Volumetric laser endomicroscopy and its application to Barrett's esophagus: results from a 1,000 patient registry.

Volumetric laser endomicroscopy (VLE) uses optical coherence tomography (OCT) for real-time, microscopic cross-sectional imaging. A US-based multi-center registry was constructed to prospectively collect data on patients undergoing upper endoscopy during which a VLE scan was performed. The objective of this registry was to determine usage patterns of VLE in clinical practice and to estimate quantitative and qualitative performance metrics as they are applied to Barrett's esophagus (BE) management. All procedures utilized the NvisionVLE Imaging System (NinePoint Medical, Bedford, MA) which was used by investigators to identify the tissue types present, along with focal areas of concern. Following the VLE procedure, investigators were asked to answer six key questions regarding how VLE impacted each case. Statistical analyses including neoplasia diagnostic yield improvement using VLE was performed. One thousand patients were enrolled across 18 US trial sites from August 2014 through April 2016. In patients with previously diagnosed or suspected BE (894/1000), investigators used VLE and identified areas of concern not seen on white light endoscopy (WLE) in 59% of the procedures. VLE imaging also guided tissue acquisition and treatment in 71% and 54% of procedures, respectively. VLE as an adjunct modality improved the neoplasia diagnostic yield by 55% beyond the standard of care practice. In patients with no prior history of therapy, and without visual findings from other technologies, VLE-guided tissue acquisition increased neoplasia detection over random biopsies by 700%. Registry investigators reported that VLE improved the BE management process when used as an adjunct tissue acquisition and treatment guidance tool. The ability of VLE to image large segments of the esophagus with microscopic cross-sectional detail may provide additional benefits including higher yield biopsies and more efficient tissue acquisition. Clinicaltrials.gov NCT02215291.

Characterization of oncocytes in deep esophageal glands.

Deep esophageal glands play a vital role in the protection and regeneration of the esophageal mucosa. Conditions such as gastroesophageal reflux disease and Barrett's esophagus have been associated with a change in the usual glands by oncocytic metaplasia. However, little is known regarding the function of oncocytes or the relevance of this metaplastic change in the human esophagus. We hypothesized that oncocytes of deep esophageal glands also express markers characteristic of a ductal epithelial phenotype because similar oncocytes have been described as part of large ductal epithelial cells in salivary glands. We used immunohistochemical stains to define structural, functional, proliferative, and potential stem/progenitor characteristics of oncocytes. Oncocytes did not express mucins or lysozyme C, two molecules found in mucous cells and used for antimicrobial defense. Oncocytes did not express CK5, a cytokeratin found in myoepithelial cells and basal epithelial cells, but expressed CK7, a cytokeratin found in intralobular ductal epithelial cells and luminal epithelial cells of the main duct. Oncocytes expressed cystic fibrosis transmembrane conductance regulator and sodium/potassium ATPase, ion channels that play a role in bicarbonate secretion. Membrane-bound beta-catenin was detected in oncocytes, but these cells did not express the proliferative marker Ki67. Approximately, a third of oncocytes expressed SOX9 and p63, transcription factors expressed in epithelial progenitor cells in multiple organs. Moreover, oncocytes expressed CD44, a transmembrane Glycoprotein expressed in cancer stem cells. Taken together, our data show that oncocytes express markers of intralobular ductal epithelial cells and luminal epithelial cells of the main duct. Additionally, our observations suggest that oncocytes act as epithelial progenitor cells and play a role in bicarbonate secretion. Since oncocytic metaplasia is associated with conditions of chronic acid injury, it is possible that oncocytes replace the mucous cells in deep esophageal glands (dEG) as an adaptive change to counteract injury from acid reflux. The marker characterization suggests that oncocytes may originate from transdifferentiation of myoepithelial and mucous cells. This transdifferentiation might lead to an overall decrease of mucins production and secretion by the dEG and a subsequent reduction of the protection conferred by the viscoelastic mucous layer.

Markers suggest reduced malignant potential of subsquamous intestinal metaplasia compared with Barrett's esophagus.

