Depth of resection using two different endoscopic mucosal resection techniques.

BACKGROUND AND STUDY AIMS: Endoscopic mucosal resection (EMR) has been carried out for high-grade dysplasia (HGD) and intramucosal carcinoma (IMCA) in Barrett's esophagus using two different cap-assisted techniques, the "inject, suck, and cut" and the "band and snare." Previous work has demonstrated comparable specimen diameters. However, the two techniques have not been previously compared with respect to depth of resection. PATIENTS AND METHODS: From a database of patients with Barrett's esophagus, we identified 40 consecutive specimens removed using EMR from patients with HGD or IMCA, 20 each from the "inject, suck, and cut" and the "band and snare" techniques. Specimens were evaluated and measured separately by two pathologists for greatest diameter and depth, and for the presence of submucosa and muscularis propria at the deepest margin of resection. Follow-up data were collected regarding clinical outcome and stricture formation. RESULTS: The mean depth of the specimens from the two techniques was not significantly different (0.51 cm vs. 0.50 cm, P = 0.76). All specimens contained substantial submucosa, allowing accurate staging of the neoplastic lesions resected. Muscularis propria was identified at the base of 65% of the "band and snare" and 50% of the "inject, suck, and cut" specimens (P = 0.52). CONCLUSIONS: The "inject, suck, and cut" and "band and snare" techniques both yield equivalent adequate depth of histological specimens from Barrett's esophagus with HGD or IMCA, and both provide accurate pathological staging.

Long-term follow-up of complete Barrett's eradication endoscopic mucosal resection (CBE-EMR) for the treatment of high grade dysplasia and intramucosal carcinoma.

BACKGROUND AND STUDY AIMS: In patients with Barrett's esophagus (BE), targeted endoscopic mucosal resection (EMR) of visible lesions of high grade dysplasia (HGD) or intramucosal adenocarcinoma (IMC) is effective, but carries the risk of leaving in place synchronous lesions and Barrett's epithelium with the potential for recurrent disease. We evaluated the safety and long-term efficacy of complete Barrett's eradication EMR (CBE-EMR) for the treatment of patients with HGD or IMC, independently of the presence of macroscopically visible lesions or surgical risk. PATIENTS AND METHODS: 26 consecutive patients with BE and HGD or IMC underwent CBE-EMRs, which were performed with the endoscopic cap suction method and/or a 2.3-mm monofilament mucosectomy snare. Endoscopic follow up after completion of resection was carried out to assess the rate of residual or recurrent BE with or without HGD or IMC. RESULTS: 24 patients completed the study. They underwent a total of 44 EMR sessions with a median of 3 pieces (range 1-8) removed per session. Two patients with immediate bleeding were successfully managed endoscopically. Three patients developed an early esophageal stricture that was completely resolved with a single endoscopic dilation. After a median follow-up of 28 months (range 15-51 months), persistent endoscopic and histologic eradication of BE was demonstrated in 21 patients (87.5 %). In two patients, Barrett's epithelium was detected beneath the neosquamous epithelium 3 months after completion of the resection. In the remaining patient, IMC was found in a nodule seen and removed by EMR at 12-month surveillance endoscopy. CONCLUSIONS: CBE-EMR is a safe and highly effective long-term treatment that should be offered to all patients with Barrett's esophagus with HGD and IMC.

Differential expression of ErbB3 and ErbB4 neuregulin receptors in dopamine neurons and forebrain areas of the adult rat.

Neuregulins have been shown to play an important role in the development of the central nervous system, but their function in adult tissues is still unclear. We investigated the expression of the neuregulin receptors erbB3 and erbB4 in the adult rat brain by in situ hybridization histochemistry. Areas with considerable expression of erbB4 receptor mRNA include cortex, amygdala, hippocampus, medial habenula, reticular thalamic nucleus, several hypothalamic nuclei, subthalamic nucleus, substantia nigra pars compacta, and ventral tegmental area. Immunostaining for tyrosine hydroxylase and dopamine depletion by 6-hydroxydopamine indicate that erbB4 is expressed in dopamine neurons in the latter two nuclei. Substantial erbB4 expression is also present in clusters of cells along the ventral and medial border of the striatum/nucleus accumbens and in the subependymal zone along the lateral and olfactory ventricles (rostral migratory stream), suggesting a role for neuregulins in adult cell proliferation. In contrast, erbB3 mRNA is mostly expressed in white matter throughout the brain and in the ependyma of the ventral half of the third ventricle (tanycytes). These results demonstrate that expression of erbB3 and erbB4 receptors is widespread in the adult rat brain and suggest a function for neuregulins into adulthood.

