Randomised controlled trial of paediatric magnetic positioning device assisted colonoscopy: a pilot and feasibility study.

BACKGROUND AND AIMS: Complete colonoscopy is critical for the evaluation of many paediatric gastrointestinal diseases. The aim of the study was to investigate the feasibility of magnetic positioning device for paediatric colonoscopy and to compare completion rate and procedure time with and without the device. METHODS: Prospective randomised controlled trial of standard colonoscopy compared to magnetic positioning device assisted colonoscopy in children and adolescents ages 7-20 years was performed. RESULTS: Analysis showed that the proportion of successfully completed colonoscopies were 19/20 (95%) in the MP arm versus 17/18 (94.4%) in the SC arm, p=NS. The median time to complete colonoscopy to the cecum was 16.5 min (range 6-52 min) in the MP arm and 12 min (range 6-33 min) in the SC arm, p=NS. CONCLUSIONS: Our preliminary data suggest that the use of magnetic positioning device for colonoscopy is feasible in paediatric patients. These data suggest that the use of magnetic positioning device may not be of benefit for experienced endoscopists who achieved very high colonoscopy completion rates without the MP device. Further studies are needed to determine its role in paediatric colonoscopy since this device may be of more benefit for physicians in training.

Novel therapeutic options in the inflammatory bowel disease world.

Advances in the understanding of the pathogenesis of inflammatory bowel disease have encouraged the development of many new therapies targeted at specific and non-specific mediators of the inflammatory bowel disease inflammatory pathway. The role of these therapies, including novel anti-tumour necrosis factor-alpha agents, anti-adhesion molecules, recombinant cytokines, myeloid growth factors, helminths, and probiotics, in the management of paediatric onset inflammatory bowel disease is promising and warrants further investigation.

Success rate and duration of paediatric outpatient colonoscopy.

BACKGROUND: The paediatric colonoscopy completion rates have rarely been reported. AIMS: We sought to evaluate colonoscopy completion rate and compare the rates using colonoscope versus enteroscope. METHODS: We prospectively investigated 60 patients who underwent colonoscopy between July 1999 and June 2001. The following data were collected: demographics, type of endoscope used, extent of colonoscopy, indication for procedure, histology, adverse events and time to reach the caecum and the terminal ileum. RESULTS: Sixty colonoscopies were performed during the study period, 30 with an enteroscope and 30 with a colonoscope. The caecum was reached in 56/60 (93%) and the terminal ileum in 50/60 (83%). An average time of 12.61 min (S.D. 7.3) was necessary to advance the instrument from the anus to the caecum, and additional 3.67 min (S.D. 3.62) to terminal ileum. There was no difference in the success rate between enteroscope and colonoscope. Six patients (10%) had definitive diagnosis established because a full colonoscopy was performed. No serious adverse events occurred. CONCLUSION: Paediatric colonoscopy to the caecum can be completed safely and expeditiously in more than 90% of procedures. Various types of instruments do not appear to influence completion rate. Full colonoscopy contributes to the establishment of a definitive diagnosis.

Extraintestinal manifestations of inflammatory bowel disease.

Inflammatory bowel disease (IBD) predominantly affects the gastrointestinal system but it is associated with a large number of extraintestinal manifestations (EIM). These extraintestinal disorders can significantly contribute to morbidity and impair the overall life quality. EIM may be diagnosed before, concurrently with, or after the diagnosis of IBD is made. The precise etiology of EIM remains unknown. It currently is believed that mucosa from the underlying bowel disease may provide associated immune responses for the inflammatory process in the extraintestinal sites. The involvement of autoimmune mechanisms has been suggested when the shared and unique epitopes in the human colon, eye, joint and biliary epithelium were detected. Recently, the presence of long-lived populations of memory lymphocytes has been discovered which arise as a consequence of bowel inflammation and express homing receptors that direct their migration not only to the gut but also to the extraintestinal sites. The most common extraintestinal disorders associated with IBD include dermatologic, ophthalmologic, musculoskeletal and hepatobiliary diseases, although virtually every organ system may be involved. If these disorders can be considered as the real extraintestinal manifestations of IBD or represent just association between different syndromes of autoimmune etiology, is still not clear. It is important to acquire knowledge on these extraintestinal manifestations of Crohn's disease and ulcerative colitis to start the respective treatment early.

