The use of dynamic positron emission tomography imaging for evaluating the carcinogenic progression of intestinal metaplasia to esophageal adenocarcinoma.

BACKGROUND: In this study, we investigate the use of PET scanning in the carcinogenic progression of reflux esophagitis to Barrett's esophagus to high grade dysplasia to esophageal adenocarcinoma, and correlate the uptake levels of 18F-FDG related to histological changes, and the rates of proliferation and apoptosis. METHODS: An established esophagoduodenal anastomsis rat model in conjunction with micro-PET scanning at 1 week, 1 month, 3 month, and 6 month after procedure was performed. RESULTS: Increased uptake levels of 18F-FDG were observed in the esophagi after EDA procedure. The higher level of 18F-FDG uptake within esophageal epithelium was identified in intestinal metaplastic transformation and esophagoduodenal adenocarcinoma by histological examination. CONCLUSIONS: Dynamic PET scanning represents a powerful tool in analyzing morphological carcinogenic transformation non-invasively in the esophagus. 18F- FDG accumulation was a sensitive marker in reflux esophageal injury carcinogenic progression from intestinal metaplasia to EAC.

Chemoprevention of carcinogenic progression to esophageal adenocarcinoma by the manganese superoxide dismutase supplementation.

PURPOSE: Oxidative stress is related to the carcinogenic pathway of reflux esophagitis to Barrett's metaplasia to esophageal adenocarcinoma (EAC). Recent studies have shown that a decreased manganese superoxide dismutase (MnSOD) level is associated with the increased incidences of Barrett's esophagus (BE) and EAC. The aim of this study was to investigate MnSOD supplementation as a chemopreventive agent to prevent oxidative injury and subsequent BE and EAC formation. EXPERIMENTAL DESIGN: Our esophagoduodenal anastomotic (EDA) model was done on rats according to our established procedure and treated with Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP; 10 mg/kg, i.p. every 3 days). Histologic changes were determined after the EDA model at 1, 3, and 6 months. Lipid peroxidation and 8-hydroxy-deoxyguanosine for DNA oxidative damage were determined by thiobarbituric acid-reactive substance assay and immunohistochemical staining. Enzymatic activities of MnSOD and Cu/ZnSOD were evaluated, and the rate of proliferation was determined by proliferating cell nuclear antigen staining. RESULTS: Severe esophagitis was seen in 100% of the EDA rats, and morphologic transformation within the esophageal epithelium was observed with intestinal metaplasia (40% of animals) and cancer (40% of animals) identified after 3 months. Decreased oxidative damage, along with the decreased degree of esophagitis and incidence of BE (20%) and EAC (0%), was found in MnTBAP-treated EDA rats comparing with the saline-treated EDA control. Decreased proliferation (46%) and increased SOD enzymatic activities (25%) were also found in the EDA rats treated with MnTBAP. CONCLUSION: MnTBAP protected rat esophageal epithelium from oxidative injury induced by EDA, and it could prevent the transformation of esophageal epithelial cell to BE to EAC by preservation of antioxidants.

Loss of manganese superoxide dismutase expression and activity in rat esophagus with external esophageal perfusion.

BACKGROUND: Manganese superoxide dismutase (MnSOD), the primary antioxidant enzyme that scavenges superoxide radicals found in the mitochondria, has been shown to protect oxygen-utilizing cells from the toxicity of the reactive oxygen species (ROS). Current studies in the animal esophageal reflux model are limited, and the reports on the relevance of protein expression level and enzymatic antioxidative activity of MnSOD in esophageal mucosal defense are controversial. Thus, the aim of this study is to investigate the role of MnSOD expression and activity in rats with esophageal perfusion injury. METHODS: We have established a novel external esophageal perfusion (EEP) animal model that allows for esophageal reflux injury. We used the model with 0.5% bovine bile as the perfusion agent in one group of rats and used saline in another group to serve as controls. The esophageal mucosal was isolated for MnSOD expression and activity analysis. RESULTS: Severe esophagitis was observed in the mucosa at 1, 2, and 4 week(s) after bile perfusion. A significant decrease in MnSOD expression with bile perfusion was demonstrated by Western blotting and immunohistochemical evaluation. Similarly, a reduction in MnSOD enzyme activity was observed in bile-perfused rats compared with the saline-perfused controls; no decrease in copper/zinc SOD enzyme activity was observed. CONCLUSIONS: MnSOD expression and activity is decreased in bile-induced esophagitis. This decrease in MnSOD expression and activity is associated with esophagitis and cell death. This study suggests that the loss of MnSOD protein contributes to the reduced level of its enzymatic activity and plays a key role in the induction of esophagitis.