Isolation and biochemical characterization of the human Dkk-1 homologue, a novel inhibitor of mammalian Wnt signaling.

In an effort to isolate novel growth factors, we identified a human protein, designated Sk, that co-eluted with Neuregulin during chromatographic separation of conditioned medium from the SK-LMS-1 human leiomyosarcoma cell line. Degenerate oligonucleotides based on amino-terminal sequence analysis of the purified protein were used to isolate the corresponding cDNA from a library generated from this cell line. Sk is a novel 266-amino acid protein that contains a signal peptide sequence and two cysteine-rich domains with no similarity to other known growth factors. A single major 2-kilobase transcript was expressed in several embryonic tissues. Transfection of mammalian cells demonstrated that the protein was secreted and expressed as a doublet of approximately 35 kDa. In vitro translation and endoglycosylase analysis indicated that this doublet, which was also observed in cells expressing the endogenous protein, arises from posttranslational modification. A search of the GenBankTM data base revealed a match of Sk with Dkk-1, which is a novel secreted protein required for head induction in amphibian embryos and a potent Wnt inhibitor. When coexpressed with Wnt-2 in NIH3T3 cells, human Sk/Dkk-1 caused reversion of Wnt-2 induced morphological alterations and inhibited the Wnt-2 induced increase in uncomplexed beta-catenin levels. These results provide biochemical evidence that human Sk/Dkk-1 antagonizes Wnt signaling upstream of its effect on beta-catenin regulation.

Epidermal growth factor and betacellulin mediate signal transduction through co-expressed ErbB2 and ErbB3 receptors.

Interleukin-3 (IL-3)-dependent murine 32D cells do not detectably express epidermal growth factor receptors (EGFRs) and do not proliferate in response to EGF, heregulin (HRG) or other known EGF-like ligands. Here, we report that EGF specifically binds to and can be crosslinked to 32D transfectants co-expressing ErbB2 and ErbB3 (32D.E2/E3), but not to transfectants expressing either ErbB2 or ErbB3 individually. [125I]EGF-crosslinked species detected in 32D. E2/E3 cells were displaced by HRG and betacellulin (BTC) but not by other EGF-like ligands that were analyzed. EGF, BTC and HRG also induced receptor tyrosine phosphorylation, activation of downstream signaling molecules and proliferation of 32D.E2/E3 cells. 32D transfectants were also generated which expressed an ErbB3-EGFR chimera alone (32D.E3-E1) or in combination with ErbB2 (32D. E2/E3-E1). While HRG stimulation of 32D.E3-E1 cells resulted in DNA synthesis and receptor phosphorylation, EGF and BTC were inactive. However, EGF and BTC were as effective as HRG in mediating signaling when ErbB2 was co-expressed with the chimera in the 32D.E2/E3-E1 transfectant. These results provide evidence that ErbB2/ErbB3 binding sites for EGF and BTC are formed by a previously undescribed mechanism that requires co-expression of two distinct receptors. Additional data utilizing MDA MB134 human breast carcinoma cells, which naturally express ErbB2 and ErbB3 in the absence of EGFRs, supported the results obtained employing 32D cells and suggest that EGF and BTC may contribute to the progression of carcinomas that co-express ErbB2 and ErbB3.

Cooperative signaling of ErbB3 and ErbB2 in neoplastic transformation and human mammary carcinomas.