Esophageal subsquamous intestinal metaplasia (SSIM) is frequently observed in patients with Barrett's esophagus (BE) and can also be found in patients after endoscopic ablative treatments for dysplastic BE. While these 'buried glands' appear identical to BE glands, features of SSIM and its malignant potency have yet to be fully elucidated. To determine differences in malignant potential between nondysplastic BE and SSIM, the Automated Cellular Imaging System was used to assess and compare changes in DNA content between SSIM and BE. Samples were further immunostained for Ki67 and Lgr5 to gauge general proliferative and possible stem cell features, respectively, in SSIM cells compared with BE glands. No significant differences were found between SSIM and BE with regards to DNA ploidy aberrance. However, significant differences were noted between SSIM and BE upon immunohistochemical analysis. SSIM was found to be negative for both Ki67 and Lgr5 while BE was positive for both markers. SSIM cells appear to be relatively quiescent and behave differently from BE, suggesting a reduced proclivity toward cancer progression.

Lgr5, an intestinal stem cell marker, is abnormally expressed in Barrett's esophagus and esophageal adenocarcinoma.

Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5), a recently discovered intestinal stem cell marker, is expressed in premalignant lesions including Barrett's esophagus (BE) and cancers including colon cancer, ovarian cancer, and hepatocellular carcinoma. It was also recently found to be expressed in tumor spheres prepared from colon cancer, suggesting that it will likely serve as a cancer stem cell marker. We sought to examine Lgr5 as a biomarker in BE-associated neoplasia. Using standard immunohistochemistry, we performed immunostaining on 81 esophageal specimens (53 biopsy specimens and 28 surgical resections) representing BE, BE-associated dysplasia, and esophageal adenocarcinoma (EAC). Each immunostain was scored based on intensity of immunostaining and percentage of positive cells. For 24 EAC cases, survival analysis was performed with expression scores and other clinicopathological variables. We found that Lgr5 expression was detected in 70% of BE cases and between 90 and 100% of advanced dysplastic lesions and EAC. The intensity of expression was significantly higher in high-grade dysplasia and EAC than BE. In EAC, high Lgr5 expression scores (> or = 5) were associated with worse survival, independent of stage, age, and neoadjuvant/adjuvant therapy (P = 0.03). Our findings suggest that Lgr5 has potential utility as a biomarker for BE-associated dysplasia and EAC.

Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor.

The mechanisms that maintain the epithelial integrity of the gastrointestinal tract remain largely undefined. The gene encoding intestinal trefoil factor (ITF), a protein secreted throughout the small intestine and colon, was rendered nonfunctional in mice by targeted disruption. Mice lacking ITF had impaired mucosal healing and died from extensive colitis after oral administration of dextran sulfate sodium, an agent that causes mild epithelial injury in wild-type mice. ITF-deficient mice manifested poor epithelial regeneration after injury. These findings reveal a central role for ITF in the maintenance and repair of the intestinal mucosa.

Three-dimensional optical coherence tomography of Barrett's esophagus and buried glands beneath neosquamous epithelium following radiofrequency ablation.

We report three-dimensional (3D) endoscopic microscopy findings in Barrett's esophagus, using an endoscopic optical coherence tomography (OCT) system in one patient before and in one patient after radiofrequency ablation (RFA). Findings were compared with those in a normal patient without Barrett's esophagus. In the normal patient,findings were of regular flat squamous mucosa with small subepithelial vessels and glands. In the Barrett's esophagus patient, findings were of large, densely packed glands with distortion of mucosal architecture. In the post-RFA case, findings were of a small number of isolated glands buried beneath 300-500 microm of neosquamous epithelium and lamina propria. Neosquamous epithelium is a marker of successful ablative therapy, while buried glands may have potential for dysplastic progression and are difficult to detect using conventional methods. These results indicate a potential role of 3D-OCT endoscopic microscopy for follow-up, including subsurface assessment, of ablative treatments for Barrett's esophagus.

Regional differences in nitrergic innervation of the smooth muscle of murine lower oesophageal sphincter.