Morphological transformation in esophageal submucosa by bone marrow cells: esophageal implantation under external esophageal perfusion.

Accumulating clinical and experimental studies indicate that Barrett's esophagus might arise through multipotential stem cells under the stress of gastroesophageal reflux. Previously, we have presented a novel external pump perfusion rat model and demonstrated that perfusion with both acid and bile can induce severe esophagitis in 1 week with a similarly pathological change seen in humans. The aim of this study was to investigate the histological changes of esophagus after bone marrow cell engraftment with bile and acid perfusion. The external pump perfusion procedure involved implantation of a microosmotic pump for esophageal perfusion. Bone marrow cells were obtained by flushing of the femur marrow, and the cell suspension was injected between the esophageal muscular and inner mucosa layer. Histological changes were determined after 4 weeks of perfusion. Proliferating cell nuclear antigen, 8-hydroxy-deoxyguanosine, manganese superoxide dismutase, and apoptosis were measured by immunohistochemical staining and TUNEL assay, respectively. Severe esophagitis was seen in both acid and bile perfusion. Bone marrow engraftment and potentiation was seen in both the acid and bile perfusion, when compared to saline controls. Glandular-like cells in submucosa, consistent with intestinal metaplasia, confirmed by Alcin Blue-PAS staining were observed after bone marrow esophageal implantation along with bile perfusion, but not with acid perfusion and controls. Bone marrow implantation in conjunction with esophageal reflux injury contributes to abnormal histological changes consistent with early Barrett's esophageal changes. Engrafted bone marrow cells proliferate under oxidative stress conditions with bile perfusion.

Cyclooxygenase-2 and epithelial growth factor receptor up-regulation during progression of Barrett's esophagus to adenocarcinoma.

AIM: To investigate the expression of cyclooxygenase-2 (COX-2) and epithelial growth factor receptor (EGFR) throughout the progression of Barretts esophagus (BE). METHODS: COX-2 and EGFR protein expressions were detected by using immunohistochemical method. A detailed cytomorphological changes were determined. Areas of COX-2 and EGFR expression were quantified by using computer Imaging System. RESULTS: The expressions of both COX-2 and EGFR increased along with the progression from BE to esophagus adenocarcinoma (EAC). A positive correlation was found between COX-2 expression and EGFR expression. CONCLUSION: COX-2 and EGFR may be cooperative in the stepwise progression from BE to EAC, thereby leading to carcinogenesis.

Rosai-Dorfman disease of the gastrointestinal tract: report of a case and review of the literature.

Rosai-Dorfman disease (RDD) is a rare, acquired disease of unknown etiology that affects primarily children and young adults. It is characterized by a proliferation of distinctive histiocytes in the lymph nodes and/or extranodal sites. Involvement of the gastrointestinal tract is rare. We report a case of RDD in a 60-year-old woman who presented with hematochezia and was found to have RDD of the rectum presenting as a rectal mass. This report highlights the current pathogenetic mechanisms, immunohistochemical markers, and the gastrointestinal manifestations of RDD.

Flt3-ligand treatment prevents diabetes in NOD mice.

The mechanism by which mixed chimerism reverses autoimmunity in type 1 diabetes has not been defined. NOD mice have a well-characterized defect in the production of myeloid progenitors that is believed to contribute significantly to the autoimmune process. We therefore investigated whether chimerism induces a correction of this defect. Mixed chimerism restored production of myeloid progenitors in NOD mice to normal levels. Notably, NOD bone marrow cells as well as donor bone marrow cells produced the mature myeloid progeny, and the level of donor chimerism was not correlated with the degree of restoration of the defect. Moreover, NOD bone marrow cells cultured with Flt3-ligand developed a heat-stable antigen-positive/Ly6C+ population comprised primarily of mature myeloid dendritic cells, suggesting that the underlying abnormality is not cell intrinsic but rather due to a block in development of mature myeloid progeny, including myeloid dendritic cells. Strikingly, treatment of NOD mice with Flt3-ligand significantly decreased insulitis and progression to diabetes and was associated with a significant increase in myeloid dendritic cells and in vivo induction of CD4+/CD25+ cells in the pancreatic lymph node. Therefore, Flt3-ligand treatment and/or the establishment of mixed chimerism in prediabetic candidates may provide a benign and novel approach to treat diabetes.

Association of manganese superoxide dismutase expression with progression of carcinogenesis in Barrett esophagus.