In the present study we demonstrate that erbB-3 and erbB-2 cooperate in neoplastic transformation. Under conditions in which neither gene alone induced transformation, they readily transformed NIH3T3 cells if co-expressed. Furthermore, at high expression levels of ErbB2 which cause transformation, ErbB3 enhanced focus formation by one order of magnitude. Synergy required an intact ErbB2 extracellular domain and tyrosine kinase activity. Cooperation between ErbB3 and ErbB2 involved heterodimerization and increased tyrosine phosphorylation of ErbB3. Signaling by the heterodimer resulted in increased PI 3-kinase recruitment as well as quantitative and qualitative differences in substrate phosphorylation. Evidence for signaling by an active ErbB3-ErbB2 heterodimer in four mammary tumor cell lines indicated relevance of this mechanism for human neoplasia. Our detection of the NDF/heregulin transcript in NIH3T3 cells implicates an autocrine loop involving this ligand in signaling by the ErbB3-ErbB2 heterodimer in the model system, whereas heregulin-independent mechanisms likely exist for cooperative signaling by ErbB3 and ErbB2 chronically activated in some human mammary carcinomas.

Efficient coupling with phosphatidylinositol 3-kinase, but not phospholipase C gamma or GTPase-activating protein, distinguishes ErbB-3 signaling from that of other ErbB/EGFR family members.

Recombinant expression of a chimeric EGFR/ErbB-3 receptor in NIH 3T3 fibroblasts allowed us to investigate cytoplasmic events associated with ErbB-3 signal transduction upon ligand activation. An EGFR/ErbB-3 chimera was expressed on the surface of NIH 3T3 transfectants as two classes of receptors possessing epidermal growth factor (EGF) binding affinities comparable to those of the wild-type EGF receptor (EGFR). EGF induced autophosphorylation in vivo of the chimeric receptor and DNA synthesis of EGFR/ErbB-3 transfectants with a dose response similar to that of EGFR transfectants. However, the ErbB-3 and EGFR cytoplasmic domains exhibited striking differences in their interactions with several known tyrosine kinase substrates. We demonstrated strong association of phosphatidylinositol 3-kinase activity with the chimeric receptor upon ligand activation comparable in efficiency with that of the platelet-derived growth factor receptor, while the EGFR exhibited a 10- to 20-fold-lower efficiency in phosphatidylinositol 3-kinase recruitment. By contrast, both phospholipase C gamma and GTPase-activating protein failed to associate with or be phosphorylated by the ErbB-3 cytoplasmic domain under conditions in which they coupled with the EGFR. In addition, though certain signal transmitters, including Shc and GRB2, were recruited by both kinases, EGFR and ErbB-3 elicited tyrosine phosphorylation of distinct sets of intracellular substrates. Thus, our findings show that ligand activation of the ErbB-3 kinase triggers a cytoplasmic signaling pathway that hitherto is unique within this receptor subfamily.

Demonstration of ligand-dependent signaling by the erbB-3 tyrosine kinase and its constitutive activation in human breast tumor cells.

The predicted human erbB-3 gene product is closely related to epidermal growth factor receptor (EGFR) and erbB-2, which have been implicated as oncogenes in model systems and human neoplasia. We expressed the erbB-3 coding sequence in NIH 3T3 fibroblasts and identified its product as a 180-kDa glycoprotein, gp180erbB-3. Tunicamycin and pulse-chase experiments revealed that the mature protein was processed by N-linked glycosylation of a 145-kDa erbB-3 core polypeptide. The intrinsic catalytic function of gp180erbB-3 was shown by its ability to autophosphorylate in vitro. Ligand-dependent signaling of its cytoplasmic domain was established employing transfectants that express a chimeric EGFR/erbB-3 protein, gp180EGFR/erbB-3. EGF induced tyrosine phosphorylation of the chimera and promoted soft agar colony formation of such transfectants. These findings combined with the detection of constitutive tyrosine phosphorylation of gp180erbB-3 in 4 of 12 human mammary tumor cell lines implicate the activated erbB-3 product in the pathogenesis of some human malignancies.

Temperature differences at periodontal sites in health and disease.