Failure of single-toxin assays to detect clostridium difficile infection in pediatric inflammatory bowel disease.

OBJECTIVES: The aims of this retrospective study were 1) to determine the ability of single-toxin assays for Clostridium difficile to detect infection among pediatric patients with inflammatory bowel disease (IBD) and 2) to determine the toxin assays routinely used by pediatric tertiary care hospitals in the United States. METHODS: Stool specimens from patients with IBD (submitted from January, 1996, to August, 1999) were evaluated for the presence of C. difficile toxin A and toxin B. Toxin profile (toxin A alone, toxin B alone, toxin A and B together) was compared in positive specimens. A phone interview was conducted with representatives from laboratories in 22 pediatric hospitals to investigate which toxin assays were routinely used. RESULTS: A total of 697 specimens were submitted from 284 IBD patients. In all, 81 IBD patients (28.5%) had at least one documented infection. Toxin A assay failed to identify 41.5% of C. difficile infections. Toxin B assay failed to detect 34.9% of C. difficile infections. Toxin profile changed in 55% of patients with multiple infections. Of the hospitals surveyed, 59% did not test for both toxins. CONCLUSIONS: Single-toxin assays for C. difficile fail to detect a significant percentage of infections. The toxins identified during one infection are not predictive of the toxins identified in subsequent infections. Despite this, many pediatric hospitals do not routinely use both toxin assays to diagnose C. difficile infection. When infection is suspected, assays for C. difficile toxin A and toxin B should be requested.

Secreted ovarian stromal substance inhibits ovarian epithelial cell proliferation.

OBJECTIVE: Determine the effects of factors secreted by normal human ovarian stroma on the proliferation of benign and malignant ovarian epithelia, in vitro. METHODS: Primary cultures of normal human ovarian surface epithelium (HOSE), human ovarian stromal tissue (HOST), and epithelial ovarian carcinomas (CSOC) were established from surgical specimens and characterized immunohistochemically using anti-cytokeratin, vimentin, and Factor VIII antibodies. Stroma-conditioned media (SCM) were collected over 3 days from confluent HOST cultures. The SCM were dialyzed, lyophilized, resuspended, and added to HOSE, CSOC, SKOV-3, and Caov-3 ovarian cancer cell cultures and growth inhibitory effects were assayed by MTS and [3H]thymidine uptake. RESULTS: SCM inhibited the growth and DNA synthesis of normal HOSE cells and cancer cells by 79-99% in > 10-cell lines studied to date. The inhibitory effect was rapid in onset with 31-82% reduction in DNA synthesis at 1 hr and approximately 50% return of activity by 23 hr following a 1-hr SCM pulse treatment. The SCM inhibitory activity was not abolished by boiling or by absorption with heparin-agarose. Size exclusion filtration places the molecular weight of the inhibitory substance between 1 and 3 kDa. Neither trypsin nor proteinase K treatments altered the inhibitory activity of SCM, while a Bligh-Dyer organic extraction placed the activity in the aqueous phase. CONCLUSION: A heat-stable, non-heparin-binding, low-molecular-weight, water-soluble substance secreted by normal ovarian stroma significantly inhibits HOSE and ovarian cancer cell proliferation. Derangements in normal ovarian stroma-epithelial interactions may contribute to growth dysregulation of the surface epithelia and result in ovarian carcinogenesis.

Cloning and characterization of the human insulin-like growth factor-I receptor gene 5'-flanking region.