BACKGROUND AND PURPOSE: Anatomical and pharmacological studies have demonstrated that the lower oesophageal sphincter (LES) is not a simple homogenous circular muscle with uniform innervation. Regional differences have been demonstrated in several species including humans. We investigated, for the first time in mice LES, regionally distinct physiological and pharmacological characteristics of the neuromusculature. EXPERIMENTAL APPROACH: Conventional intracellular recordings and pharmacological techniques were employed to evaluate electrical properties and functional innervation of smooth muscle cells. Results from CD1 (control), nNOS((-/-)) and eNOS((-/-)) genetic knockout mice were compared. KEY RESULTS: Smooth muscle of sling and clasp LES displayed unitary membrane potentials of 1- 4 mV. Transmural nerve stimulation produced a monophasic inhibitory junction potential (IJP) in the sling, whereas in the clasp a biphasic IJP, consisting of a brief IJP followed by a long-lasting slow IJP (lsIJP), was induced. Pharmacological interventions and genetically modified mice were used to demonstrate a monophasic apamin-sensitive (purinergic) component in both LES regions. However, the nitrergic IJP was monophasic in the sling and biphasic in the clasp. Unitary membrane potentials and IJPs were not different in CD1 and eNOS((-/-)) mice, suggesting no involvement of myogenic NOS. CONCLUSION AND IMPLICATIONS: These data in mouse LES indicate that there are previously unreported regional differences in the IJP and that both the apamin-resistant monophasic and biphasic IJPs are mediated primarily by nitrergic innervation.

Neuronal constitutive nitric oxide synthase is involved in murine enteric inhibitory neurotransmission.

UNLABELLED: Mice lacking neuronal nitric oxide synthase gene (ncNOS) were used to determine the enzymatic source of nitric oxide (NO) and its relationship with other putative inhibitory neurotransmitters. Inhibitory junction potentials (IJP) of circular smooth muscle of gastric fundus were studied. The IJP in the wild-type mice consists of overlapping components, the fast and slow IJPs. NOS inhibitor L-NA or VIP receptor antagonist VIP(10-28), blocks the slow IJP but not the fast IJP. The fast UP is blocked by alpha-beta methylene ATP tachyphylaxis, by reactive blue 2, and by apamin. The IJP in the ncNOS-deficient [ncNOS(-)] mutant is of short duration and is abolished by blockers of the fast IJP, but is unaffected by blockers of the slow UP. Exogenous VIP produces membrane hyperpolarization in strips from wild-type but not ncNOS(-) mice. The hyperpolarizing action of VIP is resistant to nifedipine but is sensitive to omega-conotoxin GVIA. IN CONCLUSION: (a) NO derived from ncNOS is an inhibitory neurotransmitter rather than a postjunctional mediator; (b) VIP is a prejunctional neurotransmitter that causes release of evanescent NO; and (c) ATP acts in parallel with the VIP/NO pathway.

Ultrahigh resolution optical coherence tomography of Barrett's esophagus: preliminary descriptive clinical study correlating images with histology.

BACKGROUND AND STUDY AIMS: Endoscopic ultrahigh resolution optical coherence tomography (UHR OCT) achieves an axial image resolution of approximately 5 microm, which is 2 - 3 times finer than standard endoscopic OCT imaging. This study investigated the capability of endoscopic UHR OCT for imaging patients with Barrett's esophagus. PATIENTS AND METHODS: Fivty volunteers previously diagnosed with Barrett's esophagus underwent UHR OCT. Imaging was performed at 1.3 microm wavelengths with approximately 5 microm axial and approximately 15 microm transverse resolutions using a 1.8 mm/diameter linear-scanning catheter introduced through the accessory channel of a standard endoscope. OCT images were compared with endoscopic diagnosis and pinch biopsy histological appearances. RESULTS: UHR OCT images of normal esophagus, Barrett's esophagus, high grade dysplasia and esophageal adenocarcinoma were evaluated. UHR OCT images of the normal esophagus exhibited characteristic layered architecture with uniform epithelium, while images of Barrett's esophagus corresponded to crypt-like glandular structures. High grade dysplasia and esophageal adenocarcinoma images exhibited more heterogeneous structures corresponding to irregular, heterogeneous tissue morphology from distorted and cribriform or villiform glandular architecture. Fine features can be discerned more clearly with endoscopic UHR OCT. CONCLUSIONS: This study evaluated new endoscopic OCT technology and demonstrated the feasibility of carrying out UHR OCT imaging in conjunction with standard endoscopy for in vivo real-time imaging of Barrett's esophagus, dysplasia, and esophageal adenocarcinoma. A survey of normal and abnormal upper gastrointestinal tissues was performed using a research prototype OCT system with the highest axial resolution to date, and can serve as a baseline for future investigation.

Failure of cell surface expression of a class I major histocompatibility antigen caused by somatic point mutation.