The purpose of this study was to determine if a thermocouple probe was capable of detecting differences in temperatures between healthy and diseased periodontal sites. Twenty-two patients, 11 with radiographic evidence of periodontitis and 11 without, were probed twice with the temperature probe, and twice with a conventional probe by two examiners. Two definitions of health and disease were used. Definition one was that any site probing 5 mm or a site that bled upon probing was considered diseased. Sites 4 mm and with no bleeding on probing were considered healthy. Mean temperature differences were calculated from a baseline sublingual temperature. Each arch and tooth demonstrated different temperatures with temperatures decreasing from posterior to anterior. Differences from baseline between healthy and diseased sites were consistently higher for diseased sites. For example, maxillary second molars were 0.72 degrees C higher than baseline while the maxillary central incisors were 1.40 degrees C higher than baseline. Mean temperature differences between healthy and diseased sites were significant (P less than 0.005) for all sites. Definition two was developed since all sites 5 mm or greater and all sites which bled may not be diseased, the data were recalculated with disease including all sites greater than or equal to 5 mm with bleeding on probing and health including all sites less than or equal to 3 mm without bleeding. With this definition the mean temperature difference between healthy and diseased sites was even greater. Maxillary second molars were 0.96 degrees C higher, while maxillary central incisors were 1.76 degrees C higher.

Duodenal ulcer and Sjogren's syndrome in patients with primary biliary cirrhosis: a casual association?

Peptic ulcer has been reported in patients with primary biliary cirrhosis (PBC), but its frequency and pathogenesis are still poorly defined. We have analyzed the occurrence of duodenal ulcer in 37 female patients affected by PBC and in 35 with chronic liver disease of various etiologies. An active ulcer was found in seven patients with PBC and in one with chronic autoimmune hepatitis. The presence of an exocrine gland defect, as indicated by clinical signs of Sjogren's syndrome (SS), was found in six patients with PBC and duodenal ulcer (85%), but in only eight (26.6%) of those without ulcer (p less than 0.02). Therefore, in our patients, duodenal ulcer occurs more often in PBC than in other types of chronic liver disease. The association of SS with PBC, significantly higher in patients with than without ulcer, supports the hypothesis that the underlying exocrine gland defect is involved in the development of duodenal ulcer.

Radioimmunoassay of epidermal growth factor in human saliva and gastric juice.

A sensitive radioimmunoassay was developed for human epidermal growth factor (hEGF) in saliva and gastric juice. This method was sufficiently sensitive for an accurate measurement of hEGF in these biological fluids. The minimal detectable concentration of EGF was 30 ng/L. The imprecision profile of EGF standard curve had a CV less than 10% in the range of 0.1-3.0 micrograms/L. Serial dilution curves of saliva and gastric juice paralleled that of standard EGF. The antibody to hEGF showed no cross-reactivity with a large excess of growth factors, such as human transforming growth factor alpha, human insulin-like growth factor I, and platelet-derived growth factor (c-sis). No detectable cross-reactivity was observed with some biological gut peptides: somatostatin, gastrin, secretin or pancreatic polypeptide. The intra-assay CV for saliva and gastric juice was less than 10%, and the recoveries were 93.9 +/- 8.7% and 93.7 +/- 11.3%, respectively for saliva and gastric juice. Gel exclusion chromatography revealed hEGF-like substances, heterogeneous in size in saliva and gastric juice, the origins and physiological functions of which are unknown.

Gastric juice immunoreactive epidermal growth factor levels in patients with peptic ulcer disease.

Immunoreactive epidermal growth factor (IR-EGF) was measured by a highly sensitive and specific radioimmunoassay in gastric juice samples obtained during endoscopy from 26 control subjects, 44 patients with duodenal ulcers, and 18 with benign gastric ulcers. In the active stage, the concentrations of the peptide were consistently reduced, compared with those found in control subjects (592.7 +/- 55.8 pg/ml), in both duodenal (262.6 +/- 21.4 pg/ml) and gastric ulcer patients (320.2 +/- 34.1 pg/ml) (p less than 0.001 and 0.01, respectively). Mean IR-EGF values distinctly lower than in the controls were still present in the gastric juice of patients with inactive duodenal ulcers (349.7 +/- 35.9 pg/ml; p less than 0.001), whereas no difference was observed in patients with healed gastric ulcers (502.2 +/- 132.3 pg/ml). Although these findings suggest a possible role for EGF deficiency in the pathogenesis of peptic ulcer disease, the pathophysiological significance of our results (if any) remains to be elucidated.