The insulin-like growth factor-I receptor (IGFIR) is a membrane-bound glycoprotein that mediates the action of insulin-like growth factors. The cDNAs for the human IGFIR have been cloned and expressed, but the structures of the gene and its promoter have not been elucidated. In this study, we isolated an IGFIR promoter clone from a human chromosome 15 library. This clone contained the promoter, first exon, and a portion of the first intron. Sequence analysis of the 5' region that contained the promoter revealed that it lacked both TATA and CAAT boxes. The promoter contained binding sites for the transcription factors Sp1, AP-2, and the epidermal growth factor receptor transcription factor (ETF). Primer extension analysis of IGFIR mRNA indicated the presence of a single transcription start site 1,012 bp upstream from the ATG. When the putative promoter was ligated into a promoterless CAT vector and transfected mto HEPG2 cells, CAT activity was expressed, indicating that promoter activity was contained in this fragment. Other constructs containing the promoter and portions of the 5' untranslated region were used in transfection studies, and indicated that the 5' untranslated regions may play a role in promoter activity. Comparison of the human IGFIR promoter with that of the rat IGFIR promoter revealed significant sequence homology. Comparison of the IGFIR promoter with that of the human insulin receptor (IR) revealed structural similarities, although the arrangement of promoter elements differed.

Regulation of insulin-like growth factor (IGF) I receptor expression during muscle cell differentiation. Potential autocrine role of IGF-II.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. The presence of specific, high affinity IGF receptors, as well as the expression of IGF peptides and binding proteins by muscle suggest that a significant component of IGF action in this tissue is mediated through autocrine and/or paracrine mechanisms. To explore autocrine/paracrine action of IGFs in muscle, we studied the regulation of the IGF-I receptor and the expression of IGF peptides during differentiation of the mouse BC3H-1 muscle cell line. Differentiation from myoblasts to myocytes was associated with a 60% decrease in IGF-I receptor sites determined by Scatchard analysis. Analysis of mRNA abundance and protein labeling studies indicated that the decrease in IGF-I receptor sites was associated with similar reductions in IGF-I receptor gene expression and receptor biosynthesis. IGF-II peptide gene expression was detected in myoblasts and increased 15-fold with differentiation; the increase in IGF-II gene expression preceded the decrease in IGF-I receptor gene expression. In contrast, IGF-I peptide gene expression was low in myoblasts and decreased slightly with differentiation. To explore the potential role of endogenous IGF-II in the differentiation-associated decrease in IGF-I receptor expression, we investigated the effects of IGF-II treatment in myoblasts. The addition of IGF-II to undifferentiated myoblasts resulted in downregulation of the IGF-I receptor which was associated with decreased IGF-I receptor biosynthesis and decreased IGF-I receptor mRNA abundance. These studies suggest, therefore, that IGF-I receptor expression during muscle cell differentiation may be regulated, at least in part, through autocrine production of IGF-II.

Regulating insulin-receptor-gene expression by differentiation and hormones.

Insulin regulates cell function by first binding to the insulin receptor (IR) localized on the cell surface. With the cloning of IR cDNA and the IR-gene promoter, the regulation of the IR gene during differentiation and by various hormones can be studied. Muscle is a major target tissue for insulin action. BC3H1 cells, a mouse muscle cell line in culture, are a model cell type for studying insulin action. Differentiation in these cells results in a 5- to 10-fold increase in IR binding and a 5- to 10-fold increase in IR content. Studies of IR mRNA by Northern and slot-blot analyses reveal a 10-fold increase in IR mRNA after differentiation. These studies indicate that there is a selective increase in IR-gene expression during muscle differentiation. A similar increase in IR-gene expression is observed for the IR during pancreatic acinar cell differentiation. Glucocorticoids increase IR content in several target tissues. Studies in cultured IM-9 lymphocytes indicate that glucocorticoids induce a 5-fold increase in IR mRNA levels. Studies of IR mRNA half-life indicate that glucocorticoids do not alter IR mRNA stability. When the transcription of the IR is measured by elongation assays, glucocorticoids directly stimulate IR transcription 5- to 10-fold. The effect is detectable within 30 min of glucocorticoid treatment and is maximal within 2 h. Therefore, these studies demonstrate that the IR gene is under the direct regulation of glucocorticoids. Insulin downregulates the IR in various target tissues. Prior studies indicate that this downregulation was partly because of accelerated IR degradation. Studying AR42J pancreatic acinar cells, we also found that insulin accelerates IR degradation. Moreover, in these cells, insulin decreases IR biosynthesis by approximately 50%. Studies of IR mRNA indicate there is a concomitant decrease in IR mRNA levels after insulin treatment. Thus, insulin decreases IR-gene expression. The genomic structure of the IR promoter has been elucidated. Primer extension and nuclease S1 analysis indicate that IR mRNA has multiple start sites. The promoter fragment was ligated to a promoterless "reporter" plasmid containing the bacterial gene chloramphenicol acetyltransferase (CAT). When this plasmid is transfected into cultured cells, CAT activity is detected, indicating promoter activity. Various portions of a genomic fragment were ligated to a promoter to study glucocorticoid regulation of the IR promoter. These studies indicate that IR-gene expression is regulated by differentiation and hormonal agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin receptor monoclonal antibodies that mimic insulin action without activating tyrosine kinase.

