomes were the findings described on EGD and capsule endoscopy, agreement between EGD and capsule endoscopy for findings, quality of the capsule recording, and interobserver agreement for capsule endoscopy quality and findings. RESULTS: EGD was normal in 34 patients and showed esophageal findings in 62 (esophagitis 28, hiatus hernia 21, varices 21, Barrett's esophagus 11, others 7). Average esophageal transit time of the capsule was 361 +/- 393 seconds. Capsule endoscopy was normal in 36 patients but detected esophagitis in 23, hiatus hernia in 0, varices in 23, Barrett's esophagus in 18, and others in 4. The positive predictive value of capsule endoscopy was 80.0 % and the negative predictive value was 61.1 %. Overall agreement per patient was moderate between EGD and capsule endoscopy for the per-patient (kappa = 0.42) and per-findings (kappa = 0.40) analyses. Interobserver agreement between capsule endoscopy readings was moderate for findings (kappa = 0.39) and quality assessment (kappa = 0.24). No adverse event was observed after either EGD or capsule endoscopy. CONCLUSION: In this study, despite artificial prevalence enrichment, capsule endoscopy showed a moderate sensitivity and specificity in the detection of esophageal diseases.

Chromosomal rearrangements in wheat: their types and distribution.

Four hundred and sixty polyploid wheat accessions and 39 triticale forms from 37 countries of Europe, Asia, and USA were scored by C-banding for the presence of translocations. Chromosomal rearrangements were detected in 70 of 208 accessions of tetraploid wheat, 69 of 252 accessions of hexaploid wheat, and 3 of 39 triticale forms. Altogether, 58 types of major chromosomal rearrangements were identified in the studied material; they are discussed relative to 11 additional translocation types described by other authors. Six chromosome modifications of unknown origin were also observed. Among all chromosomal aberrations identified in wheat, single translocations were the most frequent type (39), followed by multiple rearrangements (9 types), pericentric inversions (9 types), and paracentric inversions (3 types). According to C-banding analyses, the breakpoints were located at or near the centromere in 60 rearranged chromosomes, while in 52 cases they were in interstitial chromosome regions. In the latter case, translocation breakpoints were often located at the border of C-bands and the euchromatin region or between two adjacent C-bands; some of these regions seem to be translocation "hotspots". Our results and data published by other authors indicate that the B-genome chromosomes are involved in translocations most frequently, followed by the A- and D-genome chromosomes; individual chromosomes also differ in the frequencies of translocations. Most translocations were detected in 1 or 2 accessions, and only 11 variants showed relatively high frequencies or were detected in wheat varieties of different origins or from different species. High frequencies of some translocations with a very restricted distribution could be due to a "bottleneck effect". Other types seem to occur independently and their broad distribution can result from selective advantages of rearranged genotypes in diverse environmental conditions. We found significant geographic variation in the spectra and frequencies of translocation in wheat: the highest proportions of rearranged genotypes were found in Central Asia, the Middle East, Northern Africa, and France. A low proportion of aberrant genotypes was characteristic of tetraploid wheat from Transcaucasia and hexaploid wheat from Middle Asia and Eastern Europe.

Double-balloon colonoscopy after failed conventional colonoscopy: a pilot series with a new instrument.

BACKGROUND AND STUDY AIMS: The endoscopes that were developed for double-balloon enteroscopy have been successfully used in cases of failed colonoscopy. This study was a pilot series in which a new colonoscope was tested that utilized this double-balloon principle. PATIENTS AND METHODS: A total of 29 patients (5 men, 24 women; mean age 54 years) in whom conventional colonoscopy had failed were included in this study. Both the failed colonoscopy and the double-balloon colonoscopy procedures were performed under general anesthesia, the usual practice in France. A prototype instrument (working length 152 cm, diameter 9.4 mm) designed to incorporate the principles of double-balloon enteroscopy was used. The completeness of colonoscopy was assessed according to conventional criteria by the achievement of a stable position in the cecum. The indicatons for the procedure, the time to reach the cecum, the need for fluoroscopic control, and adverse events were recorded. RESULTS: The previous colonoscopy failed due adhesions (n = 16), or to long or fixed loops (n = 13). Complete colonoscopy using the balloon method was achieved in 28/29 patients, taking an average time of 18 +/- 14 minutes; a long sigmoid loop limited the examination to the left flexure in one patient. Balloon colonoscopy using double-balloon methodology was used in 24 patients and the instrument was used without an overtube (i. e. using a single-balloon technique) in five patients. Fluoroscopy was used in 16 patients to monitor endoscope progression. No complications were reported. CONCLUSIONS: Double-balloon colonoscopy enables full colonic examination in almost all patients with a previous incomplete colonoscopy. The overtube should be used in most cases. The use of fluoroscopic assessment of scope progression could be reduced further with increasing experience.

Molecular dynamics of the DNA-binding domain of the papillomavirus E2 transcriptional regulator uncover differential properties for DNA target accommodation.