Cigarette smoking increases gastric luminal prostaglandin F2 alpha and thromboxane B2 in healthy smokers.

Pentagastrin-stimulated gastric luminal prostaglandin E2 (PGE2), 6-keto-PGF1 alpha, PGF2 alpha and thromboxane B2 (TxB2) were measured using a second antibody solid-phase enzyme immunoassay before, during and after cigarette smoking in healthy smokers. Smoking significantly increased PGF2 alpha and TxB2 concentration and output; in contrast no significant changes were found for PGE2 and 6-keto-PGF1 alpha levels. In addition, cigarette smoking caused a significant reduction in gastric juice volume and acid output but did not alter intragastric acidity. These findings may suggest a possible role of prostanoids in the response of the stomach to cigarette smoking.

The effectiveness of a counter-rotary action powered toothbrush and conventional toothbrush on plaque removal and gingival bleeding. A short term study.

Initial investigations have demonstrated the effectiveness of a new contra-rotary powered electric toothbrush in removing plaque supragingivally, subgingivally, and interproximally following a single use. The purpose of this study was to evaluate the effectiveness of a counter-rotary toothbrush following 1) one time instruction, 2) reinstruction and 1 week practice; and 3) a third instruction and 3 weeks of practice and home use. Twenty-four patients were studied; 12 using the counter-rotary toothbrush and 12 using a conventional toothbrush. Using O'Leary and Turesky plaque indices, both brushes significantly reduced supragingival plaque from baseline at all intervals. The counter-rotary brush, however, was more efficient than the conventional brush at all intervals (P less than 0.01). Using a Surface Area Plaque Index, both brushes significantly reduced supragingival plaque from baseline at all intervals but there were no significant differences between brushes. A timed bleeding index showed significant reduction in gingival bleeding following 28 days of brushing with both brushes. Again, the counter-rotary toothbrush was superior to the conventional toothbrush (P less than 0.01).

Enzyme immunoassay of gastric luminal prostaglandin E2 in duodenal ulcer disease.

A highly sensitive enzyme immunoassay was used to determine gastric juice prostaglandin E2 (PGE2) levels in control subjects with or without gastritis and in both active or inactive duodenal ulcer patients. Mean pentagastrin-stimulated PGE2 concentration was significantly lower in patients with duodenal ulcer than in control subjects considered as a whole group (with or without gastritis). However, no such difference was found between duodenal ulcer patients and controls showing histologically normal gastric mucosa. On the other hand, controls with chronic superficial gastritis had PGE2 levels significantly higher than those of histologically normal subjects and duodenal ulcer patients. Therefore, it seems unlikely that an absolute gastric PGE2 deficiency is involved in the pathogenesis of duodenal ulcer disease. However, the possibility that PGE2 synthesis could be deficient in relation to the prevailing level of mucosal inflammation cannot be excluded.

A second antibody solid-phase enzyme immunoassay of prostaglandin E2 in human gastric juice.

A second antibody solid-phase enzyme immunoassay for the determination of prostaglandin E2 in human gastric juice was developed using acetylcholine esterase as label, covalently coupled to the eicosanoid. The assay was performed on 96-well microtiter plates coated with the second antibody (swine antirabbit IgG antibody). The enzyme-labeled and unlabeled prostaglandin E2 were allowed to react in a competitive manner with the immobilized specific antibody (rabbit anti-prostaglandin E2 serum). After addition of the enzyme substrate, the specifically bound acetylcholine esterase was determined at 414 nm by means of a colorimetric assay and the enzyme activity was correlated with the amount of unlabeled prostaglandin E2. According to the calibration curve, prostaglandin E2 was determined in the range of 0.5-250 pg/ml. The minimal detectable concentration of prostaglandin E2 was 1.9 +/- 0.2 pg/ml. The intraassay coefficient of variation was less than 10%. Most prostaglandins and their metabolites tested showed a cross-reactivity of less than 1%.