HYPOTHESIS: The down-regulation of manganese superoxide dismutase (MnSOD) expression plays a role in the progression of Barrett esophagus (BE). DESIGN: An evaluation of 92 esophageal samples, including 17 patients with normal esophagus, 22 with intestinal metaplasia, 22 with indefinite/low-grade dysplasia, 16 with high-grade dysplasia (HGD), and 15 with esophageal adenocarcinoma were evaluated for MnSOD expression. We evaluated MnSOD expression using immunohistochemistry and graded it separately on a 2-category ordinal scale in relation to the mucosa and submucosa that ranged from 0 (no staining) to 3 (strong staining). The total grading score of MnSOD immunoreactivity was the addition of mucosa and submucosa intensity, from 0 (no immunoreactivity in any of the anatomic sites) to a maximum score of 6 (strong staining reaction in both of the histoanatomic sites). SETTING: Study subjects were recruited from the Barrett's Esophageal Registry at the University of Louisville, Louisville, KY. MAIN OUTCOME MEASURE: Manganese superoxide dismutase expression in established groups of progressive BE. RESULTS: Ninety-two samples were evaluated for MnSOD expression. The expression of MnSOD was found to be significantly reduced in samples with specialized intestinal metaplasia (mean score, 1.8), low-grade dysplasia (mean, 2.2), high-grade dysplasia (mean, 2.4), and esophageal adenocarcinoma (mean, 2.4) when compared with normal esophagus (mean, 3.9; P = .002). Manganese superoxide dismutase expression was similar for esophageal adenocarcinoma and high-grade dysplasia. CONCLUSIONS: Manganese superoxide dismutase expression is significantly reduced in patients with BE with high-grade dysplasia and esophageal adenocarcinoma. Manganese superoxide dismutase is related to the progression of BE and may represent one of the primary factors in oxidative stress protection. Further evaluation within genotypic expression and the role of antioxidants is needed in the effective screening and treatment of BE.

Association of metallothionein expression and lack of apoptosis with progression of carcinogenesis in Barrett's esophagus.

Barrett's esophagus is the transformation of normal esophageal squamous epithelium to specialized intestinal metaplasia (SIM). Among the Barrett's specialized cells, those that can develop protective mechanisms against apoptosis may have potential to become malignant. Studies have shown that overexpression of metallothionein (MT), low molecular protein that protects cells from apoptotic stimuli, appears to be associated with more advanced, highly malignant tumors. We thus investigated the relationship between MT expression and apoptosis in different stages of Barrett's carcinogenesis. Terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling and immunohistochemical dual-staining assay were performed in human biopsy samples of normal, SIM, dysplasia, and adenocarcinoma. Apoptotic index and MT expression were quantified by using an image system to analyze the converted digital data. A negative correlation between MT expression and apoptotic index was found. MT expression was significantly increased along with the histologic progression towards adenocarcinoma. This study thus suggests that MT may contribute to cytoprotection, thereby inhibiting apoptosis and leading to carcinogenesis of Barrett's esophageal cells.

Significant increase in IgG4+ plasma cells in gastric biopsy specimens from patients with pernicious anaemia.

AIM: To investigate the presence of IgG4+ plasma cells in gastric mucosal biopsy samples from patients with atrophic gastritis (AG) and a history of pernicious anaemia (PA) (AG+PA+). METHODS: Gastric mucosal biopsy specimens from 46 patients with AG+PA+ were investigated. As controls, we evaluated specimens from patients with AG but no history of PA (AG+ PA-) (n=25), normal histology (n=25), mild chronic inactive gastritis (MCIG) (n=25) or Helicobacter pylori gastritis (HP) (n=25). IgG4+ plasma cells were detected by two immunohistochemical methods: (1) using a monoclonal antibody, the average of the three most cellular high-power fields was counted in areas with the highest density of IgG4+ plasma cells; (2) using a dual-chromagen stain for both IgG4 and CD138 (plasma cell marker), the number of IgG4+ cells per 200 CD138+ plasma cells was counted. The latter was used to ensure that the number of IgG4+ cells was not simply related to the degree of inflammation (density of plasma cells). RESULTS: Identical results were obtained with the two staining methods. Increased numbers of IgG4+ plasma cells were present in 37% of patients with AG+PA+, but in none with AG+PA-, MCIG, HP or normal gastric biopsy results (100% specific, p=0.0001). CONCLUSION: IgG4+ plasma cells may play a role in the pathogenesis of PA and may be a useful marker for its diagnosis.

Alterations in manganese superoxide dismutase expression in the progression from reflux esophagitis to esophageal adenocarcinoma.