The ability to down-regulate major histocompatibility complex class I antigen expression on allografts prior to transplantation would be expected to improve their survival in immunocompetent recipients. In order to identify genetic mechanisms that mediate attenuation of MHC class I antigen expression, we have begun characterizing H-2Kb surface null somatic cell variants derived from an H-2 heterozygous tumor cell line (H-2b X H-2d). These variants have sustained a modification in cell surface MHC phenotype, as evidenced by their failure to be recognized by both anti-H-2Kb antibodies and cytotoxic T lymphocytes. The mutant phenotype for one such variant (designated 69.9.15) was marked by the expression of abundant H-2Kb mRNA and immuno-precipitable H-2Kb protein in cell lysates. The failure in cell surface expression of the H-2Kb antigen was caused by a single base change (G to A transition) in exon 3, encoding the second external domain (alpha 2) of the H-2Kb molecule. The mutation resulted in the substitution of Tyr for Cys at amino acid position 164, thereby disrupting an intrachain disulfide linkage formed between Cys 101 and 164. In contrast to the wild-type H-2Kb gene, DNA-mediated transfer of the mutant H-2Kb gene into mouse L cell fibroblasts failed to result in cell surface expression of the H-2Kb antigen, although both the wild-type and mutant genes were transcribed to equivalent levels. These data indicate that a genetic event as limited as somatic point mutation can abrogate expression of a MHC class I antigen and provide support for the hypothesis that protein folding plays an important role in the cell surface expression of MHC class I molecules.

Hemagglutination with porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) virus grown on MARC-145 cell cultures was tested for hemagglutination (HA) with erythrocytes from a variety of species at 4 degrees C, room temperature and 37 degrees C. HA was observed at all temperatures with mouse erythrocytes but not with cattle, sheep, goat, horse, swine, guinea pig, mongolian gerbil, goose and chicken erythrocytes. The HA activity was enhanced by treatment of virus materials with Tween 80 followed by treatment with ether. The HA titer and HA pattern of virus materials treated with Tween 80 and ether (TE) were 4- to 8-fold higher and more clear than those of the virus materials without TE treatment. The optimum conditions consisted in pretreatment of virus material with Tween 80 at a final concentration of 0.06-0.125% (v/ v) for 15-60 min followed by treatment with ether at a concentration of 50% (v/v) for 5-15 min in ice bath with continuous shaking. The curve of active virus production in intra- and extracellular virus samples resembled that of HA production although it rose somewhat earlier in intracellular virus samples. The HA reaction was inhibited by specific antiserum. HI antibody titers of individual pig sera showed a significant positive correlation with their neutralizing antibody titers.

A pedunculated giant esophageal liposarcoma: a case report and literature review.

INTRODUCTION: The majority of esophageal tumors arise from the mucosal layer; only 5 % are of mesenchymal origins. Of the latter, barely 0.5 % are liposarcomas. We present a case of an esophageal liposarcoma with a review of the literature. CASE REPORT: A 64-year-old male was referred with 5 years of progressive dysphagia. Preoperative evaluation initially suggested a leiomyoma. The polypoid lesion was then resected through a cervical esophagotomy, once endoscopic resection proved to be not feasible. The definitive pathologic diagnosis confirmed a well-differentiated liposarcoma. LITERATURE REVIEW: Esophageal liposarcomas are very rare and only 40 such cases have been reported in the literature. Most patients were male (80 %), the median age was 62 years (range 38-83 years), and the most common symptom was dysphagia (85 %). Only in two cases was a liposarcoma detected on preoperative biopsy. The most common histological subtype was well-differentiated liposarcoma. Overall, 77.5 % of the patients were successfully treated with surgery, 20 % endoscopically, and 2.5 % were ablated with CO2 laser. CONCLUSION: Esophageal liposarcoma is an extremely rare tumor. The majority of patients are males; dysphagia is the most common initial symptom, and preoperative biopsy is unreliable. Because these tumors are pedunculated, well-circumscribed, and well-differentiated, they can be safely resected locally. All patients need long-term follow-up as this disease can recur many decades after treatment.

The lower esophageal sphincter.