HTC rat hepatoma cells were transfected with human insulin receptor cDNA to a level of 40,000 receptors/cell. In these cells, as well as in nontransfected cells, insulin stimulated the uptake of alpha-aminoisobutyric acid. Two monoclonal antibodies directed against the human insulin receptor alpha subunit, like insulin, stimulated amino acid uptake in transfected HTC cells, but not in nontransfected HTC cells. The antibodies, in contrast to insulin, failed to stimulate insulin receptor tyrosine kinase activity, both in intact transfected cells and in cell free extracts prepared from them. These data suggest, therefore, that activation of insulin receptor tyrosine kinase may not be an obligatory step in all of the transmembrane signaling mechanisms of the insulin receptor.

Sequence and analysis of promoter region of human insulin-receptor gene.

The promoter region of the human insulin-receptor (HINSR) gene was isolated from a human chromosome 19 bacteriophage library. With S1 nuclease mapping and primer-extension analysis, we identified multiple transcription-initiation sites. Dexamethasone, a known inducer of HINSR transcription, enhanced transcription of all major transcription-initiation sites. DNA sequence analysis indicated that the HINSR promoter has neither a TATA box nor a CAAT box. The HINSR promoter region contains six GGGCGG sequences that may be binding sites for the transcription factor Sp1. In addition, there were three TCCC sequences that were putative promoter regulatory regions. The HINSR gene promoter has structural similarity to the epidermal growth factor receptor gene promoter and has some features of the promoter of the meglutol (hydroxymethylglutaryl, HMG) CoA reductase gene and the early promoter of simian virus 40.

Expression of human salivary protein genes.

Human proline-rich proteins (PRPs) are polymorphic, homologous in sequence, and linked in a cluster called the human salivary protein complex (SPC). Recently this complex was localized to human chromosome band 12p13.2 (Mamula et al., Cytogenet. Cell Genet. 39:279, 1985). We have isolated a PRP cDNA, EO27, from a human parotid gland library, identified it by DNA sequencing, and used it to study the molecular and cellular biology of PRP production. Cell-free translation and mRNA characterization with EO27 indicate that the numerous PRPs seen in saliva are produced from relatively few, large precursors, probably by posttranslational cleavage. This supports an hypothesis originally proposed by Friedman and Karn in 1977 (Am. J. Hum. Genet. 29:44 A; Biochem. Genet. 15:549) and later supported by biochemical studies (Karn et al., Biochem Genet. 17:1061, 1979) and molecular studies (Mamula et al., Fed. Proc. 43:1522, 1984; Maeda et al., J. Biol. Chem. 260:1123, 1985). EO27 was also used in this study to localize PRP mRNA production to the acinar cells of the parotid gland by in situ hybridization.

Localization of the human salivary protein complex (SPC) to chromosome band 12p13.2.

In situ hybridization of a 3H-labeled probe containing a fragment from PRP-1, a genomic clone with human salivary proline-rich protein gene sequences, revealed significant labeling on the short arm of human chromosome 12 in metaphase preparations from two individuals. Fifty-three percent of metaphases exhibited labeling on one or both chromosomes 12. Additional cells scored at the 850-1,000 band level revealed a significant proportion (52% [32/61] grains, p less than 0.005) of the labeled sites on chromosome 12 to be on band 12p13.2. This probe for a human salivary proline-rich protein gene fragment, probably PMS, is from a cluster of 13 linked genes designated as the human salivary protein complex (SPC). Studies of the DNA of human-mouse somatic-cell hybrids have assigned the SPC to chromosome 12, but have not provided a regional localization (Azen et al, 1985). This paper reports the localization of the SPC to a specific chromosomal band, 12p13.2.