BACKGROUND: Comprehensive understanding of the basic mechanisms in the progression of esophagitis, Barrett esophagus (BE), and esophageal adenocarcinoma (EAC) is urgently needed to develop a management strategy for an effective screening of BE and management of EAC. The aim of this study is to provide a detailed insight of the histology and the cellular and molecular events associated with the genesis of BE and EAC under the esophagoduodenal reflux conditions. METHODS: Esophagoduodenal anastomosis (EDA) was performed on rats. Animals were weighed weekly and killed after 1, 2, 3, 4, 5, and 6 months. The entire esophagi were examined for macroscopic and microscopic changes and for manganese superoxide dismutase (MnSOD) expression, and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay was performed. RESULTS: Morphological transformation from esophagitis (100% of animals) to BE (66% of animals) to EAC was observed after 3 months. There was marked loss of MnSOD expression in animals with esophagitis and BE at 1 and 2 months, with an increase in expression during the transformation to dysplasia and EAC. Increased proliferation and apoptosis was observed and reached a peak at months 1 and 2. Greatly increased levels of 8-hydroxy-deoxyguanosine was found during the progression to EAC. CONCLUSIONS: The morphological transformation of the esophageal mucosa is an adaptive process, and it is an important foundation for the transdifferentiation of BE and cancer. The significant loss of MnSOD expression to achieve BE and then the adaptive increase in expression to achieve dysplasia and EAC during this transformation may represent a predictive marker in identifying patients who will progress from BE to EAC.

A novel external esophageal perfusion model for reflux esophageal injury.

The current animal models of esophagitis and Barrett's esophagus consist of surgeries that divert the gastroduodenal contents to the esophagus. The limitations of these models are the inability to control the amount and concentration of the refluxate and the causing of significant postoperative stress and morbidity. Eighteen adult rats were cannulated at the upper esophagus and connected to a subcutaneous osmotic micropump to perfuse the esophageal lumen with bile and acid. Animals were sacrificed after 7 days of perfusion. Histological changes were determined. Cell proliferation, apoptosis, lipid peroxidation, and glutathione were measured. Histopathological changes in the bile- or acid-perfused esophagus were consistent with the findings associated with reflux esophagitis. Enhanced proliferation and apoptosis were seen, along with increased oxidative stress. The external esophageal perfusion model enabled precise control of the injurious agent. It induced the histologic and cellular injury of reflux esophagitis after 7 days.

Preconditioning of NOD mice with anti-CD8 mAb and costimulatory blockade enhances chimerism and tolerance and prevents diabetes, while depletion of alpha beta-TCR+ and CD4+ cells negates the effect.

Bone marrow transplantation blocks diabetes pathogenesis and reverses autoimmunity in nonobese diabetic (NOD) mice. However, there is a greater barrier to engraftment in the context of autoimmunity. In the present study, we characterized which recipient cells influence engraftment in prediabetic NOD mice, with the goal to replace myelotoxic conditioning with antigen-specific deletion of reactive host cells. Preconditioning of NOD mice with anti-CD8 and anti-CD154 monoclonal antibodies (mAbs) synergistically enhanced engraftment and significantly reduced the minimum total body irradiation (TBI) dose for engraftment. Strikingly, preconditioning with anti-CD4 mAb significantly impaired engraftment, negating the beneficial effect of anti-CD8, and resulted in a requirement for more TBI-based conditioning compared with controls conditioned with TBI alone. Similarly, more TBI was required when anti-T-cell receptor beta (TCRbeta) mAb was administered as preconditioning. The addition of anti-CD152 to CD154 preconditioning abrogated the engraftment-enhancing effect of anti-CD154. Taken together, these data indicate a role for CD4+ regulatory T cells in vivo which require signaling via CD152 in the induction of chimerism and tolerance in NOD recipients. Notably, disease prevention and reversal of autoimmunity was absolutely correlated with the establishment of chimerism. These studies have important implications for the design of novel clinical approaches to treat type 1 diabetes.

Esophageal injury with external esophageal perfusion.