The lower esophageal sphincters (LES) together with the crural diaphragm are the major antireflux barriers protecting the esophagus from reflux of gastric content. However, reflux of gastric contents into the esophagus is a normal phenomenon in healthy individuals occurring primarily during episodes of transient lower esophageal sphincter relaxation (TLESR), defined as LES relaxation in the absence of a swallow. Transient lower esophageal sphincter relaxation is also the dominant mechanism of pathologic reflux in gastroesophageal reflux disorder (GERD) patients. Frequency of TLESR does not differ significantly between healthy individuals and those with GERD, but TLESRs are more likely to be associated with acid reflux in GERD patients. Understanding the mechanisms responsible for elicitation of a TLESR, using recently introduced novel technology is an area of intense interest. Pharmacologic and non-pharmacologic manipulation of receptors involved in the control of TLESR has recently emerged as a potential target for GERD therapy.

Lessons from genetically engineered animal models. IV. Nitric oxide synthase gene knockout mice.

Nitric oxide is a ubiquitous molecule implicated in a variety of biological processes. The specific action of nitric oxide depends on its enzymatic sources, namely neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), each having distinct tissue localization. Conventional pharmacological antagonists could not distinguish these enzymes or provide models of chronic nitric oxide depletion in whole animals. Several lines of knockout mice have been generated to distinguish the roles of nitric oxide from each enzyme: nitric oxide from nNOS is a major inhibitory neurotransmitter, nitric oxide from eNOS regulates blood flow under physiological conditions, and nitric oxide from iNOS causes hypotension during severe inflammatory conditions. Moreover, the nitric oxides from each isoform have different roles in tissue injury and inflammation. Studies of NOS-deficient animals have also identified redundant and compensatory pathways and revealed the consequences of life-long deficiency of these enzymes. The nNOS-deficient mice develop gastric dilation and stasis, the eNOS-deficient mice develop hypotension and lack vasodilatory responses to injury, and iNOS-deficient mice are more susceptible to inflammatory damage but more resistant to septic shock.

Gastric stasis in neuronal nitric oxide synthase-deficient knockout mice.

BACKGROUND & AIMS: Nitric oxide (NO) is a major inhibitory neurotransmitter in the gut. This study aimed to identify the effect of chronic deprivation of NO derived from neuronal (nNOS) or endothelial (eNOS) nitric oxide synthase on gastric emptying. METHODS: nNOS-deficient (knockout) mice were compared with wild-type mice for gastric size, fluoroscopic appearance after gavage of contrast, and histology of the pyloric sphincter. Wild-type mice treated with the NOS inhibitor N(omega)-nitro L-arginine (L-NA) and eNOS-deficient mice were also compared with wild-type and nNOS-deficient mice for liquid and solid gastric emptying. RESULTS: nNOS-deficient mice showed gastric dilation. Fluoroscopy showed delayed gastric emptying of radiologic contrast. There was no marked localized hypertrophy or luminal narrowing at the pyloric sphincter by histology of relaxed wild-type, nNOS-deficient, and eNOS-deficient tissues. Gastric emptying of both solids (28% +/- 27%) and liquids (22% +/- 18%) was significantly delayed in nNOS-deficient mice compared with control wild-type mice (82% +/- 22% for solids; 48% +/- 17% for liquids). eNOS-deficient mice showed no significant difference from wild-type mice (74% +/- 28% for solids; 47% +/- 23% for liquids). Wild-type mice treated acutely with L-NA showed delay in emptying of solids (43% +/- 31%) but not liquids (39% +/- 15%). CONCLUSIONS: Chronic depletion of NO from nNOS, but not eNOS, results in delayed gastric emptying of solids and liquids.

Structure and expression of murine intestinal trefoil factor: high evolutionary conservation and postnatal expression.

Intestinal Trefoil Factor (ITF) is a member of a family of gastrointestinal tract peptides with region-specific expression which are enhanced at sites of injury and repair. In the present study, the murine homologue gene of ITF was molecularly cloned in order to characterize the structure and expression of this peptide in mice. Murine ITF exhibited 78, 95 and 94% nucleotide homology respectively in exons I, II and III, with overall 90% predicted amino acid identity when compared to the rat ITF. Murine ITF exhibited 70% inferred amino acid identity compared with human ITF. Northern blot analysis of various adult mouse tissues demonstrated that ITF is expressed abundantly in the intestine and colon, and minimally in stomach, but not in brain, lung, spleen, kidney, uterus, pancreas, liver, heart or thymus tissues. Expression of ITF appeared to occur as a post-natal event: antibody specific for ITF stains intensely goblet cells in the intestine and colon of three-day old and older mice, but not in the gastrointestinal tract of younger mice or embryos.