Papillomaviruses are small DNA tumor viruses that infect mammalian hosts, with consequences from benign to cancerous lesions. The Early protein 2 is the master regulator for the virus life cycle, participating in gene transcription, DNA replication, and viral episome migration. All of these functions rely on primary target recognition by its dimeric DNA-binding domain. In this work, we performed molecular dynamics simulations in order to gain insights into the structural dynamics of the DNA-binding domains of two prototypic strains, human papillomavirus strain 16 and the bovine papillomavirus strain 1. The simulations underline different dynamic features in the two proteins. The human papillomavirus strain 16 domain displays a higher flexibility of the beta2-beta3 connecting loop in comparison with the bovine papillomavirus strain 1 domain, with a consequent effect on the DNA-binding helices, and thus on the modulation of DNA recognition. A compact beta-barrel is found in human papillomavirus strain 16, whereas the bovine papillomavirus strain 1 protein is characterized by a loose beta-barrel with a large number of cavities filled by water, which provides great flexibility. The rigidity of the human papillomavirus strain 16 beta-barrel prevents protein deformation, and, as a consequence, deformable spacers are the preferred targets in complex formation. In contrast, in bovine papillomavirus strain 1, a more deformable beta-barrel confers greater adaptability to the protein, allowing the binding of less flexible DNA regions. The flexibility data are confirmed by the experimental NMR S2 values, which are reproduced well by calculation. This feature may provide the protein with an ability to discriminate between spacer sequences. Clearly, the deformability required for the formation of the Early protein 2 C-terminal DNA-binding domain-DNA complexes of various types is based not only on the rigidity of the base sequences in the DNA spacers, but also on the intrinsic deformability properties of each domain.

The auxin-inducible GH3 homologue Pp-GH3.16 is downregulated in Pinus pinaster root systems on ectomycorrhizal symbiosis establishment.

In an attempt to determine whether auxin-regulated plant genes play a role in ectomycorrhizal symbiosis establishment, we screened a Pinus pinaster root cDNA library for auxin-upregulated genes. This allowed the identification of a cDNA, Pp-GH3.16, which encodes a polypeptide sharing extensive homologies with GH3 proteins of different plants. Pp-GH3.16 was specifically upregulated by auxins and was not affected by cytokinin, gibberellin, abscisic acid or ethylene, or by heat shock, water stress or anoxia. Pp-GH3.16 mRNAs were quantified in pine roots inoculated with two ectomycorrhizal fungi, Hebeloma cylindrosporum and Rhizopogon roseolus. Surprisingly, Pp-GH3.16 was downregulated following inoculation with both fungal species. The downregulation was most rapid on establishment of symbiosis with an indole-3-acetic acid (IAA)-overproducing mutant of H. cylindrosporum, which overproduced mycorrhizas characterized by a hypertrophic Hartig net. This indicates that, despite being auxin-inducible, Pp-GH3.16 can be downregulated on establishment of symbiosis with a fungus that releases auxin. By contrast, Pp-GH3.16 was not downregulated in pine root systems inoculated with a nonmycorrhizal mutant of H. cylindrosporum, suggesting that the downregulation we observed in mycorrhizal root systems was a component of the molecular cross-talk between symbiotic partners at the origin of differentiation of symbiotic structures.

Outcome of capsule endoscopy in determining indication and route for push-and-pull enteroscopy.

BACKGROUND AND STUDY AIM: Video capsule endoscopy and push-and-pull enteroscopy (PPE), both allow a complete examination of the small bowel in patients with suspected intestinal disorders. Due to the invasiveness of PPE, indications should probably be selective. The aim of the present prospective study was to evaluate the outcome of an approach whereby capsule endoscopy was used to select patients in whom PPE was indicated. PATIENTS AND METHODS: 164 patients were included (90 men; age 54+/-18 years) with various indications for small-bowel investigation, such as obscure bleeding (n=88), suspected Crohn's (n=14) or celiac (n=12) disease, or known or strongly suspected localized diseases such as neoplasms (n = 18) for biopsy. Four patients with a suspected intestinal stenosis underwent PPE without prior capsule endoscopy. In the remaining 160 patients, who had a capsule endoscopy, PPE was selected if there were lesions requiring biopsy or angiodysplasias to be treated by argon plasma coagulation (APC). Regarding the insertion route, an anal PPE was indicated if the capsule transit time from ingestion to arrival at the lesion was >or= 75 % of the total time from ingestion to arrival at the cecum. After gut cleansing, PPE was performed with general anesthesia, and the small bowel was examined until the lesion was reached or the scope could not be advanced further. If the suspected lesion had not been reached, a second procedure was performed through the alternative route, under the same conditions. RESULTS: The diagnostic yield of capsule endoscopy was 75 %. According to the indications, 47 PPE procedures were performed in 42 patients, including 33 through the oral route, 4 through the anal route and 5 combined ones. Indications were: suspicion of intestinal tumor (n=13), celiac disease with chronic bleeding (n=4), suspicion of Crohn's disease (n=3), treatment of significant arteriovenous malformations (AVMs) (n=10), diffuse enteropathies (n=3), nonsteroidal anti-inflammatory drug (NSAID)-related conditions (n=2), and obscure digestive bleeding (n=3). Lesions detected by capsule endoscopy were reached by PPE in all but two cases. The positive predictive value (PPV) of capsule endoscopy to make a correct indication for PPE was 94.7 % and the negative predictive value (NPV) was 98.3 %. The PPV and NPV of a time index of > 0.75 to start via the anal route were 94.7 % and 96.7 %. No complications were observed and all patients were discharged the day after the procedure. Follow-up at 9 months showed that capsule endoscopy followed by PPE had positively influenced the management of 90.5 % of the patients. CONCLUSIONS: The use of capsule endoscopy as a filter for PPE results in effective management of patients with various intestinal diseases. Capsule endoscopy can also direct the choice of route of PPE; a time index of > 0.75 appears to reliably indicate an anal route so that a double procedure is required in only about 12 % of cases.