BACKGROUND: External esophageal perfusion (EEP) with the idea that esophageal perfusion can be controlled with a single ingredient at a constant rate and concentration, might be used to dissect the injurious role of gastro-duodenal secretions for the progression from esophagitis to Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). This study is to evaluate the EEP rat model for esophagitis induced by using a micro-osmotic pump with bile perfusion. METHODS: Eighteen adult rats underwent the EEP procedure. Bile (0.5% bovine bile, pH 7.4) was used as perfusion agent and three types of perfusions were performed: 1 week perfusion, 2 weeks perfusion, and 4 weeks perfusion compared to saline perfusion and sham operation. Histological changes, cell proliferation, apoptosis, 8-hydroxy-deoxyguanosine (8-OH-dG) and Manganese superoxide dismutase (MnSOD) were observed after perfusion and compared. RESULTS: The bile perfusion for 1 week, 2 weeks, and 4 weeks induced mucosa infiltration of inflammatory cells, basal cell hyperproliferation, and papillae hypertrophy in all animals. Histopathology and cellular changes consistent with the findings associated with reflux esophagitis. The apoptotic index, the proliferating index, and expression of 8-OH-dG were significantly increased in the esophageal mucosa compared to controls. MnSOD expression was decreased with bile perfusion compared to saline controls. CONCLUSIONS: The external esophageal perfusion model enabled precise control of the injurious agent. It induced the typical histological injury and cellular changes seen in severe reflux esophagitis. The cellular changes in apoptosis, proliferation and anti-oxidant defense make this model unique for reflux esophagitis studies. Further studies are needed to induce Barrett's esophagus and esophageal adenocarcinoma.

p-Aminophenol-induced hepatotoxicity in hamsters: role of glutathione.

p-Aminophenol (PAP) is a widely used industrial chemical and a known nephrotoxin. Recently, it was found to also cause hepatotoxicity and glutathione (GSH) depletion in mice. The exact mechanism of liver toxicity is not known. The aims of this study were to determine whether PAP can cause acute hepatotoxicity in hamsters and to further investigate the role of GSH in PAP-induced toxicity. PAP was administered ip to hamsters in doses of 200-800 mg/kg. Liver damage at 24 h after PAP administration was assessed by elevations in plasma enzyme activities and histopathologic examination. GSH and cysteine (Cys) levels in liver at 4 h were determined by HPLC. PAP decreased hepatic GSH concentration to 8% and Cys to 30% of vehicle control values. It increased plasma glutamic pyruvic transaminase (GPT) activity by 47-fold and sorbitol dehydrogenase (SDH) activity by 113-fold. PAP also caused severe centrilobular hepatocellular necrosis. 2(RS)-n-Propylthiazolidine-4(R)-carboxylic acid (PTCA), a Cys precursor, attenuated the PAP-induced decreases in hepatic sulfhydryl levels; GSH and Cys were 39% and 78% of vehicle controls, respectively. PTCA also attenuated the PAP-induced elevations in plasma enzyme activities and hepatic necrosis. It was concluded that PAP hepatotoxicity is associated with depletion of hepatic GSH and can be prevented by PTCA.

Diverse antioxidants protect against acetaminophen hepatotoxicity.

The reactive oxygen species-sensitive transcription nuclear factor-kappaB (NF-kappaB) plays a pivotal role in the development of acetaminophen (APAP) hepatotoxicity. We investigated the efficacy of a diverse series of antioxidants in preventing APAP-induced hepatotoxicity. BALB/c mice were divided into four groups and provided with antioxidants incorporated into chow as follows: (1) control diet; or diet supplemented with (2) S-adenosylmethionine (SAMe); (3) green tea polyphenols (GrTP); or (4) (RS)-n-propylthiazolidine-4(R)-carboxylic acid (PTCA). After 5 days on these diets, the animals were further subdivided into (A) given an IP injection with APAP (750 mg/kg), or (B) kept as untreated controls. The animals were sacrificed at 0, 4 h, and 24 h following APAP administration. PAP/vehicle induced marked decreases in hepatic reduced glutathione (GSH) levels and endogenous SAMe concentrations (46%) when compared to controls. APAP also caused severe centrilobular necrosis and marked increase in serum enzyme ALT activity (38-fold). Oral administration of antioxidants significantly attenuated the APAP-induced liver damage and depletion of hepatic GSH. There were profound increases in serum TNF-alpha levels at 4 h following APAP administration in nonsupplemented compared to antioxidant-treated animals, but no significant differences noted after 24 h. Serum amyloid A increased in APAP-challenged mice irrespective of antioxidant treatment. Finally, hepatic SAMe concentrations were drastically decreased 24 h following APAP administration, and these decreases were attenuated by pretreatment with antioxidants. In conclusion, these orally administered antioxidants with dissimilar properties provided protection against liver damage, supporting the potential use of antioxidant therapy in patients with APAP toxicity. This is the first report that GrTP and oral administration of PTCA and SAMe can provide protection against APAP injury in this model.